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P-1 A Comparative Study of Sample Preparation Methods for 2D PAGE Analysis of Plant Tissue

S. C. Carpentier, B. Panis and R. Swennen K.U.Leuven Department of Applied Plant Sciences, Laboratory of Tropical Crop Improvement, Leuven Belgium

More and more genomes are being sequenced thereby generating a lot of useful information. Proteomics is seen as a necessary complement between the genomic sequence information and the actual protein population of a specific tissue, cell or cellular compartment. Post- translational modifications - critical for the understanding of the physiological protein function - can not be analysed based on nucleic acid sequences and changes in mRNA transcript levels do not automatically imply corresponding changes in protein amount or activity. Appropriate sample preparation is still one of the most critical steps in 2D electrophoresis and is absolutely essential for good results. Plant material does not provide a ready source of proteins for investigation by 2 D gel electrophoresis. Plant cells generally have a low protein content and contain high concentrations of proteases and other interfering compounds (e.g. salts, organic acids, phenolics, lignins, pigments, terpenes, etc…). Usually, sample preparation involves the removal of non-protein sample components, the complete dissociation of protein interactions and arrest of protease activity. The plant species under investigation, Musa spp (banana), is known to be exceptionally rich in polyphenolic compounds. Four different approaches were tested to extract total proteins: the common method TCA precipitation in acetone (i) without and (ii) with fractionation, (iii) extraction with phenol followed by precipitation with ammonium acetate and (iv) a no-precipitation extraction procedure with fractionation. Precipitation of sample proteins is very efficient in removing interfering compounds and concentrates proteins in low abundance, both resulting in high- quality gels. However these methods (i,ii, iii) result in an uncontrolled loss of proteins: there is a chance that not all proteins precipitate, a number of the precipitated proteins are difficult to redissolve and proteins can be lost during the washing steps. The phenol/precipitation method is the most efficient in removing interfering substances but it contains an extra risk of losing glycosilated and hydrophilic proteins as they reside preferably in the water phase, yet only the phenolic phase is used onwards for analysis. Fractionation (ii, iv) distributes the many different proteins of one extract over 2 gels, which increases the resolution. The first fraction contains predominantly soluble proteins, whereas the second fraction contains hydrophobic proteins and resolved complexes. Some proteins are represented in both fractions. The uncontrolled loss of proteins is minimized in the no-precipitation extraction procedure (iv) by omitting the extra steps. The absence of the purification steps is reflected in the somewhat lower quality of the image. Varieties extremely rich in interfering compounds show a higher but still acceptable background at the acidic pH. Each method has its pros and cons. The no-precipitation method (iv) can be considered as the most simple one and assures a minimal loss and thus the largest pool of proteins. POSTER SESSION

P-2 Effect of Different Protein Precipitation Methods on Results in Proteome Analysis of Human Blood Platelets

W. Winkler1, M. Zellner1, I. Miller2, M. Chang3 and R. Oehler1 1 Surgical Research Laboratories, University of Vienna, 2 Institute of Medical Chemistry, University of Veterinary Medicine Vienna, 3 Institute of Applied Microbiology, University of Agricultural Sciences Vienna, Austria

Protein precipitation followed by re-solubilisation is a frequently used procedure either to separate sample proteins from contaminating compounds or for concentration of diluted samples. The present study compares the usability of three different methods for protein precipitation in proteome analysis of human platelets. Platelets are non-nucleated cellular particles which play an essential role in blood clotting. They are strongly involved in main health problems such as cardiovascular diseases and diabetes. Platelet proteins were precipitated using either ethanol (EtOH), trichloroacetic acid (TCA) or the also TCA-based PlusOne 2D clean-up Kit (Amersham). The precipitate was solubilised in sample buffer containing urea, thiourea and CHAPS and analysed by 2D-electrophoresis (1st dimension: 24 cm Immobiline Dry strips pH 4-7, 2nd dimension: 11% SDS-PAGE). The protein spots were visualised either by silver stain after the gel run or by binding of fluorescent CyDyes before the gel run. The images were scanned using a Typhoon scanner and processed with an appropriate imaging analysis software. Precipitation with ethanol showed by far the best protein recovery, significantly less protein (40-70% of EtOH precipitated amounts) was recovered by the two methods using TCA. The 2D protein spot patterns of TCA-precipitation and the PlusOne 2D clean-up Kit both showed about 1000 protein spots and were nearly identical. However, ethanol precipitated proteins showed a completely different spot pattern which contained 20% more protein spots. Although EtOH precipitated samples contained less high MW proteins, they displayed a much higher number of protein spots (50%) below 60 kDa. Only about 50% of the spots could be found in all three samples. In addition, EtOH precipitated sample proteins showed a better separation of the single spots throughout the gel. Taken together, our results confirm that the precipitation method has a strong effect on the results of 2D-electrophoresis. It influences which and how many spots are detectable. EtOH precipitation resulted in the best separation patterns of human platelet proteins. POSTER SESSION

P-3 Specific Removal of Multiple High Abundance Proteins from Human Sera

G. R. Nicol1, N. Zolotarjova1, J. Martosella1, B. Boyes1, C. Sauber2 and F. Mandel2 1Agilent Technologies, Wilmington, U.S.A.; 2Agilent Technologies, Waldbronn, Germany

Introduction: Analysis of the protein components of complex protein samples often require the specific removal of very high abundance proteins for the detection and characterization of lower abundance proteins in the sample. A high abundance protein may have mass representation in the sample of 2–60 % of the total protein present. The challenges of analyzing complex protein samples are obvious in the characterization of proteins present in plasma or serum. In such samples, proteins and peptides of interest are present in abundances ranging from 55 % of total protein (e.g., serum albumin) to levels less than 10 orders of magnitude of total protein. Although standard interactive LC methods, such as ion exchangers, have been used to fractionate complex protein solutions, a preferred approach is the use of specific interaction media for the selective removal of target high abundance proteins.

Methods: Specific removal of high abundance target proteins can be accomplished by immunoaffinity chromatography (for example, using resins produced by immobilization of antibodies specific to HSA and other high abundance proteins). We have developed an immunoaffinity column that selectively removes six of the highest abundance proteins (HSA, transferrin, haptoglobin, IgG, IgA and anti-trypsin) from human serum. To compare the specificity of Cibacron Blue and Immunoaffinity column approaches for proteomic sample preparation, we have identified the proteins that bind non-specifically to Cibacron Blue, by the use of affinity isolation, followed by gel electrophoretic separation, band excision, proteolytic digestion and MALDI-TOF MS, or by digestion followed by multidimensional LC/MS.

Preliminary Data: The immunoaffinity column we have developed can be used to resolve proteins in serum or plasma samples more than 200 times with minimal or no loss of binding capacity. We find that this immunoaffinity chromatographic method shows less non-specific binding than other methods for removing high abundance proteins, such as the widely used Cibacron Blue resin (CB-resin). Although it is generally acknowledged that CB-resins bind proteins other than serum albumins, little information is available on the identities and quantities of serum proteins bound by CB.

We have demonstrated that a variety of serum proteins, in addition to serum albumin, bind to CB-resin. Serum proteins were bound to CB-resin, the resin washed extensively, and the CB- resin binding proteins eluted. The eluent was passed over an immunoaffinity resin specific for human serum albumin. The proteins which were unretained by the immunoaffinity column were collected and analyzed using gel electrophoesis and multidimensional LC/MS/MS. A wide variety (>75) of serum proteins were found to bind CB-resin. The amounts and identities of these serum proteins suggest that using CB-resin for sample preparation prior to proteomic analysis will result in loss of useful information for a wide range of serum proteins. This problem is not apparent with appropriate immunoaffinity methods. POSTER SESSION

P-4 Prefraction of Samples for Two-dimensional Gel Electrophoresis with Gel Filtration

K. Katsuta1, T. Ueda1, M. Toriyama1, T. Mori1 and N. Inagaki1,2 1Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma, Nara, Japan, and 2Recognition and Formation, PRESTO, JST, Kumamoto, Japan

One of the goals of proteomics is to detect and identify as many proteins as possible expressed in cells and tissues. The combination of two-dimensional gel electrophoresis (2- DE) and mass spectrometry (MS) is a core technology of proteomics. However, it is still not easy to detect and identify low abundant proteins by these procedures, because amounts of proteins in spots on 2-DE gels are not enough. Here we applied gel filtration chromatography prior to 2-DE in order to improve spot recovery on 2-DE gel. Insoluble protein fraction of postnatal 14 day rat brain was solubilized by a buffer containing 4.9 M urea, 1.4 M thiourea, 2.8 % CHAPS, 0.35 % TritonX-100, 0.0435 % SDS, 200 mM Tris-HCl (pH 7.5) and 1.5 M NaCl. Then protein extracts were separated by gel filtration chromatography (Superdex prep grade gel filtration column, Amersham Bioscience) for prefractionation, precipitated by 2D clean-up kit (Amersham Bioscience), resolubilized by 7 M urea, 2 M thiourea, 4 % CHAPS, 0.5 % TritonX-100, 0.0625 % SDS, 100 mM DTT and 0.5 % IPG buffer, and applied onto 2-DE. First, high amounts of protein extracts (1 - 3 mg) were applied onto 2-DE without prefractionation. When samples more than 2 mg were applied, proteins aggregated during IEF and less protein spots were detected on 2-DE gels. Next, protein extracts were fractionated by gel filtration. SDS-PAGE confirmed that protein bands were enriched by gel filtration according to their molecular weights in each fraction. When the prefractionated samples were applied onto 2-DE gels, improved spot recovery as well as an increased number of protein spots were observed as compared with the unfractionated samples. Consistently, remarkably increased peptide peaks were detected in MALDI-TOF-MS spectra from spot samples prefractionated by gel filtration. These results suggest that sample prefractionation by gel filtration enables to enrich proteins according to their molecular weights and are effective for detection and identification of low abundant proteins by 2-DE and MS. There are several advantages of the prefraction by gel filtration. First, proteins are enriched according to their molecular weights. Therefore, it is easy to predict fractions in which a protein of interest is exists. Second, molecular-weight- based sample enrichment also enables to reduce the total amounts of the protein application onto IEF. This is important for successful 2-DE, because sample overloads to IPG stripe disturbs the IEF procedure. Third, this method enables to enrich high amounts of protein extracts (over 70 mg) in a single filtration run. Finally, gel filtration is possible to combine with other prefractionation methods, such as, subcellular separation [1], isoelectric focusing [2] and reverse phase chromatography [3] for further enrichment of sample proteins.

References [1] Krapfenbauer, K., et al. (2003) Electrophoresis 24: 1847-70. [2] Gorg, A., et. Al. (2002) Proteomics 2: 1652-7. [3] Van Den Bergh, G., et. Al. (2003) Electrophoresis 24: 1471-81. POSTER SESSION

P-5 Sample Prefractionation with Sephadex-IEF

A. Köpf, O.Drews, W. Weiss and A. Görg Technische Universität München, Fachgebiet Proteomik, Freising-Weihenstephan, Germany www.wzw.tum.de/proteomik

Different electrophoretic procedures for enrichment of low-abundance proteins have been reported, most of which are based on electrophoretic prefractionation according to isoelectric point (pI) in the liquid phase, such as preparative isoelectric focusing (IEF) in a rotating, multichamber device, free flow electrophoresis, or multicompartment electrolyzers with isoelectric membranes (Righetti et.al). This type of prefractionation allows the loading of much higher protein concentrations on narrow IPG-IEF gels and allows detection of low-abundance proteins because major interfering proteins such as albumin can be removed. However, the major drawbacks of electrophoretic prefractionation procedures in the liquid phase are that (1) sophisticated instrumentation is required, (2) the sample solution is usually diluted during or after the separation process, and (3) protein precipitation cannot always be avoided. To circumvent the formentioned obstacles, we developed a simple, cheap, and fast prefractionation procedure based on flat-bed IEF in granulated gels. We have adapted flatbed IEF in granulated gels (described by Radola in 1973 for the separation of enzymes) for sample prefractionation before loading on 2-D gels with narrow pH ranges. Complex sample mixtures are prefractionated in flat-bed Sephadex gels containing urea, and/orthiourea, zwitterionic detergents (e.g. CHAPS), dithiothreitol, and carrier ampholytes. After IEF, up to ten Sephadex fractions alongside the pH gradient are removed with a spatula and directly applied onto the surface of the corresponding narrow-range IPG strips for the first dimension of 2D-PAGE. Using pre-stained low molecular weight pI markers, the pH gradient can be measured directly in the Sephadex gel with the tray still on the cooling plate. The major advantages of this technology are the highly efficient electrophoretic transfer of the prefractionated proteins from the Sephadex IEF gel fraction into the IPG strip without any sample dilution, and the full compatibility with subsequent IPG-IEF: Fractionated samples have not to be eluted, concentrated or desalted, nor does the amount of the carrier ampholytes in the Sephadex gel fraction interfere with subsequent IPG-IEF. Moreover, in contrast to applying unfractionated samples onto narrow pH range IPG gels, this method not only allows the detection of a much higher number of proteins, but also helps to save samples which are available in only limited quantity, since no proteins are lost by precipitation outside the pH interval applied. This technology has been successfully applied for the prefractionation of mouse liver proteins with sample loads in mg amounts (Görg et al., Proteomics 2002, 2: 1652-1657). POSTER SESSION

P-6 Utilization of Subcellular Proteome Extraction to Monitor Regulatory Protein Redistribution

A. Aboldzade-Bavil, J. Anders and R. Hendriks Merck KGaA, Life Science Products R&D MDA, Darmstadt, Germany, www.merck-lsp.de

Background: Identification of less abundant proteins is facilitated significantly by prefractionation schemes that reduce the protein complexity prior to analysis. There for, w e have developed an all-in-one subcellular proteome extraction scheme for reproducible fractionation of samples prior to analysis.

Method: SOPs were developed for serial sample preparation of complex protein mixtures according to differences in subcellular localization. Subcellular fractionation was monitored using immunofluorescence microscopy taking advantage of tracking dyes specific for numerous subcellular compartments and GFP. Proteins were separated by 1DGE or 2DGE, visualized and analyzed in more detail by immunoblotting (IB). The respective subcellular fractions were also assayed for the activity of marker enzymes.

Results: Fluorescence microscopy indicated that the sequential extraction seemed selective on the subcellular level, a finding that was confirmed on the protein level by IB with antibodies directed against appropriate marker proteins. Furthermore, the protein patterns of the respective fractions are clearly distinct. Thus, stepwise extraction yields four different fractions enriched in (1) cytosolic proteins, (2) proteins of membranes and organelles, (3) soluble and DNA-associated nuclear proteins and (4) cytoskeletal proteins. Activity profiling show the compatibility of the extraction procedure with enzyme assays and demonstrated the separation of the cell components according to their subcellular localization in their native and functional state. The procedure was successfully used to monitor redistribution events of NfkappaB upon stimulation of cells.

Conclusions: As substantially more protein spots can be identified in 2DE gel arrays that lack most soluble and high abundant proteins, sequential extraction of proteins according to their subcellular localization (using the ProteoExtract™ Subcellular Proteome Extraction), raises the chance to visualize low-abundant proteins. The ProteoExtract™ Subcellular Proteome Extraction Kit allows linkage of expression profiling by 2D gel electrophoresis with functional data from e.g. enzyme assays. In addition, it enables redistribution analysis of regulatory proteins upon influence of disease/ experimental factors. POSTER SESSION

P-7 Towards Subcellular Fractionation Procedures for Small Brain Tissues

H.G. Nothwang, J. Schindler, I. Guillemin, M. Becker and E. Friauf Sinnes- und Entwicklungsbiologie, Abt. Tierphysiologie, Universität Kaiserslautern

A subcellular proteome analysis is crucial for the understanding of physiological processes as it assesses the molecular repertoire of functional entities such as organelles or different membrane compartments. Furthermore, it alleviates the functional characterisation of unknown proteins, because its location is a major determinant of a protein’s function. This analysis, however, faces the difficulty that current protocols do not keep pace with the progress made in protein identification by mass spectrometry. The latter technique allows the analysis of minute amounts of protein samples, whereas compartment-specific protein isolation still requires a huge amount of starting material. To overcome this drawback, we currently develop novel, rapid fractionation protocols that cope with the increased sensitivity in protein identification and that can be applied to small tissue samples. One protocol will be presented for a rapid two-step procedure using differential centrifugation in a 1.5 ml reaction tube that can be scaled down to minute amounts of tissue (e.g. twenty punches (diameter 300 µm) from 300-µm-thick brainstem slices). It yields three fractions, highly enriched for nuclei, cytoplasm, and membranes (including mitochondria, the Golgi- apparatus (Golgi), the endoplasmatic reticulum (ER), and the plasma membrane, as judged by immunoblot analysis of marker proteins). This protocol can be applied to both fresh and frozen tissue, with higher discriminatory power when using frozen material. The three fractions are currently characterized by 2-D gel electrophoresis and mass spectrometry to gain further insight into their composition. To efficiently isolate plasma membrane proteins which play a pivotal role in brain function, a previously published aqueous two-phase affinity partitioning system for liver proteins is currently being adapted to brain tissue. This procedure is based on the selective affinity of plasma membranes to dextran-linked wheat-germ agglutinin. Immunoblot analysis demonstrated that Golgi, ER, and nuclear proteins were almost completely removed and that the mitochondrial protein content was reduced by more than two-thirds. Concerning plasma membrane enrichment, controversial data were obtained. Immunoblot analysis indicated only a 2-3fold enrichment, whereas the analysis of the fraction by 16-BAC-SDS gel electrophoresis and mass spectrometry revealed that seven out of 12 spots analysed had previously been found in plasma membrane protein extracts such as Na+/K--ATPase, neurofilaments and tubulin; three proteins were of mitochondrial origin, and two from the cytosol. This points to a higher degree of enrichment for plasma membrane than indicated by the immunoblot analysis. Quantitative analysis indicates a recovery rate of ~50% for plasma membrane proteins. Immunoblot analysis of the different steps revealed that 30% of the plasma membrane were lost in the interphase of the initial two-phase partitioning system. We currently investigate whether this loss can be reduced by an additional reextraction step. In summary, these novel fractionation protocols represent a first step towards subcellular proteome analysis of small brain tissue samples. Further refinements are required to get a resolution that can cope with the many subcellular compartments. POSTER SESSION

P-8 Simplified 2-D Electrophoresis with DeStreakTM

K. Larsson, B. Bjellqvist, S. Bourin, S. Cetinkaya, J. Goscinski, J.J. Hedberg and I. Olsson Discovery Systems, Amersham Biosciences, Uppsala, Sweden

Isoelectric focusing (IEF) of proteins in the basic pH-range has always been problematic, when used as first dimension in 2-D electrophoresis. The reason for poor resolution and lack of reproducibility is mainly the difficulties to maintain the proteins in a reduced state. A major drawback with DTT as reducing agent in basic IPG-strips is the transport of DTT from the basic part towards the anode during focusing. Thereby oxidation reactions can take place generating inter and intra peptide disulfide bridges. The result is streaking and changes of the spot pattern over time. The solution to these problems is to include DeStreak [hydroxyethyldisulfide] in the rehydration step preceding focusing. Instead of trying to keep the proteins in a reduced state the cysteinyl groups are specifically oxidized by the use of DeStreak to ”mixed disulfides”.

In earlier work with DeStreak the ordinary equilibration procedures were used i.e. strips were either only reduced or alternatively reduced and then alkykated prior to transfer to the second dimension gel. In the present work different equilibration procedures, which maintain the disulfides generated in the focusing step also during the second dimension run have been tested. Resulting 2-D maps have been compared to those generated with cysteinyl groups either reduced or alkylated prior to the second dimension run. Well-polymerized second dimension gels, containing none or small amounts of acrylamide, resulted in marked vertical streaking when only the reducing equilibration step was used. Maintaining the cysteinyl groups as "mixed disulfides”, as well as alkylation prior to the second dimension run eliminate this streaking. Destreak enables the equilibration between the first and the second dimension to be simplified. Since DeStreak in the strip gives proteins with already protected cysteines all that is needed for equilibration is in many cases a short rinse in a buffer solution. POSTER SESSION

P-9 Protein Carbamylation – An Actual Problem in 2-D Electrophoresis?

L. Thoenes, O. Drews, A. Görg and W. Weiss Technische Universität München, AG Proteomik, Freising-Weihenstephan, Germany www.wzw.tum.de/proteomik

The preparation of protein samples for denaturing 2-D electrophoresis in proteome analysis typically requires high concentrations of urea (8 - 9 M) to ensure that protein complexes are disrupted and unfolded into individual polypeptides. In aqueous solution urea is in equilibrium with ammonium cyanate, which can react with the amino groups of proteins and modify their isoelectric points (pI), and it has been discussed controversially whether this reaction also happens under 2-D electrophoretic conditions. The present study has been undertaken to analyse the degree of protein carbamylation under typical 2-D electrophoresis conditions. Different parameters (singly or in combination) such as concentration (0-8 M), storage time (24 - 72 h) and pH (7 and 9) in urea solution, as well as the effect of carrier ampholytes (which may act as as cyanate scavengers) at temperatures up to 37°C have been investigated by one-dimensional isoelectric focusing and 2-D electrophoresis using the two model proteins myoglobin and carbonic anhydrase. To verify the critical parameters bordered with these model protein experiments, a complex cell lysate of Lactococcus lactis was then analyzed with high resolution 2-D electrophoresis. The results of the model protein experiments revealed (i) that different proteins may have distinct "sensitivity” to carbamylation, and (ii) pI modification can even occur at moderate temperatures (20°C), given that their storage time in highly concentrated urea solutions in absence of carrier ampholytes exceeds 24 h. On the other hand, carrier ampholytes (1 - 2%), which are included in most standard 2-D PAGE protocols, inhibit carbamylation very effectively, even after incubating the samples in urea solutions at temperatures up to 37°C for a period up to 24 h. These results were confirmed by analyzing the complex Lactococcus lactis cell lysate. No differences were observed in the 2-D spot patterns between the controls versus samples incubated at 20°C for 24 hours. Higher temperatures (37°C) in absence of carrier ampholytes produced "spot trains”, i.e. rows of spots with identical molecular weights but different pIs, but, again, these "spot trains” were prevented with high effeciency by adding 2% of carrier ampholytes to the sample solubilization buffer. In conclusion, protein carbamylation under typical 2-D electrophoretic conditions does not seem to occur, since carrier ampholytes, which are included in most sample solubilization buffers, efficiently preclude carbamylation of proteins, even in concentrated urea solutions, for periods of up to 24 hours at temperatures of 37°C, and at least up to 72 hours at 20°C. POSTER SESSION

P-10 Cell Isolation and Subsequent Fractionation of Glomeruli Monitored by Cy 2, 3, and 5 Dyes

C. Mayrhofer1,2, S. Krieger2, G. Allmaier1, D. Kerjaschki2 1Institute of Chemical Technologies and Analysis, Vienna University of Technology, Vienna, Austria, and 2Institute of Clinical Pathology, University of Vienna, Vienna, Austria

Kidney function depends on an intact glomerular filtration unit, allowing the excretion of potentially toxic low molecular weight compounds but retaining essential macromolecules. The permeselectivity of the glomerular filter is defined by a fenestrated endothelial cell layer, the glomerular basement membrane and visceral glomerular epithelial cells or podocytes. Glomerular diseases remain the main cause of renal failure. Damage to the filtration barrier is associated with a loss of the filtration slits, formed by podocytes, and change of the foot processes, leading to proteinuria. But the molecular mechanisms of diseases leading to proteinuria are poorly understood. (J Am Soc Nephrol 13: 1586-1594, (2002) Rantanen M. et. al.). Therefore the analysis of the proteom, especially a sub fraction, namely the membrane proteom, of glomerular cells by means of 2-dimensional gel electrophoresis and MALDI mass spectrometry based identification is of general interest. Cell isolation and subsequent fractionation of cell lysates are important and the most critical factors in clinical proteomics. We tested the CyDye DIGE Fluors (Amersham, Uppsala, S) for labeling of intact cells, in order to observe if it is possible to stain the cell surface and to detect by this means plasma membrane proteins. In our first experiments intact human primary osteogenic sarcoma (HTB-85) cells, in PBS pH 8.0, were labelled with Cy5 fluor for 45 minutes. Following labeling (the reaction was monitored by fluorescence microscopy) they were subsequently lysed and proteins were separated in a membrane and a cytosolic fraction. By applying 2-dimensional gel electrophoresis (Multiphor II (IEF) and Ettan DALT six (SDS-GE), Amersham) with fluorescence scanning (Storm or Typhoon 9400, Amersham) as well as silver staining we were able to observe the labelled proteins. In the membrane fraction a similar pattern of fluorescence labeled and silver stained spots could be observed, although not all fluorescent labeled proteins were silver stainable and vice versa. In the cytosolic fraction, only a few spots were Cy5 fluor labeled in comparison to silver staining. Further experiments were done with all three CyDye fluors (2, 3 and 5). The application of Cy5 fluor showed the strongest signal intensities and was most comparable with the silver-staining pattern. Afterwards MALDI mass spectrometric analysis was performed by in-gel digestion of the fluorescence marked protein spots for identification. After optimisation of the whole strategy the method sequence was applied to intact mouse podocytes, grown either at 33°C or at 37°C (mouse podocytes harboring a temperature- sensitive SV40 large T antigen under the control of a g-interferon inducible promotor (Exp Cell Res 236: 248-258 (1997) Mundel P. et. al.) and the obtained results will be presented. Furthermore, glomeruli were isolated from kidneys of either healthy or puromycin aminonucleoside (Puromycin aminonucleoside nephrosis is widely used as a model of nephrotic syndrome (Am J Pathol 161: 1597-1606 (2002) Yaoita E. et. al.)) treated male rats and a similar strategy was applied.

Part of this work was supported by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung (SFB F005F507) POSTER SESSION

P-11 Current 2D Electrophoresis based Technologies in Stress Response Assessment: Fluorescence- (DIGE) & Radioactive Pulse-Labeling

O. Drews and A. Görg Technische Universität München, Fachgebiet Proteomik, Freising-Weihenstephan, Germany

One major endeavor of proteomic analyses is the assessment of differences between proteomes. These differences facilitate the discovery of clinical markers, therapeutic targets or information about cellular mechanisms, for example in response to stress. Recently, a new fluorescence based technology was introduced to improve such analyses. The special feature of this technology is the possibility to compare two proteomes in the same two- dimensional gel by tagging different fluorescence dyes to the proteins prior to the electrophoresis. A well-established and very sensitive approach to detect differences is radioactive pulse-labeling of the proteins at the moment of a changed condition. In the present study different stress conditions applied to Lactococcus lactis were characterized by these two technologies. In course of this, the potentialities of each method in a differential analysis became apparent. Lactococcus lactis was grown in chemically defined media under strictly consistent conditions to exclude other influences than the chosen stress condition. For radioactive pulse- labeling 55 µCi Promix™ was added per ml medium. Protein extraction was standardized and optimized to achieve highly reproducible protein patterns and concentrations were determined. Of each condition at least three individual extracts were produced to exclude biological and methodical variances. For difference in-gel electrophoresis (DIGE) proteins were labeled with different CyDyes™. Similar protein amounts of each extract were loaded onto a customized immobilized pH-gradient from 3.5 to 7.5. After the second dimension, gels were directly scanned with the Typhoon 9400 or after drying and exposition to phospho-imaging screens. The obtained protein patterns were analyzed with the Image Master 2D or the DeCyder software (Amersham Biosciences), dependent on the protein labeling. The protein pattern after fluorescence-labeling was comparable to that after silver staining of the same gel. Radioactive pulse-labeling expectedly revealed differences due to the fact that only proteins are visualized during the pulse duration of the growth time of the bacteria. At first, for the evaluation of each technology, a well investigated condition was compared to reference extracts: heat shock. With both methods major alterations in the overall protein pattern were observed. The most influenced protein by heat shock is DnaK. The determined increase by DIGE was about 2,7–fold and by radioactive pulse-labeling about 3,5–fold. GroEL, another well known protein, was also highly affected. Most of the proteins are decreased in their intensity. This is already indicated by the scintillation count of the extract of heat shocked cells, which is in average approximately 55% of the extract of untreated cells. Here, radioactive pulse-labeling gives additional information about the synthesis capacity of the cells. Finally, the response of L. lactis to high hydrostatic pressure was determined. For this, cultures were exposed to 60 and 90 MPa. In general, the change in the protein pattern after 60 MPa was weaker than after 90 MPa. Furthermore, most of the proteins with altered expression after 60 MPa were stronger influenced by 90 MPa. Complete statistical data of a comparative analysis of these conditions after fluorescence- as well as radioactive pulse- labeling will be presented. POSTER SESSION

P-12 Combined Use of Laser MicroDissection and 2-D DIGE for the Identification of Differential Protein Expression Across the Cortical Layers of Cat Visual Area 17

S. Jacobs, E. Van der Gucht, G. Van den Bergh, S. Clerens and L. Arckens Lab. of Neuroplasticity and Neuroproteomics, Katholieke Universiteit Leuven, Belgium

The 6 layers of the neocortex of mammals contribute differently to the functional diversity within the cortical circuitry. We compared the protein expression patterns between cortical layers within cat primary visual area 17. Up till now, manual collection of cortical gray matter seriously hampered such a detailed laminar analysis, since the neocortex is only 2 mm thick. Application of Laser MicroDissection (LMD) allowed the separate procurement of tissue samples from the granular (IV), infra- (V-VI) and supragranular (II-III) layers of cat area 17. To this end a correct demarcation of the six cortical layers was achieved by a histochemical methylene blue staining, revealing specific cell characteristics like size and morphology. The effects of tissue preparation and staining have been investigated and were rendered compatible with subsequent protein analysis. Combination of LMD, fluorescent 2-D difference gel electrophoresis and mass spectrometry identified a first set of layer-enriched proteins in mammalian neocortex, when comparing the protein expression between supra- and infragranular cortical layers of area 17. POSTER SESSION

P-13 Protein Recovery after Separation by 2D-PAGE

E. Traxler, E. Bayer, W. Mosgöller and C. Gerner Institute of Cancer Research, University of Vienna Medical School, Vienna, Austria

Separation of proteins by 2D-PAGE is the most common technique for proteome analysis. Currently we establish quantitative proteome databases listing amounts, synthesis rates and half-lives of a large number of proteins, which reflect important biological characteristics of the investigated cells. For correct protein quantification, it is obligatory to avoid unspecific protein loss during separation. Due to the complex behaviour of protein mixtures, however, the protein quantity recovered in a 2D-spot may be less than the corresponding protein amount, which was present in the original protein sample. Therefore, we determined protein quantities recovered after separation using isoelectric focussing tube gels with carrier ampholytes in comparison to immobilised pH gradients (IPGs) for the first dimension, and SDS-PAGE for the second dimension. Separated radiolabelled proteins were quantified by fluorography and subsequent autoradiography. We loaded 1-, 3-, 9- and 27-fold amounts of a cytosolic protein fraction per gel and determined whether spot intensities of a gel series followed the same measure. Indeed, the intensities of well-focussed protein spots from tube-gels showed a proportion similar to the original protein load, quantification of smears was rather inaccurate. Generally, protein recovery in case of IPGs was substantially less than that obtained with tube gels. Our data indicated that varying protein amounts were lost during equilibration of IPG strips. We conclude that protein separation based on isoelectric focussing with carrier ampholytes results in optimal protein recovery allowing accurate protein quantification. POSTER SESSION

P-14 Use of a Soluble IPG Matrix in Proteomic Analysis of Small Sample Amounts

A.R. Goodall1, H.B. Gutstein2 and B. Herbert1 1Proteome Systems, North Ryde, Sydney, Australia, and 2Departments of Anesthesiology and Molecular Genetics, UT-MD Anderson Cancer Center, Houston, TX, USA.

Fractionation has become a key complexity reduction step in proteomics, especially isoelectric fractionation when downstream 2-D separation is used. However, two key issues arise when dealing with small samples; protein losses can become a major problem and the fractionation instruments and techniques applicable to large samples are not appropriate.

Here we demonstrate the use of a soluble IPG matrix that addresses some of the drawbacks of conventional fractionation and IPGs and enables 2-D based study of small samples. Firstly; IPGs which can be completely solubilized post-focusing enabling isoelectric fractionation of very low protein loads. Secondly; partial solubilization of the IPG matrix during transfer provides increased protein release from the IPG strip to the second dimension gel.

The fractionation capacity of dissolvable IPGs is demonstrated with plasma and rat brain and the enhanced transfer to the second dimension is demonstrated with membrane proteins from yeast. POSTER SESSION

P-15 Description of an Alternative Silver Staining Protocol for Detection of Proteins in Large Two-dimensional Gels

M. Roncoletta1, P.H. Franceschini2 and C.R. Esper2 1Lagoa da Serra Ltda. Sertãozinho/SP – Brasil and 2FCAVJ/UNESP

The silver staining procedure for detecting proteins in 2D gels has been simplified and its costs have been reduced, maintaining stability, controllability and being faster than previous silver staining methods. This proposed method retains its sensitivity to protein detection and reproducible staining patterns. The costs were reduced by re-using some staining solutions, and the protocol is quicker because it works with a special plastic staining tray set that allows staining of up to 10 large gels per repetition. Protein samples from seminal plasma, sperm membrane and lysed oocytes were used. A Multiphor II Electophoresis Unit was used by IEF, and focusing was performed in individual IPG dry strips 3mm wide and 180mm long, with a pH range from 4-7 for seminal plasma proteins and from 3-10 for sperm membrane proteins and lysed oocyte proteins. Prior to IEF, IPG dry strip were rehydrated overnight with a solution containing 40mM TRIS, 8M urea, 2% CHAPS, 0.25% DTT and 0.2% IPG buffer. The rehydrated strips were then placed into the cooling plate of an electro-focusing chamber, covered with Dry Strip Cover Fluid and sample cups were placed on the surface of the gel strips. Samples were diluted to 75µg of total protein in 100ml of solution containing 40mM TRIS, 9M urea, 2% CHAPS, 0.25% DTT and 0.2% of a specific IPG buffer (pH 4-7 or 3-10). A low voltage gradient was applied (0-3500 V) for 90minutes, and then 3500V for 7 hours. Following IEF the strips were equilibrated in 2,5ml of 1,5M TRIS-HCl pH 8.8, 6M urea, 30% glycerol and 2% SDS for 25 minutes. The equilibrated IPG strips were loaded on top of vertical setups with large gel plates with 20x25cm and 1,5mm wide. 10, 13 and 17% SDS PAGE were used for oocytes, sperm membrane and seminal plasma samples, respectively. The equilibrated IPG gel strips were embedded in sealing solution (0,5% agarose, 25mM TRIS, 192mM Glycine and 0,1% SDS). Vertical setups were used for 10 large gel plates using 90 V constant overnight. Silver staining was performed as suggested by Blumm et al. (1987), with some alterations. These alterations were: (1) Solutions 50% ethanol; 0.02% sodium thiosulfate and 120mM silver nitrate containing 0,75% formaline were storage in plastic containers at 4oC to be re-utilized, (2) the fixation soaking time were bigger than 48h (3) developer solution without sodiun thiosulphate, (4) use a special plastic staining tray sets, that can stain 10 large gels per time; (5) use large volume for solutions – 600mL/gel. The staining procedure, used 1200ml of each solution for two large gels, in the same tray, in constant and gentle agitation. The solution baths were done in special plastic staining tray sets. Each set was composed by two plastic trays, in which the upper one was perforated. To stain the 10 gels simultaneously, 5 sets of these containers were piled up. In total, 1500 large gels were stained. The results showed that sit is possible re-use solutions until 8 times or used the same soluitons to staining 80 gels, without loss sensitivity. Use more than 48h of soaking time was necessary to avoid background staining because of the diffusion of ampholytes from the gels to the fixation solution, showing an overall cleaner gel. The background could be minimized using developer solution without sodium thiosulfate too. This protocol showed to be applicable to 2D large gels, using an enormous number of gels, a small amount of protein, a reduced complexity and costs in the silver staining. ACKNOWLEDGMENTS - This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo – FAPESP No. 00/03758-1 e 99/03153-3. POSTER SESSION

P-16 New Ruthenium II Tris (Bathophenantroline Disulfonate) Staining and Destaining Protocol for a Better Signal to Background Ratio and Improved Baseline Resolution

A. Lamanda1, A. Zahn1, D. Röder2 and H. Langen2 1Departement für Chemie und Biochemie, Universität Bern, Bern, Switzerland, and 2F.Hofmann-La Roche, Ltd, Basel Switzerland

The visualisation of proteins in polyacrylamide gels is a central step in proteomics. Recently, non-covalent fluorescent stains have been introduced such as SYPRO Ruby, and staining with Ruthenium II tris-bathophenantroline disulfonate (RuBP) as described by Rabilloud [Proteomics, 1, 2001, 699-704]. The linear dynamic range of detection using fluorescent stains is superior to colorimetric or reverse stains therefore fluorescent detection of proteins in electrophoresis gels is gaining popularity. Unfortunately, fluorescent staining of SDS and 2 DE gels suffer from the drawback of relatively high background, weak signals and bad signal to noise ratio. Whereas SYPRO Ruby is a ready to use formula the RuBP-staining according to Rabilloud could be optimised by varying the reagent concentration, and solvent composition for the staining step. An new and crucial destaining step was added. Here we present the new improved staining/destaining procedure that results in strong signals, superior signal to background behaviour, better linearity and improved baseline resolution. POSTER SESSION

P-17 New Ruthenium II Tris (Bathophenantroline Disulfonate) Staining and Destaining Protocol Part II: Aditional Procedures

A. Lamanda1, D. Stalder1 D. Molina2 and A. Schaller2 1Departement für Chemie und Biochemie, Universität Bern, Bern, Switzerland and 2Department of Clinical Research, 3010 Bern, Switzerland

Staining of proteins with Ruthenium II tris (bathophenantroline disulfonate) in polyacrylamide gels using a new destaining protocol is a routine procedure at the University of Berne. The new technique produces strong signals, superior signal to background ratio, better linearity and improved baseline resolution than current available fluorescent staining techniques. This was demonstrated by Lamanda et al. [Proteomics, 3, 2003, in press]. Here we present 6 additional procedures to:

1. Identify proteins out of RuPB stained gels with MALDI-TOF and PMF. 2. Dry and scan RuPB stained gels (two methods). 3. Blot RuBP stained Protein on nitrocellulose and/or PVDF membranes. 4. Identify protein/RuBP complexes on nitrocellulose membranes with antibodies. 5. Wash out RuBP from a gel (towards total destaining). 6. Sequentially silver stain a RuPB stained gel.

With the help of these procedures we demonstrate the broad application range and the flexibility of the new Ruthenium II tris (bathophenantroline disulfonate) staining and destaining protocol. POSTER SESSION

P-18 2-DE and Liquid Chromatography of Intact Proteins are Complimentary Approaches for Proteomic Analysis of Heart Failure in Swine

I. Neverova and J. E. Van Eyk Department of Physiology, Queen’s University, Kingston, Ontario, Canada

Current proteomic techniques are limited in their ability to analyze complex proteomes. Development of more efficient protein separation and identification methodologies enhances our ability to reveal the diversity and complexity of the protein changes including post- translational modifications (PTM) and isoform switch that occur during the onset and progression of heart disease, such as heart failure (HF). One way to approach the analysis of a complicated proteome, such as cardiac, is to dissect it into distinct subproteomes. We implemented this approach by preparing the specific subproteome using an in-house extraction procedure. The separation of cardiac tissue proteins during this extraction procedure is based on their solubility at various pH. Since the molecular mechanisms underlying HF can be elucidated by identifying the protein alterations in diseased vs. healthy tissue, we investigated protein profiles of left ventricular tissue from an in vivo ischemic- induced HF model in swine. Here we present a complimentary analysis of cardiac subproteomes by 2-DE and RP-HPLC. For instance, in myofilament protein enriched subproteome we have identified more than 50 of medium to low abundance proteins representing mitochondrial, cytoskeletal and nuclear types of proteins using a combination of 2-DE and MS. The separation of intact proteins by reversed phase (RP) HPLC and further peptide mass fingerprinting (PMF) of each collected HPLC fraction allowed to identify up to 100 additional proteins. Although the pI range of identified proteins was within the same range (4.6 to 9.9), the proportion of proteins with pI above 8.0 was 20% greater with RP HPLC separation compared to 2-DE. Moreover, LC separation of proteins greatly improved detection of proteins with molecular weight above 100kDa. The detection of protein isoforms and PTMs using the two approaches is supporting the need for as many complimentary methods as possible to apply. All these protein IDs, in addition to the ones identified by 2-DE, are providing a groundwork for the creation of a comprehensive database of cardiac proteins from swine – a species not well represented in protein databases. A full analysis of disease-induced protein changes includes not only tissue analysis but biological fluids as well. The technical limitation in the analysis of serum is the extent of the dynamic range of protein concentrations. To undertake the analysis of high degree complexity proteome such as serum we applied the same approach of multiple complimentary separation techniques. Extending the proteome coverage and detection of PTMs in tissue and serum is the key to the understanding of driving forces in HF POSTER SESSION

P-19 ICPL - A Novel Approach for Quantitative Proteomics

A. Schmidt, J. Kellermann, C. Ciosto, H. Sarioglu and F. Lottspeich Max-Planck-Institute of Biochemistry, Martinsried, Germany

Quantitative proteome analyses usually are accomplished by 2D-elelctrophoresis (2DE) followed by mass spectrometric protein identification. Although this method is well established, accurate quantitative determination and high reproducibility of 2D-gels need good expertise and experience. Recent developments, like the ICAT reagent [1] or GIST [2] methodology have shown to be powerful alternatives to comparative 2D-gel imaging analysis. Nevertheless, these methods also have their limitations. Here we describe a new method termed Isotope Coded Protein Label (ICPL) which is based on isotopic labeling of all free amino groups in proteins. After multiplexing, any protein separation method may be adopted for reduction of complexity on the protein level (e.g. 1D-, 2D-electrophoresis, FFE, chromatography, etc.) followed by cleavage and high throughput LC-LC-MS-MS identification and quantification using an AB 4700 Proteomics Analyzer. Several amino-reactive reagents have been evaluated concerning separation quality, MS sensitivity and MS-MS fragmentation behavior. Some of these increased MS-response by far and lead to lower detection limits and improved MS-sequencing. For increased throughput, a multiplexed strategy was developed to simultaneously compare 3 samples in one single experiment. Having the same analytical effort, the information obtained could be doubled. The efficiency (e.g. dynamic range, sensitivity, speed) of the method is demonstrated by comparative analysis of two E. coli - proteomes spiked with different amounts of standard proteins. All added proteins could be identified having their correct quantities and high sequence coverage between 50 and 90% after separation by 2DE. Although this approach is preferably designed for separation techniques based on chromatography, it offers several advantages in combination with 2DE compared to the analysis of unlabeled proteins. First, multiple proteins located under the same 2D-spot can be correctly quantified, which is not possible by 2D-gel image analysis. Second, almost every protein is becoming more acidic after labeling, making basic proteins (pI > 10) easier accessible when using 2D-gels. Finally, the effectiveness of peptide mass fingerprint (PMF) searches is greatly improved by selecting labeled peptides only, excluding interfering peaks and increasing search results’ confidence by checking identified peptides for the correct amount of free amino groups. Compared to the ICAT reagent, that modifies the low abundant amino acid cysteine in proteins, higher sequence coverage and thus more information about posttranslational modifications, isoforms and cysteine lacking proteins are obtained with ICPL. Using the GIST approaches isotope labeling of peptides is performed after enzymatic cleavage of the proteins. Although almost every peptide is modified using this strategy, the highly demanded quantitatively controlled separation dimension on the protein level is lost. With the opportunity of using any separation method, this new strategy is suitable to challenge comprehensive quantitative proteome analysis.

[1] Gypi, S. P., et al. Nat. Biotechnol. 1999, 17, 994-99 [2] Chakraborty A., et al. J Chromatogr A 2002, 949(1-2), 173-84 POSTER SESSION

P-20 Shotgun Sequencing of the Human Cerebrospinal Fluid Proteome using 2D Chromatography in Combination with Linear Ion Trap Mass Spectrometry

A. F. Hühmer1, R. G. Biringer1, H. Amato1 and M. Harrington2 1Thermo Electron, San Jose, CA, U.S.A., and 2Huntington Research Institutes, Pasadena, CA, U.S.A.

The human cerebrospinal fluid (CSF) proteome provides a readily accessible window into the health state of the human central nervous system (CNS). Diseases involving the CNS markedly and characteristically alter the concentrations and isoform patterns of CSF proteins. We have recently used 2D-PAGE methods in combination with nanospray LC/MS/MS to identify more than 40 proteins in CSF and to determine significant changes in protein levels associated with acute migraine attacks (Harrington et al., 2003). However, analysis of CSF by multidimensional-LC MS/MS-based methods provide the opportunity for the detection and quantification of lower abundance proteins potentially allowing us to detect additional changes associated with acute migraine attacks. The goal of this study was to utilize LC/MS/MS-based technologies to identify low-abundance proteins from CSF using a multidimensional separation approach in combination with linear ion trap mass spectrometry for their detection and identification. Cerebrospinal fluid was concentrated on a VivaSpin (5000MWCO) ultrafilter separating proteins from the low molecular CSF components. Proteins were subsequently reduced, alkylated and trypsinized for 2D- nanospray LC/MS/MS analysis on a ProteomeX (Thermo Finnigan, San Jose, CA). More than 200,000 tandem MS spectra were acquired during a 2D run, of which many could be assigned with high confidence to proteins in the Swiss-Prot database. Over 100 proteins that were detected in human CSF were identified by two or more peptides. An additional 300 proteins were identified by only one peptide. Due to the high ion capacity storage, the fast scan rates and the efficient tandem analysis capabilities of the linear ion trap, proteins known to be present at the lower µg/L range were detected with high confidence. The results show that we were able to detect many proteins using the 2D-nanospray LC/MS/MS approach previously undetected in human CSF .

Reference: Harrington, MG, Fonteh, AN, Stochaj, WA, Biringer, RG, Amato, H, Hühmer, AFR, Kilian, SC, Pogoda, JM, Chequer, RS, and Cowan, RP. A molecular catastrophe in migraine directs an understanding of pathophysiology and potential treatments. (submitted) POSTER SESSION

P-21 Analysis of Complex Protein Mixtures by Liquid Chromatography in Combination with High Resolution FTICR Mass Spectrometry

C. Ihling and A. Sinz Biotechnological-Biomedical Center, University of Leipzig, Leipzig, Germany

The basic problem of complexity poses a significant challenge for proteomic studies. We are developing gel-free approaches on the basis of nano-HPLC and high resolution FTICR (Fourier Transform Ion Cyclotron Resonance) mass spectrometry. FTICRMS offers several advantages for the analysis of biological samples including excellent resolution and ultra-high mass accuracy [1]. We successfully applied nano-HPLC in combination with nano-ESI FTICR mass spectrometry to analyze proteins separated by 2D-gels [2]. The greatest potential of nano- HPLC / nano-ESI-FTICRMS however lies in the possibility to analyze hundreds of peptide masses in parallel and to characterize highly complex protein mixtures, such as complete cell lysates. A nuclear extract from HeLa cells was first separated by strong cation-exchange (SCX) chromatography to reduce the complexity of the mixture. For the collected fractions different sample preparation procedures were tested for optimum compatibility with subsequent enzymatic digestion and analysis of the peptide mixtures on a nano-HPLC system coupled to a 7 Tesla FTICR mass spectrometer equipped with nanoelectrospray source. Nano-HPLC w a s performed using reversed phase C-18 columns for preconcentration of the samples and reversed phase C-18 capillary columns for separation using water/acetonitrile gradients. Database searches were performed with the Mascot and the Profound software. In this work, we demonstrate that a combination of strong cation-exchange chromatography on the protein level followed by reversed phase nano-HPLC separation and high resolution FTICR mass spectrometry of the enzymatically digested fractions has the potential to be a powerful method for analyzing complex protein mixtures. Strengths and limitations of this approach will be discussed.

References: [1] Quenzer, T. L., Emmett, M. R., Hendrickson, C. L., Kelly, P. H., Marshall, A. G. (2001) Anal. Chem. 73, 1721-1725. [2] Ihling, C., Berger, K., Höfliger, M.M., Führer, D., Beck-Sickinger, A., Sinz, A. (2003) Rapid Comm. Mass Spectrom. 17, 1240-1246. POSTER SESSION

P-22 Peptide Mapping of Human Plasma by Multidimensional LC/MALDI-TOF MS with Integrated On-line Sample Clean-up Using Restricted Access Materials (RAM)

E. Machtejevas1, T. Miliotis2, D. Lubda3, R. Hendriks3 and K.K. Unger1

1Institute fuer Anorganische Chemie und Analytische Chemie, Mainz, Germany; 2Cell Biology and Biochemistry, AstraZeneca Mölndal, Sweden; 3Life Science Products, Merck KGaA, Darmstadt, Germany

Columns with Restricted Access Materials (RAM) have been successfully introduced in automated sample clean-up of peptides and proteins of human hemofiltrate by multidimensional liquid chromatography (LC) off-line coupled to MS [1]. Later we have implemented novelly developed strong cation exchange (SCX)-RAM columns in an automated LC system off-line coupled to MALDI-TOF MS for peptide mapping in urine samples.[2]. In this study we report on the integration of SCX-RAM columns for automated sample clean up into 2D Nano-LC systems off-line coupled to MALDI-TOF-TOF MS-MS. Human plasma w a s used as a sample and up to 800 peptides could be identified with a molecular weight ranging between 700 and 3, 500 Dalton . The system tested provides a powerful technology platform to substantially enhance selectivity, sensitivity, throughput, and robustness for the identification of target peptides and proteins out of biofluids.

References [1] K. Wagner, T. Miliotis, G. Marko-Varga, R. Bischoff, K.K. Unger, Anal. Chem. 65, 12 (2001) 434R. [2] E. Machtejevas, K.K. Unger, C. Lindberg, G. Marko-Varga. Sample clean up of urine using restricted access strong cation exchange columns for protein and peptide mapping. 26th International Symposium on High Performance Liquid Chromatography and Related Techniques, HPLC’02, Montreal, Canada, oral presentation, Abstract Book, page. 118. POSTER SESSION

P-23 LC-based Multi-dimensional Separation of Mouse Brain Proteins

K. Marcus1, C. Lohaus1, O. Schmidt1, H. Schaefer1, A. Nolte1, G. Koerting2, M. Blueggel2 and H.E.Meyer1 1Medical Proteom-Center, Ruhr-University Bochum, Bochum, Germany, and 2Prot@gen AG, Dortmund, Germany

The project ‘Development of Technologies for Functional Proteome Analysis’ – funded by the BMBF – applies new technologies most relevant and efficient for functional proteome analysis focusing on the human and mouse brain. The ability to comprehensively characterize a complex protein sample by mass spectrometry (MS) depends on the power and sensitivity of the separation technique employed prior to the MS analysis. The high complexity involved in analyzing protein mixtures, and the large dynamic range that is associated with this complexity, make a multi-dimensional separation necessary. A number of two dimensional and higher order separation strategies have been developed to assist in protein identification including 2D gels, 1D gels with LC/MS/MS and 2D HPLC. Our sub-project deals with the characterization of the human and mouse brain proteome using multi-dimensional separation techniques such as polyacrylamide gel electrophoresis and nano-HPLC coupled to mass spectrometric approaches. The results of two-dimensional peptide separations with cation- exchange chromatography (SCX) followed by offline reversed-phase (rp) nano-LC-MS/MS are compared with results from three-dimensional (3D) approaches with a protein separation by 1D PAGE prior to SCX/rp-nano-LC-MS/MS. Preliminary results show a significant improvement for the 3D separations over the 2D LC-MS/MS approach. Additionally, a method is shown for the reduction of memory effects caused by sample pre-concentration via small C18- pre-columns. POSTER SESSION

P-24 Differential Comparison in Gel-Free Techniques Based on Matching Liquid Chromatography Electrospray Ionization Mass Spectra

U. Bauer, C. Baumann, and J. Schwarz Proteome Sciences plc, Coveham House, Downside Bridge Road, Cobham, Surrey, UK.

In gel-free proteomics the most prominent applications involve either extensive cataloguing of expressed proteins using the MudPIT technology [1] or the analysis of differentially expressed proteins based on the ICAT reagent [2], or both of it, using the PST®/TMT® technology [3]. For quantification purpose, the latter ones make use of chemical tags attached to the peptides in one of the two samples, thus inducing a mass shift in corresponding peak pairs at the MS and MS/MS level, respectively. Nevertheless , due to mass spectrometric considerations, both techniques for accurate quantification of parent peptide amounts require, that samples are mixed and objected concurrently to mass spectrometric analysis.

This approach has major drawbacks. First of all, comparing expression levels in multiple samples requires a complex pair-wise combination of samples. Second, the decision to trigger a tandem MS analysis has to be made on-line, to avoid re-injection of the samples. Therefore we developed a computational method to analyse differences between two samples based on de-isotoped and deconvolved mass profile spectra (compare abstract "Deisotoping and Deconvolution of Liquid Chromatography Electrospray Ionisation Mass Spectra”) of complex peptide mixture generated by the PST technology.

We demonstrate that regulations of parent peptides reported on the basis of this algorithm are accurate and within the same range of accuracy as for 2D gels using a series of peptide mixtures spiked into a complete tryptic digest of yeast. Also first examples of real life applications will be shown.

References: [1] Washburn M. P., Wolters D., Yates III J. R. (2001), Nature Biotechnology, 19, pp 242-247. [2] Gygi S. P., et. al. (1999), Nature Biotechnolgy,17, pp 994-999. [3] Thompson A. et al. (2003), Anal. Chemistry 75(8), pp 1895 – 1904. POSTER SESSION

P-25 Fast Screening for Pparameters for the Chromatographic Purification of Proteins with a 96-well Chromatography System

J. Thiemann, S. Kurzawski and H. Schlüter Inst. für Toxikologie, Freie Universität Berlin, Berlin, Germany

For the purification of proteins, where it is desired to maintain biological activity (enzymatic activity), non-denaturating conditions are required. Liquid chromatography meets these requirements. As for each protein a specific and individual strategy for purification usually is necessary, initial tests should be performed before deciding on a final purification protocol. For a target protein with unknown properties, a 'stability window' should be determined, to avoid protein inactivation during purification. It is advisable to check the target protein stability window at least for pH and ionic strength as well as for the compatibility with stationary phases of the chromatographic media. Further parameters, which can affect stability and activity, are detergent requirement, co-factors, protease sensitivity, sensitivity to metal ions and redox sensitivity. In order to find the optimum conditions a large number of initial empirical experiments are useful. Because these experiments are time-consuming, we developed a system, which helps to speed up the search for the appropriate protein purification conditions. The system is based on chromatography experiments in the batch mode. These experiments are performed in parallel in 96-deepwell plates and therefore can be automated. With the 96- well format 96 different chromatographic parameters are possible. Every well contains a defined amount of a chromatographic medium, which is equilibrated to the individual sample application buffer. E.g. for ion-exchange chromatography, 8 different buffers with 8 different pH-values and 12 different ion concentrations can be chosen, resulting in 96 individual sample application buffer conditions. Aliquots of the sample are applied to each of the 96 wells. The proteins, which do not bind to the medium, will be removed by several washing steps. The adsorbed proteins are desorbed with an eluent, which has a high elution strength (e.g. in the case of ion-exchange-media: 2 mol/l NaCl). The 96 individual eluates can be analyzed for activity, protein concentration and protein composition. The 96-well-protein-purification- scouting system will be demonstrated with a mixture of standard proteins as well as with protein extrats, which are applied to ion-exchange gels. In summary, with the 96-well-protein-purification-scouting system fast, automated screening for parameters for the chromatographic purification of proteins from crude extracts is possible. POSTER SESSION

P-26 Polymer-Chip Based 2D-Electrophoresis

A. Griebel, S. Rund, W. Dörner, F. Schönfeld and R. Konrad Institut für Mikrotechnik Mainz GmbH, Mainz, Germany

A microstructured, credit-card sized electrophoresis chip developed at IMM combines the advantages of immobilized pH gradients, such as high resolution and long supportability for the separation in the 1st dimension, with the speed of highly parallel SDS capillary gel electrophoresis (CGE) in the 2nd dimension. The chip (88 mm x 56 mm x 2 mm) contains a cavity for an especially prepared IPG strip and two buffer reservoirs for SDS-CGE. These buffer reservoirs are physically connected via 300 parallel channels (50 µm x 50 µm cross section, 64 mm long). The cavity for the IPG strip and the 300 parallel channels are arranged perpendicular to each other and are connected via a small gap (50 µm wide). Microfabrication technologies like UV lithography, advanced silicon etching and electroforming have been employed to manufacture a nickel mold insert. Hot embossing or injection molding were applied to produce polymer chips containing the microstructures for SDS-CGE. Closed channels were created by sealing with PMMA sheets by solvent bonding. Channel wall surfaces were coated in cooperation with an industrial partner (Serva Electrophoresis GmbH, Heidelberg, Germany) to reduce electroosmotic flow in the presence of SDS and to prevent protein interactions with the channel walls. A handling system comprising programmable power supplies with electrodes for IEF and SDS- CGE, cooling units, a laser as a fluorescence excitation light source and a CCD camera serves to operate the chip. At first, the functionality of the developed chip device was validated both by results of protein separation of IEF and SDS-CGE separately. This poster summarizes our most recent achievements concerning the transfer of the proteins from the first to the second dimension.

This project is part of the BMBF Verbundprojekt "Proteom-Analyse des Menschen" (FKZ 01GG9836). POSTER SESSION

P-27 Identification of Tumor Markers in Toxicology by Non-gel Based Proteomic Approaches

K. Fella, Y. Walter, P.-J. Kramer and M. Kröger Institute of Toxicology, Toxicoproteomics, Merck KGaA Darmstadt, Germany

The application of proteomic technologies in industrial toxicology is becoming widely accepted and two dimensional gel electrophoresis (2DE) remains the major tool for displaying the proteome. In spite of technical improvements, 2DE is still very time consuming and has problems regarding reproducibility. For this reason, researchers are looking for alternative proteomic approaches. In order to identify cancer associated markers, rat liver tumors were tested on an Antibody Microarray and a BD PowerblotTM, now available from BD Biosciences. Male Wistar rats were treated with the well characterized carcinogen N-Nitrosomorpholine (NNM) according to the NNM stop model. After 25 weeks the animals had developed liver tumors which were removed for further analysis. The Antibody Microarray was performed with 500 spotted antibodies per chip, and for the PowerblotTM Western array 120 antibodies from different research areas were chosen. Microarray data analysis was carried out with Gene Spring, the Power BlotTM results were visualized and analysed using the gel imaging softwares PDQuest (BioRad) and ProteomWeaver (Definiens) in parallel. Many cancer related proteins were differentially expressed in the tumourous liver tissue. MEK 1, ERK 1 and ERK 2, all belonging to the MAPkinase pathway and known to play an essential role in tumor progression and metastasis, were significantly upregulated. GST-pi, a widespread used histological carcinogenic marker, and the tumor suppressor E-Cadherin were also found to be upregulated. In addition, proteins belonging to the cell cycle and apoptosis pathways or cell adhesion were differentially expressed. Some of the changes observed were verified by immunoblotting. Our results show that the Antibody Microarrays, as well as the BD PowerblotTM, are potentially useful alternative platforms to identify proteins which are related to carcinogensis and tumor growth. POSTER SESSION

P-28 Functional Proteome Analysis by a Novel System Using Immobilized siRNA Expressing Adenoviruses Immobilized on Microarrays

H. Volkmer, M. Niere, F. Weise, T. Pruss and H. Herrmuth NMI an der Universität Tübingen, Reutlingen, Germany

Functional analysis of proteins is most relevant in a cellular environment. A major caveat is the possibility to analyze the function of many different proteins in small scale culture formates. We develop microarrays that enable the miniaturized and parallelized transduction of primary cells by highly efficient immobilized adenoviruses to inhibit endogenous biosynthesis by siRNA expression. A proprietary technique is used to immobilize recombinant adenovirus on microarrays. As adenoviruses are natural cellular ligands, cells are trapped on adenoviral spots where they adhere. Immobilization is performed in a way that adenoviruses remain infectious. Therefore, cells not only adhere to the viral spots but also get infected. We show that an immobilized Green Fluorescent Protein transducing recombinant adenoviral vector transduces 62% of adhered rat primary microglia cells. Such cells cannot be addressed by alternative approaches using immobilized plasmid vectors by reverse transfection. Transduced cells are then available for cellular assays on the microarrays. The use of adenoviral gene transfer is associated with problems that have been solved in our system. 1. Preparation of recombinant adenoviruses is time-consuming, 2. Full length cDNA is sometimes hard to recombine, 3. If only a subfraction of cells is infected on the array, infected cells need to be identified. We developed a PCR-based screening procedure in combination with prokaryotic recombination technology to speed up the identification of successfully recombined adenoviruses. The application of siRNA technology is adapted to the adenoviral transduction to circumvent the need of full length cDNA for the functional analysis . In addition to the siRNA expression cassette, α β-galactosidase gene is introduced as a marker gene to identify infected cells. As an example we studied the impact of the neuronal cell adhesion molecule neurofascin on neurite outgrowth. Candidate siRNA sequences were cloned into plasmid vectors to identify a siRNA that efficiently inhibits target mRNA after transient transfection. To this end, a cell line was transfected that expresses neurofascin under the control of the strong CMV promoter. Knock-down was verified by analysis of neurofascin mRNA and protein expression. Subsequently, a successful siRNA expression cassette was recombined into the adenoviral genome. Neurite outgrowth of primary neurons was examined after adenoviral infection to express neurofascin specific siRNA. Neurite outgrowth was efficiently blocked by specific siRNA expressing adenovirus but not by a control vector. POSTER SESSION

P-29 Development of a DNA-Protein Microarray for the Analysis of Eukaryotic Transcription Factor Activation

J.K.G. Crean*1, D. Finlay*2, R. O’Leary*3 and F. Martin3 Departments of Medicine and Therapeutics1 and Pharmacology3, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Mater Misericordiae Hospital and The Dublin Molecular Medicine Centre, Belfield, Dublin 4, Ireland. 2Current address, The Burnham Institute, La Jolla, California 92037, USA. *These authors contributed equally to the work described.

Sequence specific DNA-protein interactions control transcription, recombination, restriction and replication. These interactions are usually measured by a variety of laborious procedures, overly complicated for the analysis of the many DNA variants. Here we describe a high throughput DNA-protein microarray assay for studying the interaction of transcription factors and their binding sites. Using conventional mutation studies and mathematical matrices based on these experiments, consensus transcription factor binding DNA oligonucelotide sequences were designed. Mutant oligonucleotides (incapable of binding a specific transcription factor and therefore used as negative controls) were designed similarly except that the essential bases necessary for transcription factor binding are replaced. Each oligonucleotide strand was synthesised commercially using a solid-phase synthesis system with one strand of each oligonucleotide pair possessing a 5’- carbon 12 amine group. This modification allows orientation specific chemical attachment to modified aminosilane slides. Double stranded oligonucleotides were obtained by annealing each consensus pair prior to arraying. Oligonucleotides corresponding to the transcription factors ATF-1, NFkb and HIF1a were arrayed on aminosilane modified slides. Nucleoproteins from stimulated and non-stimulated human mesangial cells were then labelled with cyanine dyes and hybridised to the double stranded oligonucleotide array. Quantitative and qualitative data on transcription factor-binding site interactions were obtained by measuring the relative fluorescence intensity using a dual laser microarray scanner. This represents a significant technical advance and will have many applications to include the measurement of the response and activity of transcription factors in normal and diseased states and high throughput screening of transcription factor responses to drugs regimens. POSTER SESSION

P-30 A Mass Spectrometric Ladder Sequencing Approach for Systematic Characterization of C-terminal Proteolytic Processing Events of Intact Proteins

B. Samyn, K. Sergeant and J. Van Beeumen University Ghent, Laboratory of Protein Biochemistry and Protein Engineering, Gent, Belgium

C-terminal proteolytic processing is a posttranslational modification (PTM) that plays a critical role in a variety of biological processes. The C-terminus of proteins is uniquely positioned to serve as a recognition signature for a variety of cell-biological processes, including protein targetting, subcellular anchoring and the static and dynamic formation of macromolecular complexes. Contemporary approaches for protein identification by mass spectrometry usually involve two- dimensional gel electrophoresis separation of the intact proteins, excision of gel spots, tryptic digestion of the excised protein(s), and identification using peptide mass fingerprinting or tandem mass spectrometry (MS/MS). Although MS can yield very detailed information about a protein, including an extensive analysis of PTMs, it is not possible to state whether or not sequence alternatives or protein modifications other than those detected are present. Characterization of protein variants, either due to genetic mutations in protein sequences or to PTMs, is considerably more difficult than the simple protein identifications typical of most current proteomic investigations. All recently developed approaches to study PTMs focus on phosphorylation and glycosylation, two of the most occuring modification events. So far, extensive studies of N- and C-terminal proteolytic events have been hampered by the lack of suitable methods. Although continuous progress with novel MS-techniques is being made, the chemical degradation techniques (N- or C-terminal) are the only methods which can directly confirm the structure of an intact protein. Amino-terminal protein sequencing by the classic technique of Edman degradation is still the method of choice to determine N-terminal proteolytic processing. Analogously, C-terminal chemical degradation, based on the thiocyanate chemistry, can be directly applied on the intact protein to yield complementary information to the Edman degradation. However, the relatively modest sensitivity attainable to date and the inability to sequence through some specific amino acids still hamper the utility of this approach. Here we present a novel method to study C-terminal proteolytic processing events. A number of test proteins, in solution or separated by 2D-PAGE were chemically cleaved with CNBr and the resultant peptide mixtures were directly incubated with carboxypeptidases and analysed by MALDI-MS. During cleavage with CNBr, Met residues are converted to homoserine lactone, in equilibrium with homoserine. Our experiments have shown that only the original C-terminal containing fragment is accessible to enzymatic degradation, eliminating the need for the specific isolation of the C-terminal peptide. This allows a high throughput approach, compatible with protein purification techniques currently used in large-scale proteomic studies (2D-PAGE, multi-LC). In order to examine the applicability of this approach at a proteomic scale this new approach was applied on a number of 2D-PAGE separated proteins from Shewanella oneidensis, a metal-reducing microbe of potential importance to the field of bioremediation. POSTER SESSION

P-31 Electrospray and Gas Phase Surface Chemical Ionization in the Analysis of Protein Spots of Human Sperm Obtained by 2D Gel Electrophoresis

S. Cristoni1, P. M. Gerthoux2, E. Gonella2, P. Mocarelli2, C. Sarto2, I. Biunno3, C. Canton1, F. Guidugli4 and L. R. Bernardi1 1Università degli Studi di Milano, Centro Interdisciplinare Studi Bio-molecolari e Applicazioni Industriali CISI, Segrate Milano, Italy; 2University Department of Laboratory Medicine, University of Milano-Bicocca, Hospital of Desio, Desio, Milan, Italy; 3CNR-ITB, , Segrate, Milano, Italy; and 4ThermoFinnigan Italia S.p.A., Rodano, Milan, Italy

In previous investigations [1-3] it has been shown that the vaporization of sample solutions in atmospheric pressure conditions, without the presence of any corona discharge, leads anyway to the production of a quite high number of ions. These experiments were performed by a commercially available atmospheric chemical ionizatio (APCI) source and this approach was called "no discharge APCI”. It must be emphasized that, in these conditions and in the case of proteins and peptides, the formation of doubly charged species is privileged and, considering their usefulness in further MS/MS studies [4] the observed phenomenon w a s considered of high interest. Furthermore, a metallic surface has been inserted into the ionization chamber of the no discharge APCI source in order to induce the ionization of the analyte neutral molecules present in gas phase. The new ionization method has been named by us "Gas Phase Surface Chemical Ionization” (GPSCI) [5,6]. This approach has been used in order to identify and characterize some protein spots of human sperm obtained by 2D gel electrophoresis (2D-GEL) and the results are reported. Moreover, a comparison with the data acquired using the ESI source is also reported showing that the GPSCI ionization source is a valid alternative to the usually employed ESI source for the characterization of 2D-GEL protein spots.

References: [1] Cristoni S, Bernardi LR, Biunno I, Guidugli F. Rapid Commun. Mass Spectrom. 2002; 16:1686-1691. [2] Cristoni S, Bernardi LR, Biunno I, Guidugli F. Rapid Commun. Mass Spectrom. 2002; 16:1153-1159. [3] Cristoni S, Bernardi LR. Mass Spectrom. Rev. In Press. [4] Cramer R, Corless S. Rapid Commun. Mass Spectrom. 2001; 15:2058-2066. [5] Cristoni S, Bernardi LR, Biunno I, Tubaro M, Guidugli F. Rapid Commun. Mass Spectrum. submitted. [6] Cristoni S, Bernardi LR, Biunno I, Tubaro M, Guidugli F. 21st Informal Meeting on Mass Spectrometry 2003; University of Antwerp (Belgium). POSTER SESSION

P-32 Silicone/Graphite Coated MALDI Targets for Improved Automated MALDI Acquisition in High Through-put Proteomics

T. Franz1, X. Li1 and M. Wilm2 1Proteomic Core Facility; 2Bioanalytical Research Group European Molecular Biology Laboratory, Heidelberg, Germany

Robotic sample preparation for MALDI-MS analysis in high throughput proteomics is a relatively challenging task. In general, robots are used for automatic in-gel digestion and spotting of the extracted peptides onto MALDI target plates. The spotting of the sample and matrix on the usual steel targets is critical and the result of the robotic application can be poor when compared with manual procedures. A second disadvantage is that the salt content of the spotted sample has to be removed not to suppress signal intensity.

At the moment there are different systems like anchor chip targets and reversed phase micro columns on the market. The danger of anchor chip targets and similar permanently coated targets is a possible carry over from sample to sample if they are not carefully cleaned after usage. Reversed phase and similar stationary phase materials in micro columns are expensive in use.

In this presentation a very cheap single-use coating for MALDI target plates is presented. A mixture of silicone/graphite is applied in a thin layer (0.2 mm) on the steel target. The hydrophobicity of the material allows very precise robotic spotting of 4 times more material than on a blank steel target. In connection with graphite on-target washing without loss of sample is possible for the removal of salt contaminations. Additionally, the new surface offers a much better, because finer crystallization. The search for a "hot spot” is not necessary anymore. We could not detect background peaks originating from the coating materials in a mass range from 500 to 3500 Da. Therefore it is ideal for automatic MALDI acquisition of mass peptide fingerprints in high throughput projects. The intensities are up to 4 times better than on steel targets, without affecting the resolution. After usage the coating can be easily removed and the target is ready for recoating for the next analysis avoiding carry over of former analyte or contaminations. POSTER SESSION

P-33 An Alternative Modifier of Cysteinyl Groups in 2D-Electrophoresis Permitting Identification by MALDI-ToF MS

J. Goscinski, S. Bourin, S. Cetinkaya, D. Haid, J.J. Hedberg, I. Olsson and B. Bjellqvist Discovery Systems, Product Development, Amersham Biosciences AB, Uppsala, Sweden

In 2-D electrophoresis, the difficulty of producing streak-free, highly reproducible protein maps on the basic side (pI >7) is of common knowledge. Traditionally, DTT has been used to reduce the proteins during isoelectric focusing. The problems arise on the basic side from which DTT is transported, thereby permitting re-oxidation of proteins in this area resulting in inter- and intra-protein disulfides. This generates streaky spot patterns and poor reproducibility. The introduction of DeStreak in the IPG strip rehydration step has solved this problem by specifically oxidizing the cysteinyl groups to a defined hydroxyethyl form. By maintaining these "mixed disulfides” throughout the SDS electrophoresis the modified cysteine-containing peptides can readily be identified by MS. Semi-preparative amounts of mouse liver proteins were analyzed on both narrow and broad range pH strips, where the cysteins were modified with DeStreak or iodoacetamide. Following tryptic digestion and peptide extraction, the proteins were identified in a MALDI-ToF MS. Notably, the DeStreak-modified proteins were identified with the same frequency as iodoacetamide-modified ones. The MS data also show that the spectral quality for the two variants is equal with regards to intensity and number of detected cysteine-containing peptides. This demonstrates the efficiency at which DeStreak modifies proteins. In conclusion, these experiments describe a simplified equilibration step, resulting in streak- free 2-D maps and reliable MS data. POSTER SESSION

P-34 Direct Molecular Imaging of Lymnaea stagnalis Nervous Tissue at Subcellular Resolution by Mass Spectrometry

A.F.M. Altelaar1, J. van Minnen2, C. R. Jiménez2, R.M.A. Heeren1 and S. R. Piersma1 1FOM-Institute for Atomic and Molecular Physics, Amsterdam, The Netherlands and 2Dept. of Molecular and Cellular Neurobiology, Faculty of Earth and Life Sciences, Vrije Universiteit, Amsterdam, The Netherlands

Mapping the dynamic state of the proteome inside a cell or tissue is an important area in biological research. One of the emerging technologies in this field is imaging mass spectrometry. Recently, MALDI-ToF (matrix-assisted laser desorption/ionization time-of flight) imaging mass spectrometry of mammalian tissue has resulted in protein and peptide specific maps at 30-100 µm resolution (Stoeckli et al, Nature Med. 2001 pp 493). In order to improve spatial resolution we have combined MALDI sample preparation methods and the imaging capabilities of ToF-SIMS (secondary ion mass spectrometry) for direct molecular imaging of molluscan nervous tissue. A thin layer of 2,5-dihydroxybenzoic acid was electrosprayed on cryostat sections of Lymnaea stagnalis cerebral ganglia yielding micron- sized matrix crystals. The energy-moderating matrix allowed subcellular imaging of the neuropeptide APGW-amide and cholesterol. Electrospray deposition facilitates analyte incorporation into the matrix and minimizes lateral diffusion over the tissue surface. Here we show the proof-of-concept that matrix-enhanced SIMS (ME-SIMS) imaging combined with electrospray matrix deposition allows high-resolution molecular imaging (resolution better than 3 µm) of peptides/small molecules in tissues and opens a complementary mass window (< 1500 Da) to MALDI imaging mass spectrometry, but at an order of magnitude higher spatial resolution. POSTER SESSION

P-35 Investigation of Proteins in Post-Mortem Human Brain Tissue by Laser-Microdissection/Pressure-Catapult and nano-LC/MSMS

C. Sauber1, B. Sägmüller2 , M. Neumann3 and H. A. Kretzschmar3 1Agilent Technologies AG, Waldbronn, 2P.A.L.M. Microlaser Technologies AG, Bernried, Germany and 3Institut of Neuropathology, Ludwig Maximilians University, Munich, Germany

The molecular mechanisms involved in most neurodegenerative disorders, such as Parkinson’s and Alzheimer’s disease, are still unclear. So far, protein expression analysis is often performed on homogenized preparations of whole tissues which do not provide any information about relevant changes in specific cell types. The aim of the following study was to examine whether laser-microdissected samples of single cell types from post-mortem brain tissue can be used for protein expression analysis. Therefore, we used haematoxylin and eosin stained frozen sections (15 µm) from human post-mortem brain tissue and collected cell material selectively by laser-microdissection and pressure catapulting (LMPC) as combined in the AutoLPC-function of a MicroBeam® instrument (P.A.L.M. Microlaser Technologies). Different amounts of material have been collected such, that the cells are readily fragmented and homogenized while being catapulted out of the brain section into the urea denaturation buffer (6M urea, 100mM Tris/HCl), which was placed in the center of a cap for sealed reaction tubes exactly on top of the desired areas for LMPC-cell- harvest. The tryptic digested protein mixture was subsequently analyzed with a nano-LC/MSMS ion trap system (Agilent Technologies). Database searching was done with SpectrumMill MS Proteomics Software. The samples were injected to a reversed-phase enrichment column, which was backflushed after 5 minutes loading time. The peptides were then eluted to a ZORBAX SB300 C18 nano column (0.075 x 150 mm) which was directly coupled to an ion trap mass spectrometer via an orthogonal nanospray source. The gradient was raised with 0.25% B per minute resulting in a total cycle time of 200 minutes. The ion trap was operated in Auto-MS2 with ActiveExclusion and SmartFrag for generating MS and subsequent MS/MS spectra of the tryptic peptides. Applying this non-contact investigation method we were able to indentify the glial fibrillary acidic protein by 25 distinct peptides, the myelin basic protein by 6 distinct peptides and a mutant beta-actin by 7 distinct peptides. POSTER SESSION

P-36 Comparison Between Manual and Automated Procedures of 2-D Electrophoresis, Protein Spot Picking and Processing for Mass Spectrometry Analysis

V. Corti1, L.B. Areces2, A. Bachi3, P. Oatey4, R. Willock4 and M. Alessio1 1Proteomics Laboratory and 3Mass Spectrometry, San Raffaele Scientific Institute, Milan, Italy; 2European Institute of Oncology-IEO, Milan, Italy and 4PerkinElmer Life Sciences, Cambridge, UK.

V. Corti and L.B. Areces contributed equally to this work

The use of the proteomics technologies allows the simultaneous analysis of a large number of proteins enabling a comprehensive analysis of biological phenomena. However, it is mandatory to restrict the size of the analysis and the complexity of the samples in order to have a manageable study, and this is a field in which many investigations has been applied. In the meantime technical solutions that increase the throughput and the number of processed samples are necessary for particular proteomics applications. One of these applications is the "differential expression proteomics" which refers to the study of a complete set of proteins that are expressed by the genome of a cell in a well-defined physiological condition compared to a different condition (i.e. pathological, activated, etc.). The "differential expression proteomics" approach usually includes the generation of 2-DE maps combined with differential image analysis, protein identification by MS analysis and data base comparisons. This is still the most powerful approach. While 2-DE is a difficult method to be automated, post electrophoretic steps are actually starting to be robotized. In fact, one of the limitations is the length and the complexity of the processing necessary for spot excision, digestion with protease, peptide elution and purification, and sample deposition on MS target. An open question is the possibility to improve the number of processed samples with the introduction of automation, without losing sensitivity, accuracy and reliability. Several different systems are commercially available for high throughput sample processing. Moreover it is possible to have either integrated robots from a single company or combined machines from different companies. We have had the opportunity to test a system proposed by PerkinElmer (Boston, MA) including the automated spot picking robot, ProXCISIONTM, and the automated liquid handling instrument, MultiPROBE® II Proteomics Workstation. The performance of the automated processing has been compared in parallel, with manually processed samples. Eighteen protein spots have been excised from twin 2-DE gels, and processed for MS analysis. Manual processing gave slightly better results with less abundant proteins, while, no discrepancies have been found in protein identification. The automated processing resulted in an acceptable performance, and can be used for the gross number of medium/high abundant spots. We believe that the conclusions and protocols that we have made will be helpful for scientists considering adopting automated solutions for gel-based proteomic studies. POSTER SESSION

P-37 Automated Batch Filtering and Recalibration of Mass Spectra for Increased Protein Identification Rates in High-throughput Proteomics

F. Levander1, T. Rognvaldsson2, J. Samuelsson2 and P. James1 1Department of Protein Technology, Lund University, Sweden and 2School of Information Science, Computer and Electronic Engineering, Halmstad University, Sweden

For successful protein identification by matrix-assisted laser desorption ionisation – time of flight mass spectrometry, removal of dirt peaks and recalibration of the mass spectra are essential parts of the process. An established approach is to use peptide peaks from trypsin autolysis as internal calibrants, remove the tryptic peaks from the peak list, and subsequently send the peak list for protein identification by peptide mass fingerprinting (PMF). However, in a batch of samples it is usual to find other contaminant peaks that potentially could be used for internal calibration of the spectra, and which should not be included in the PMF. It is a time consuming process to find those peaks but nevertheless it is often a requirement for maximum protein identification rates. Here we present a piece of software that automatically finds potential contaminant peaks by a statistical method, identifies the peaks for calibration by matching to a database, and finally generates a filter for removal of dirt peaks and recalibration of the spectra. The filter can be used in an automated flow from peak extraction to PMF protein identification. The batch filter software has been tested on a several sets of spectra of different origin with sometimes remarkable improvements in protein identification rates. Example results will be shown and discussed. POSTER SESSION

P-38 Protein Identification Expert System for SDS-PAGE/MALDI-ToF Proteome Profiling Procedure

P. G. Lokhov and A. I. Archakov Orekhovich Institute of Biomedical Chemistry, Moscow, Russia

MALDI-TOF MS is most conventional technique for proteomics. The available search systems can significantly identify proteins using MALDI-TOF mass-spectra of their proteolytic digests. However, the prior high performance protein separation, such as 2D-PAGE, is a necessary condition for automatic high-throughput analysis. A substitution of two-dimensional separation for more simple technique, e.g. SDS-PAGE, encounters a need of mass-spectra analysis for proteolytic digest of protein mixture. Interpretation of mixed digest mass-spectra is time- consuming and labor-intensive even for skilled artisans. The development of high-throughput system for proteome research using simple separation methods remains very actual objective. It is also important because SDS-PAGE is a choice method for membrane proteins study in proteomics. The goal of our project is to develop the software for proteome profiling based on one- dimensional electrophoretic protein separation combined with mass-spectrometric protein identification. The database search is suitable for protein identification from complex mixtures and it uses the signal-processing algorithm implemented in MSA program (http://www.ibmh.msk.su/proteomics/ms_projects.htm). MSA performs the database search using several algorithms and score functions, such as Bayesian, MOWSE, Z-score, correlations between calculated and actual peptide length fractional abundance, the probability of protein digest pattern in peptide fingerprint following the data processing by neural network to increase identification success rate by improving the signal-noise separation. The power of MSA algorithm was tested on Helicobacter pylori proteome profiling after preceding protein separation by SDS-PAGE. With the purpose to make progress in solving the problem of minor component identification, the additional score function will be added to database search algorithm. Peptide mass fingerprint peak intensity prediction in mass-spectrum by classification and/or regression methods will be used to increase efficiency of the protein identification algorithm for sample minor components due to reduction of the peptide number needed for significant identification. POSTER SESSION

P-39 Complementarity of de-novo and MS/MS Search Programs for High-troughput Identification of Peptides using LC/MS-MS Fragmentation

B. Cañas1, D. López1, R. Navajas1, M. Campillos1, A. Marina1, F. Martín-Maroto2 and J. Vázquez1 1Protein Chemistry and Proteomics Lab. Centro de Biología Molecular "Severo Ochoa", CSIC-UAM.Madrid. Spain, and 2Thermo-Finnigan, San Jose, CA., USA.

Peptide fragmentation spectra obtained by MS/MS in an Ion/Trap mass spectrometer are automatically assigned using programs as Sequest or Mascot, provided that the proteins, which originate these peptides, are included in the database. Each of these programs use their own scoring system showing the probability of finding in a database a peptide identical to that producing a particular spectrum. Clear-cut results are produced in many cases with their scoring systems but, frequently, an expert is needed to take an eye on the spectra to be sure about the results. This is not compatible with high-throughput peptide and protein identification. Spectra from peptides unmodified or with known modifications constitute the ideal conditions for peptide identification and facilitate retrieving results with a high scoring and no doubt about authenticity. Nevertheless, depending on the quantity of the peptide and on the averaging time, poor quality spectra are often produced, precluding their automatic identification.Also Peptides may fragment in a non-homogeneous way giving poor sequence coverage. This effect is pronounced in spectra taken from non-tryptic peptides with internal basic residues, particularly when Arg residues are localized in the middle of the molecule are present. Finally, the existence of unknown modifications in peptides precludes absolutely their identification using these searching engines.. We have used LC-MS/MS to identify peptides class II MHC-bound peptides. We were able to find tens of peptides retrieved with good scoring with Sequest, but there were still many interpretable spectra giving low score results. We found that the use of DeNovoX was extremely useful to distinguish among bad and good Sequest matches. When there was a coincidence between the peptide sequence retrieved by Sequest and a short sequence (5-6 amino acids) retrieved by DeNovoX, there was absolutely no doubt about the Sequest result. To validate these results, dozens of peptides from different sources were sequenced manually and results compared with those obtained with Sequest and DeNovoX. Since these programs work in a completely diverse way any significant similitude in their results cannot be assigned to chance alone. POSTER SESSION

P-40 Large-scale Protein Identification by Bidimensional Chromatography-ion Trap Mass Spectrometry: Development of Second-Generation Algorithms for Unambiguous Assignation of Peptides from MS/MS Spectra

1D. López, 1M. Campillos, 1B. Cañas, 2F. Martín-Maroto and 1J. Vázquez. 1Centro de Biología Molecular "Severo Ochoa", CSIC-Univ. Autónoma de Madrid. Madrid. Spain and 2Thermo-Finnigan, San Jose, CA. USA.

We have used SCX chromatography in combination with LC-Ion Trap Mass Spectrometry for the large-scale analysis of unseparated protein mixtures. We have studied several proteomes, including the nuclear proteome from jurkat human cells. We typically collect more than 100.000 MS/MS spectra, and identify several thousands of peptides; we automatically identified 774 proteins in only 100 ug of nuclear cell extracts. While database searching of these huge amounts of data using conventional engines, such as Sequest, allows the identification of most the peptides, but a large fraction of these peptides were not conclusively identified. We have recently reported that DeNovoX, an intelligent algorithm, for large-scale "de novo” interpretation of MS/MS spectra, developed in our laboratory, can discriminate with very good statistical confidence the correct assignations. Here we demonstrate that conventional criteria produce the incorrect assignation of peptides in an unexpectedly large fraction of cases, and that the automated comparison of sequences deduced by DeNovoX with those obtained by Sequest allows a very accurate discrimination of correct peptide matchs. We have thus developed a second-generation algorithm for database searching which combines some of the intelligent, probability-driven scoring schemes of DeNovoX with the enhanced database- indexing and searching capabilities of Sequest. This algorithm allows a much more robust assignation of correct peptide sequences, and opens the road to the automated identification of postranslational modifications. POSTER SESSION

P-41 Screening Protein Libraries for Enzyme Activities with the MES-system

H. Schlüter1, J. Rykl1, S. Kurzawski1, J. Thiemann1, L. a´Brassard2, J. Oster2, M. Altgott3, J. Feldhusen3, T. Pohl4 and B. Wittmann4 Free University of Berlin1, Chemagen AG2, IKT-RWTH Aachen3,WITA GmbH4, Berlin, Germany

With the MES-system unknown enzymes with defined enzymatic properties according the catalysed reaction can be detected, purified and identified. The MES-system is an automated platform technology, which integrates the fractionation of protein extracts by multi-dimensional chromatography (I), conversion of the fractions into protein libraries (II), screening the libraries for enzymatic activities (III) and identifying the active proteins (IV). The proteins are converted into the protein library by immobilizing them covalently to magnetic, activated affinity- chromatography particles. The enzymatic activities of individual fractions from the library are detected with a MALDI-mass spectrometer. Therefore the immobilized protein fractions are incubated with a reaction specific probe. Aliquots of the reaction mixture are removed and spotted to a MALDI-target. The enzyme of interest is present in a protein fraction, if the mass signal of the expected reaction product can be observed [1]. In this case the active fraction is passed to the 2D-electrophoresis and the resulting protein spots are identified by the tryptic- peptide fingerprint MALDI-MS analysis and, if necessary, with denovo-MS/MS sequencing. The mass spectrometry-assisted detection of enzymatic activities presents the advantage to use authentic probes. In contrast to conventional enzyme-assay-probes the MES-probes are not modified with chromophores or fluorophores and are not labelled with radioactive isotopes. The determination of the molecular weight of the reaction products enables the investigator to evaluate the identity of the reaction product. Furthermore additional information about other enzymes present in the analysed fraction can be recognized. In comparison to UV- and fluorescence-based enzyme assays the MES-system is some orders of magnitudes more sensitive. The MES-system covers a broad application range: It can be used for the search for therapeutic relevant enzymes, for discovering enzymes of signalling cascades (kinase cascades, proteolytic cascades such as apoptotic signalling) but also for enzymes, which are useful for biotechnological processes. Furthermore the MES-system will be a tool for differential-display approaches. With the MES-system enzymatic activities of biological systems in two different states can be compared. This comparison will be possible in many cases with complex mixtures of proteins, thus avoiding time-consuming fractionation procedures.

[1] Jankowski, J., Stephan, N., Knobloch, M., Fischer, S., Schmaltz, D., Zidek, W. & Schlüter, H. (2001) Mass-spectrometry-linked screening of protein fractions for enzymatic activities-a tool for functional genomics. Anal. Biochem. 290, 324-9. POSTER SESSION

P-42 An Optimized Method for the Isolation and Identification of Membrane Proteins

I. Lehner, M. Niehof and J. Borlak Fraunhofer Institute of Toxicology and Experimental Medicine, Center for Drug Research and Medical Biotechnology, Hannover, Germany

The purpose of this study was to develop a protocol suitable for membrane protein extraction from limiting starting material and to identify appropriate conditions for 2-D gel electrophoresis. We used A549 cells, a human alveolar type II cell line and evaluated three protein extraction methods based on different separation principles, namely protein solubility, detergent-based and density-based organelle separation. Detergent-based extraction achieved the highest yield with 14.64% ± 2.35 membrane proteins but sequential extraction with 7.35% ± 0.78 yield and centrifugal extraction with 4.1% ± 0.54 yield produced the purest fractionation of membrane proteins. Importantly, only the sequential and the detergent-based extraction proved suitable for small volumes of starting material. We identified annexin I + II, electron transfer flavoprotein beta chain, H+ transporting ATP synthase, mitofilin and protein disulfide isomerase A3 as membrane and cytokeratin 8 + 18, actin and others as soluble proteins using MALDI-TOF analysis and started to map the A549 cell proteome. Our data suggest that membrane proteins can be extracted efficiently from small samples using a simple sequential protein extraction method. They can be separated and identified successfully using optimized conditions in 2-D gel electrophoresis. The presented methods will be useful for further investigations of membrane proteins of alveolar and bronchial carcinomas. POSTER SESSION

P-43 Proteome Analysis of Peribacteroid Membrane Preparations from Pisum sativum and Lotus japonicus Root Nodules

G. Saalbach and S. Wienkoop* Risø National Laboratory, Plant Research Department, 4000 Roskilde, Denmark; *Present Address: Max-Planck-Institute of Molecular Plant Physiology, Golm, Germany

The peribacteroid membrane (PBM) forms the structural and functional interface between the legume plant and the rhizobia and mediates the exchange of metabolites and signals between the symbionts. At first, PBM was prepared from root nodules of Pisum sativum (pea), and the proteins were separated by two-dimensional gel electrophoresis. Eighty-nine spots were excised from the gels and analysed by MS. Fourty-six of these spots were identified by database screening but represented mainly bacteroid proteins. In addition, several proteins from the plant endomembrane system were found. The results showed that these symbiosomes are not suitable for PBM preparation (high bacteroid contamination!). Thereafter, the proteome studies were concentrated on the use of advanced methods for the analysis of PBM proteins from Lotus japonicus, a model legume in genetic studies. The PBM was purified from root nodules by an aqueous polymer two-phase system. Extracted proteins were subjected to a global trypsin digest. The peptides were separated by nanoscale liquid chromatography and analysed by tandem mass spectrometry (MS/MS). Searching the non- redundant protein database and the green plant EST database using the MS/MS data identified approx. 94 proteins, a number far exceeding the number of proteins reported for the PBM hitherto. In particular, a number of membrane proteins like transporters for sugars and sulfate; endomembrane-associated proteins such as GTP-binding proteins and vesicle receptors; proteins involved in signalling, for example receptor kinases, calmodulin, 14-3-3 proteins and pathogen response-related proteins including a so-called HIR protein were detected. Preliminary results from experiments with GFP fusion proteins confirmed the localisation of the HIR protein. Several ATPases and aquaporins were present indicating a more complex situation than previously thought. In addition, the unexpected presence of a number of proteins known to be located in other compartments (like cytoplasmic and secreted proteins) was observed. Two characteristic protein complexes obtained from native gel electrophoresis of total PBM proteins were also analysed. Together, the results identified specific proteins at the PBM involved in important physiological processes and localised proteins known from nodule- specific EST databases to the PBM. POSTER SESSION

P-44 Identification of Thylakoid Integral Membrane Proteins from Chlamydomonas reinhardtii by Peptide Mass Fingerprinting and MALDI-MS

S. Rexroth1, J. Meyer zu Tittingdorf1, F. Krause1, H. Seelert1, R. Schlichting2 and N.A. Dencher1 1Physical Biochemistry and 2Institute of Botany, Darmstadt University of Technology, Darmstadt, Germany

Analysis of the membrane proteome is mainly dependent in the ability of protein separation. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a technique, so far mainly applied to mitochondrial respiratory chain, capable of efficient membrane protein separation. Applying BN-PAGE to thylakoid membranes after mild solubilisation with digitonin, we identified extremely hydrophobic subunits of the photosystem complexes with 5 - 11 transmembrane helices by trypsination and subsequent matrix assisted laser desorption / ionization - mass spectronomy (MALDI-MS). All photophosphorylation complexes, as well as their supercomplexes, from thylakoids of C. reinhardtii were resolved in a single gel, providing an analytical tool suitable to characterize composition of membrane protein supercomplexes in different physiological states. Using this technique significant differences in supercomplex composition from C. reinhardtii grown under photoautotrophic and photomixothrophic conditions can be observed [1].

[1] Rexroth, S., Meyer zu Tittingdorf, J., Krause, F., Dencher, N. A., Seelert, H., Electrophoresis 24 (2003) (in press). POSTER SESSION

P-45 Enrichment of the Membrane Proteome of C. glutamicum and Analysis by LC-ESI

Frank Fischer1, Daniela Schlüsener1, Dirk Wolters2 and Ansgar Poetsch1 1Plant Biochemistry and 2Analytical Chemistry, Ruhr-University Bochum, Germany

The aim of our project is the development of methods to analyze the complete membrane proteome of Corynebacterium glutamicum. The development of such methods is part of a nationwide BMBF-project that is focused on the proteome and metabolome of C. glutamicum. C. glutamicum is a biotechnologically very important bacterium, used for the industrial production of amino acids (e.g. L-glutamate and L-lysine) by our industrial partner, the Degussa AG. The genome of C. glutamicum is completely sequenced and is coding for about 3000 proteins, about 650 of these are predicted to be located in the membrane. Membrane proteins are usually underrepresented in proteomic studies. This has also been the case in a previous study of the C. glutamicum proteome . One reason for this underrepresentation is the relative low abundance of most membrane proteins. Therefore, we developed a protocol to enrich the membrane proteins in one fraction prior to the further separation by nano-RP-LC or by 2D- PAGE. C. glutamicum wild type and L-lysine producing strain were cultivated in shaker flasks or fermenter, respectively. The bacteria were lysis by French press passage and the cell membrane was isolated. In order to enrich only the integral membrane proteins, different membrane washes were tested. The largest amount of membrane-associated proteins was removed with NaBr washes, while preserving the integrity of the membrane. The washed membranes were either treated with proteases for analysis by LC-ESI or were solubilized by detergents for 2D-PAGE. Upon sequential digestion of the washed membranes with CNBr and trypsin, peptides were separated by nano-LC and identified by ESI using the SEQUEST algorithm. With the current digestion strategy about 100 proteins were unambiguously identified. Yet more than 80% of these are soluble proteins or peripheral membrane proteins. In the case of integral membrane proteins, our preliminary analysis shows that only peptides originating from soluble domains were identified. A prerequisite for separation of the membrane proteome by 2D-PAGE is a highly efficient protein solubilization in a buffer that is compatible with the IEF step. Different detergents have been tested to solubilize the membrane proteins. The best solubilization was obtained with the detergents ASB-14 and lauryl- maltoside. Furthermore, these solubilization conditions were suitable for protein separation by 2D-PAGE. To assess the solubilization efficiency, proteins from the detergent extract and the insoluble pellet were separated by SDS-PAGE and were subsequently identified using MALDI- TOF peptide mass fingerprinting. The extract contained integral membrane proteins with one or more transmembrane segments, and some soluble proteins. The 20 most intense protein bands from the insoluble pellet did not correspond to integral membrane proteins. Our results show that fractionation of the proteome to reduce sample complexity and enrich low abundance proteins is an important step for the further analysis of the membrane proteome. We have established techniques to separate intact membrane proteins and their peptide fragments with the goal to maximise the coverage of the membrane proteome of C. glutamicum. POSTER SESSION

P-46 Membrane Proteome Analysis of the Green Sulfur Bacterium Chlorobium tepidum

M. V. Aivaliotis1, C. Corvey2, I. Tsirogianni1, M. Karas2 and G. Tsiotis1 1Division of Biochemistry, Department of Chemistry, University of Crete, Heraklion, Greece, and 2Institut für Pharmazeutische Chemie, Instrumentelle Analytische Chemie, Johann Wolfgang Goethe Universität, Frankfurt am Main, Germany

Chlorobium tepidum is a photosynthetic, unicellular, strictly anaerobe, green sulfur bacterium which utilize sulfur compounds as electron donors. Similar to other Gram-negative bacteria, Chl. tepidum has an envelope consisting of an outer membrane, a peptidoglycan layer, and a plasma membrane [1]. The outer membrane represents an effective permeability barrier in controlling the permeation of substrates and antibiotics as well the export of substances. The role of the green sulfur bacteria’s plasma membrane as an energy-transducing membrane is also crucial since a large number of biological processes are coupled to transmembrane ion potential or dependent on membrane-bound ATPases as well as the respiratory chain. In addition, these organisms have a distinct intracellular membrane system, the chlorosomes, which are energy-collecting membranes. The complete genome of Chl. tepidum was determined recently and represents the first genome sequence from the phylum Chlorobia [2]. Proteomic studies require, apart from the total genome knowledge, that each cellular compartment can be isolated without cross-contamination. As a crucial first step toward the analysis of the composition and function of the Chl. tepidum membranes, procedures were developed for their biochemical purification. In the present work we have used different protocols for the selective extraction of the outer membrane proteinsand the whole membrane proteins. In our studies we have used the extracted proteins in proteomic studies based on two-dimensional gel electrophoresis [3], trypsin treatment of excised spots, and MALDI-TOF analysis with database identification [4].

References [1] Oelze, J.a.G., J. R., Membranes and chlorosomes of green bacteria: structure, composition and development. Anoxygenic photosynthetic bacteria, ed. R.E. Blankenship, Madigan, M. T. and Bauer, C. E. (eds). 1995, Dordrecht: Kluwer Academic Publishers. 259- 278. [2] Eisen, J.A., Nelson, K. E., Paulsen, I. T., Heidelberg, J. F., Wu, M., Dodson, R. J., Deboy, R., Gwinn, M. L., Nelson, W. C., Haft, D. H., Hickey, E. K., Peterson, J. D., Durkin, A. S., Kolonay, J. L., Yang, F., Holt, I., Umayam, L. A., Mason, T., Brenner, M., Shea, T. P., Parksey, D., Nierman, W. C., Feldblyum, T. V., Hansen, C. L., Craven, M. B., Radune, D., Vamathevan, J., Khouri, H., White, O., Gruber, T. M., Ketchum, K. A., Venter, J. C., Tettelin, H., Bryant, D. A. & Fraser, C. M., The complete genome sequence of Chlorobium tepidum TLS, a photosynthetic, anaerobic, green-sulfur bacterium. Proc. Natl. Acad. Sci. U S A, 2002. 99: p. 9509-9514. [3] O'Farrell, P.H., J. Biol. Chem., 1975. 250: p. 4007-4021. [4] Pappin DJ., H.P., Bleasby JA., Curr. biol., 1993. 3: p. 327-332. POSTER SESSION

P-47 Proteomic Analysis of B. anthracis Cell Membrane Proteins

T. Chitlaru, E. Elhanany, N. Ariel, A. Zvi, M. Lion, B. Velan and A. Shafferman Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, Israel

In the context of defining B. anthracis surface constituents potentially associated with the induction of anti-Anthrax protective immunity, a partial proteomic analysis of a B. anthracis membranal sub-cellular fraction has been carried-out. This high-through-put global approach, involving the concomitant inspection of a large number of bacterial proteins was facilitated by the recent advent in the DNA sequencing [1, 2] and gene annotation [3] of the B. anthracis chromosome. B. anthracis V770-NP1-R (pXO1-,pXO2-) cells from a HBI-media-stationary phase culture were subjected to sub-cellular fractionation and a protein mixture highly enriched in membranal proteins was subjected to 2-Dimensional Electrophoresis (2DE) generating a highly reproducible protein signature. The 2DE map was subsequently complemented with protein analysis by tryptic-digest and MALDI-TOF mass spectrometry. More than 80 individual protein spots representing the most abundant membranal proteins of the sub-cellular fraction under study, were unequivocally identified by comparison of their tryptic fingerprint spectrum with that generated by the annotated ORFs in the chromosomal B. anthracis in-house data base. These 80 protein spots represent 30 distinct proteins as most of the proteins exhibit a multitude of isoelectric forms, indicative of intense post-translation modification activities. The vast majority of the proteins belong to the category of membranal proteins, based either on membranal localization amino acid domains, or membranal nature of their orthologs. The proteomic data indicate the prevalence of S-layer proteins on the surface of B. anthracis. The S-layer protein Extractable-Antigen 1 (EA1) represents 80% of the membranal protein mass. The S-layer associated protein (Sap) is present only in minor quantities (100fold less than EA1) stemming from the stationary nature of the culture under study [3]. Five additional proteins containing S-layer homology (SLH) domains were identified for the first time in this study.

References: [1] T. Read et al., 2002, Science 296, 2028. [2] T. Read et al., 2003, Nature 423, 81. [3] N. Ariel et al., 2003, Infection and Immunity, 71, 4563. [4] T. Mignot et al., 2002, Molecular Microbiology, 43, 1615. POSTER SESSION

P-48 Characterization of Plasma Membrane Proteins of Brain Microvascular Endothelial Cells

M. Jäger, T. Oppolzer, T. Bangsow, B. Pelzer and S. Wolf Esplora GmbH, c/o Institute of Biochemistry, Darmstadt, Germany

The aim of proteome analysis is the quantitative description of protein expression patterns of a cell type, an organism or body fluids at strictly defined conditions. Although mass spectrometry is not a quantitative method it has become a powerful tool to characterize molecules at the protein level. Routinely the methods used for quantification and identification are mainly based on 2D-PAGE followed by a mass spectrometric analysis. This combination shows some limitations, because very large proteins as well as membrane proteins are only poorly separated by 2D gels. More recently, alternative approaches avoiding the use of a gel have been established. A particularly powerful technique is the combination of liquid chromatography (LC) and mass spectrometry (MS). These techniques combined with the use of isotope-coded affinity tags allow relative quantification of proteins between two samples (Gygi et al. (1999) Nat Biotechnol. 17, 994- 999). This approach involves differential labeling of proteins in two samples with affinity reagents differing in mass. Cellular communication is essential for the function of complex networks in mammalian tissue. The first to react after external perturbations are the proteins located at the cell surface. In our group we are looking at the changes in the protein level of brain microvascular endothelial cells (BMEC). Here, new reagents for isotope-coded affinity labeling are presented and their ability to be used for quantitative mass spectrometry is shown. They allow a selective labeling of cell surface proteins because they are not membrane permeable. In contrast to other reagents w e choose amino-reactive reagents because of the lack of sulfhydryl groups on the cell surface. We show the use of the light variant of ESP-TagTM for a selective labeling of the cell surface proteome of cultured brain microvascular endothelial cells. After labeling the proteins were solubilized using detergent and the labeled proteins were isolated using affinity chromatography. The eluted fraction was separated on a SDS-Page and the lane was cut in pieces. A tryptic digest was performed and the resulting peptides were analyzed by MALDI- MS. We could identify 45 proteins of which 50 % were described as membrane, membrane associated or secreted proteins. About 30 % of the identified proteins were unkown so that function and localization remains unclear. 5 of the identified proteins were chosen for further characterization. For these proteins, one member of the multi drug resistance family, one Cu2+-transporter, Plexin B1, membrane metallo proteinase 10 and one unkown protein, expression pattern were obtained using RT PCR and real time pcr, resp. In addition western blot analysis and immune fluorescence staining of cultured BMEC were performed. POSTER SESSION

P-49 Isolation of the Plasma Membrane from T-cells for Protein Analysis

S. Helling1, P. Lutter2, P. Weingarten2, C. Hüls2, H.E. Meyer1 and K. Marcus1 1Medizinisches Proteom-Center, Ruhr-Universität Bochum, Germany, and 2Protagen AG, Dortmund, Germany

The cellular immunity is mainly regulated by different types of T-cells, which are able to stimulate or suppress an immune response against antigens. T-cells differentiate through induction by metabolites like cytokines and antigens or by cell-cell contacts. The initiating factor of this signal transduction process is the binding of cell surface proteins to their counterparts. To analyze the different cell states with proteomic tools the isolation and purification of the plasma membrane (PM) is necessary. An optimal sample preparation includes the separation of the PM from other cell-compartments like the nuclei, the endoplasmatic reticulum or microsomes. Indeed, the isolation of the PM is problematic due to vesicle formation after cell- lysis or due to co-sedimentation of membranes from other organelles. In the present study, two methods are described for the PM isolation including a strategy for the quality control of plasma membrane fractions. To optimize the methods, the sample preparation was performed with Jurkat cells, a human leukemia T-cell line. Prior to lysis, the cells where surface-biotinylated and washed. The isolation was performed by either differential or density-gradient centrifugation. For the quality control the plasma membrane containing fraction was checked first for biotinylated proteins by an immuno-blot. Afterwards, the extent of contamination by soluble cytosolic proteins was determined using two dimensional gel electrophoresis (2D-PAGE) and MALDI-TOF MS. The last step of the quality control was immuno-blotting with antibodies specific for known T-cell membrane proteins CD3, CD4 and the T-cell receptor (TCR) to ensure specific extraction of T-cell characterising plasma membrane proteins with the applied extraction buffer POSTER SESSION

P-50 Proteomic Tools to Identify Thylakoid Components

G. del Riego, H.R. Lascano, L. M. Casano and M. Martín y B. Sabater Departamento de Biología Vegetal, Universidad de Alcalá de Henares, Madrid, Spain

Plastids probably contain more than 2000 different polypeptides, most of them encoded by nuclear genes. Although the genes for the major polypeptides of plastid thylakoids are well identified, a large number of minor thylakoid polypeptides and their correspondent genes are not identified. In addition, uncertainties remain in respect to N- and C- terminal processing and other post-translational modifications of major thylakoid polypeptides. We are mainly interested in the chloroplast Ndh complex (homologous to the respiratory complex I) which is a minor component of thylakoids. Supposedly, the Ndh complex is composed of chloroplast and nuclear encoded polypeptides. However, little is known on the putative nuclear genes and on the possible post-translational processing of the primary translation products of the chloroplast and nuclear genes involved. Proteomic analysis could provide significant advances to reach a full description of the thylakoid proteins. The best proteomic approach requires an organism with entirely sequenced genome and rending intact thylakoids, without contamination by other cellular fractions. Starting from the experience of our group on barley thylakoid and the running programme to sequence the complete genome of this plant, we are investigating for the identification of minor thylakoid proteins of barley, specially the Ndh complex. 2D-electrophoresis is the most powerful separation tool to deal with complex samples like the thylakoid membranes. Anyway, major components make difficult the detection of minor components. In order to reduce the number of polypeptides in each 2D-electrophoresis w e assayed previous fractionation of thylakoid components by two different approaches: gel filtration and sequential solubilization of thylakoid polypeptides. For the first approach, we solubilized barley thylakoid membranes with Triton X-100 and separated the components in a Sephacryl S-300 HR column. The fraction with the highest specific activity of the Ndh complex was analysed by 2D-electrophoresis. In the sequential solubilization, we first used increasing concentrations of sodium chloride (0.15, 0.5, 0.75 and 1 M) and later, three concentrations of Triton X-100 (0.1%, 0.5% and 1%). In this approach, we firstly expect to identify known polypeptides as markers of each fraction by 2D-electrophoresis and MALDI-TOF. The first polypeptides identified in different fractions corresponded to peripheral subunits of major thylakoid complexes, such as the _ subunit of the ATP synthase or some subunits of the oxygen evolving complex. None of the polypeptides identified so far corresponded to intrinsic membrane polypeptides, which presumably should be present in the different samples. Tryptic peptide profile of many of 2D-electrophoresis spots did not correspond to known gene sequences and, while more sequences of barley genome will be available, our immediate purpose will be to determine the amino acid sequence of selected peptides. We are also assaying new modifications of the solubilization and electrophoresis protocols to improve the yield in highly hydrophobic polypeptides. POSTER SESSION

P-51 Targeted Proteome Analysis of GPI-Anchored Proteins in Human and Plant Cell Membranes

F. Elortza1, L. J. Foster1, T. S. Nühse2, A. Stensballe1, S. Peck2 and O. N. Jensen1

1Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark and 2Sainsbury Laboratory, Norwich Research Park, Colney, Norwich UK.

In eukaryotic cells, a subset of proteins are attached to the external leaflet of the plasma membrane by a glycosyl-phosphatidylinositol (GPI) anchor. There is substantial evidence suggesting that these GPI-anchored proteins are clustered in sphingolipid-sterol microdomains or lipid rafts. Rafts play a crucial role in many cellular functions including membrane traffic, cell signalling and human diseases. We have combined detergent-based phase separation methods and phosphatidylinositol specific phospholipase C (PI-PLC) enzyme treatment, to enrich and isolate GPI-proteins from HeLa cell lipid rafts, and GPI-proteins from membrane fractions isolated from Arabidopsis thaliana. The isolated protein samples were concentrated by precipitation, separated by SDS-PAGE and protein bands were detected by two complementary methods. Firstly, silver staining was used to visualize the proteins recovered in the aqueous phase. Secondly, western blotting using anti cross reacting determinant (CRD) antibody specific for the C-terminal of PI-PLC treated GPI-anchored protein demonstrated that GPI-APs were highly enriched in the aqueous phases after PI-PLC treatment. Protein bands were excised for in-gel digestion and the resulting peptides were subsequently analyzed by nanoscale liquid chromatography coupled online to electrospray Q-TOF tandem mass spectrometry (nLC-MS/MS). Tandem mass spectra were interpreted automatically using the MASCOT sequence database search engine. With this combination of methods we have identified 6 already known GPI-AP in HeLa cell rafts, and more than 40 GPI anchored proteins in A. thaliana cell membranes, which validates this proteomic approach for analysis of GPI anchored membrane proteins. The strategy presented is expected to be generally applicable and highly useful for studying membrane proteins, for example for investigations of molecular disease markers on the cell surface from different organisms.

This research was supported by a post-doctoral fellowship to F.E. from the Basque Government and by resources provided by the Danish Biotechnology Instrument Center. POSTER SESSION

P-52 Investigation of Hydrophobic Proteins by a Novel Gel-free Technology: Protein Sequence Tag (PST®)

T. Prinz, K. Kuhn, J. Müller, C. Baumann, J. Korder, M. Sonnentag, T. Neumann and C. Hamon Proteome Sciences plc, Coveham House, Cobham, Surrey UK

Mass spectrometric detection coupled to liquid chromatography analysis of peptide digests is emerging as a powerful gel-free alternative tool for the analysis of complex protein mixtures, which has the potential to overcome the limitations of 2-D gel electrophoresis with respect to hydrophobic proteins [1]. Methods of ‘sampling’ protein digests have great value if they can provide sufficient information to identify substantially all of the proteins in the sample while reducing the complexity of the sample to maximise the efficient usage of LC-MS/MS capacity [2]. Here we describe a novel method for the isolation of protein sequence tags to identify proteins in a complex mixture of hydrophobic proteins [3]. The PST® (Protein Sequence Tag) technology deals with the isolation and LC-MS/MS based identification of one N-terminal peptide from each polypeptide fragment generated by cyanogen bromide cleavage of a mixture of proteins. We have applied PST® sampling to an enriched mitochondrial fraction from baker’s yeast. The method presented here combines effective sample preparation with a novel peptide isolation protocol involving chemical and enzymatic cleavage of proteins coupled to chemical labelling and selective capture procedures. Thereby, this method substantially reduces the complexity of a protein digest by ‘sampling’ the peptides present in the digest, which increases the efficiency of the LC-MS/MS based protein identification. Our experimental data demonstrate the high reproducibility and reliability of the PST® protocol to identify, beside known soluble proteins, all classes of hydrophobic proteins, including transmembrane proteins with a-helix or b-barrel structures and membrane associated proteins. As other gel-free approaches, we were able to study even proteins with very high or very low molecular weights and proteins with pI values at the extreme ends of the pI range. Noteworthy, this technology was shown to be applicable to mitochondrial membrane fractions as well. The majority (~ 80%) of the found proteins were integral membrane proteins or membrane-associated proteins. Thus, the overall process has been very successful for the analysis of complex mixtures of hydrophobic proteins, particularly membrane proteins.

References: [1] D. Lin, Tabb D.L., and Yates J.R. Biochim Biophys Acta 2003, 1646, 1-10 [2] S.P. Gygi, Rist B., Griffin T.J., Eng J., and Aebersold R. J. Prot. Res. 2002, 1, 47-54 [3] K. Kuhn, Thompson A. , Prinz T. , Mueller J., Baumann C., Neumann T., and Hamon C. J. Prot. Res. 2003, In press POSTER SESSION

P-53 Cell Surface Proteome: Labeling and Comparing Cell Surface Proteins

M. Beaumont, C. Sheppard, F. Vega, M. Mattison, M. Oft and C. Hannum DNAX Research, Palo Alto, CA, USA

We have developed a method to selectively label cell surface proteins, correlate the total cell 2D protein pattern to the cell surface 2D protein pattern and compare cell surface proteins across various cell lines. The example used to demonstrate this new method is a comparison of cell surface proteomes between EP2 cells (Ras-transformed mammary gland epithelial cell line) and EP2 cells treated with TGFb. Treatment by TGFb induces a switch from an epithelial to mesenchymal type morphology in these cells. More precisely, the method involves labeling live cells with biotin, lysing the cells, and labeling the total cell lysate with Cy5. For signal enrichment, a membrane preparation step can be added before Cy5 labeling. The Cy5-labeled total lysate, containing biotinylated cell surface proteins, is then spread out by 2D electrophoresis, and the 2D gels are blotted onto PVDF membranes. Membranes are then probed with streptavidin-HRP, detected with ECL+, and scanned for both Cy5 and ECL+ on a Typhoon 9400. Dual scans Cy5/ECL+ are obtained from membranes with EP2 cells and EP2 treated with TGFb, and image pairs are compared for differences using DeCyder software. Spots of interest are then robotically excised from a parallel Sypro Ruby-stained preparative gel and their identity determined by nanospray LC-MS/MS. POSTER SESSION

P-54 Identification of Nuclear Proteins by a Single-Step Method using Mass Spectrometry

J. M. Estanyol, M. Ventura and O. Bachs Department of Cell Biology, Faculty of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain

Proteomics became one of the most powerful techniques to characterize complex protein samples and to make comparison studies of protein expression in different cells, differrent cell state conditions and different diseases. Today, several methods to approach comparison from different cell protein state by proteomics were developed. 2D electrophoresis and characterization proteins by MALDI TOF and 2DLC MS-MS are the most powerful. We tried a simple home method to characterize protein content from nucleus compartment from mouse hepatocytes by "off line” MS-MS mass spectrometry without HPLC instrument. Different works from our laboratory determined different protein levels and protein complexes present in the nucleus of hepatocytes in normal state and after partial hepatectomy by several strategies. We compare previous results with results after analysis with mass spectrometry. Firstly w e isolate nucleus from mouse liver hepatocytes. Samples were checked by light microscopy to determine good nuclei isolation and were digested by trypsin. Salts were taken off by loading peptides in a Zip-Tip (Millipore) and eluted in different concentrations of acetonitrile. Resulting peptides from each extraction were loaded in a home-made gold coated glass tip and sprayed into a nano-ESI LCQ-DECA XP mass spectrometer (ThermoFinnigan) in different steps. Sequences of peptides were determined by SEQUEST software with normal parameters. With these conditions, we can determine about 500 proteins that we can divide in structural proteins, cell cycle proteins, transcription factors, DNA regulation proteins etc. These results are in consonance with results using other techniques, but with mass spectrometry using simple step isolation, we can find in one assay the most abundance proteins in this compartment.

References: Mann M, Hendrickson RC, Pandey A. Analysis of proteins and proteomes by mass spectrometry. Annu Rev Biochem. 2001;70:437-73. Review. Jaumot M, Estanyol JM, Serratosa J, Agell N, Bachs O. Activation of cdk4 and cdk2 during rat liver regeneration is associated with intranuclear rearrangements of cyclin-cdk complexes. Hepatology. 1999 29:385-95. Pujol MJ, Jaime M, Serratosa J, Jaumot M, Agell N, Bachs O. Differential association of p21Cip1 and p27Kip1 with cyclin E-CDK2 during rat liver regeneration. J Hepatol. 2000 33:266-74. POSTER SESSION

P-55 Histone Modifications in the Estabilishment and Maintenance of Epigenetic State

T. Bonaldi1, A. Imhof2, J. Boeke2 and J. Regula1 1Zentrallabor für Proteinanalytik, Adolf Butenandt Institut, 2Histone Modifications Group, Adolf Butenandt Institut, Ludwigs Maximilians Universität, München, Germany

Histone tails on the nucleosome are subject to enzyme-mediated posttranslational modifications of selected amino acids, such as lysine acetylation, lysine and arginine methylation, serine phosphorylation and attachment of the small ubiquitin peptides. These modifications, singly or in combination, are thought to generate an epigenetic code that specifies different patterns of gene expression and silencing. Such information is essential for cells to establish and ‘remember’ specific programmes of gene expression, set during embryonic development, corresponding to different cell type identities. Recent work has provided new insights into how single histone modifications affect chromatin function. However, to fully decipher the information storage function of histone tails, a better understanding of how specific combinations of tail modifications are generated and how the modification of one residue can affect that of another is essential. The main tools presently used to study the epigenetic code are antibodies against specific histone modifications. Unfortunately this approach has several limitations when it comes to the problem of detecting combinations of several modifications: as a matter of fact, often the affinity of antibodies against one modification is dramaticaly altered by a modification close by, making it very difficult to distinguish between the disappearance of a modification and its masking due to the presence of an adjacent one. Moreover it is extremely difficult to generate antisera even to single histone modifications with enough specificity to distinguish between very similar modifications: e.g. di-methylation versus trimethylation on the histone molecule. Mass spectrometry (MS) offers a potential solution for these important limitations, thanks to the recent development of the techinque to investigate protein posttranslational modifications. The approach we developed uses high resolution mass spectrometry on chromatin subdomains purified form different cell cultures and from embryos at different developemental stages. The model system for chromatin modifications will be Drosophila melanogaster. We developed a reliable protocol for the chromatographic separation of the histones: they were separated by reversed phase HPLC into 3 fractions, containing Histone H2B, a mixture of Histone H4 and H2A and Histone H3. Two main reasons for the use of reversed phase HPLC instead of SDS-PAGE to separate individual proteins is based on the higher efficiency of the proteases in solution compared to their in gel activity. A second advantage is the higher recovery of peptides, essential to obtain the highest sequence coverage. The separated histones were then digested in solution by the proteases Arg-C, Asp-N (singly or in combination) instead of Trypsin because of the the high content in R and K residues of histones. The resulting peptides were first analyzed with MALDI mass spectrometry to map potential modification sites. Peptides of interest were further characterized and mapped by sequencing with nano electrospray mass spectrometry. We are applying this method to the analysis of different samples of chromatin, with a specific interest in comparing the modification patterns of consitutive heterochromatin domains with those of euchromatic regions. A crucial question with respect to heterochromatin is which is the specific modification pattern of how this is established maintained and controlled. Recently, diverse methods for the isolation of heterochromatin domains have been developed, based on specific sonication of nuclei followed by differential centrifugation; we apply these protocols to enrich heterochromatin from different Drosophila cells or embryos at different cell stages that will be then analyse by MassSpec. The aim is to full characterize the heterochromatic modification pattern. POSTER SESSION

P-56 Do You Really Know Where Your Protein Starts? A Proteomic Approach

D. Bellido1, S. Paytubi2, A. Juárez2, A. Carne3 and E. de Oliveira1 1Proteomic Facility of Scientific and Technical Services-Science Park of Barcelona, University of Barcelona, Spain; 2Department of Microbiology, Faculty of Biology, University of Barcelona, Spain, and 3Proteomika, Science Park of Barcelona, Spain

Most expressed proteins are posttranslationally modified to become active. Many kinds of modifications are very well known such as phosphorylation and glycosylation and also, some proteins are synthesised as the precursor and undergo a cleavage of the signal peptide. In general, this peptide participates in the recognition of the protein and is cleaved forming the functional polypeptide. The equilibrium of the reaction is directed towards active protein, but it can be altered by some special in vivo or in vitro conditions. In such cases, it can be crucial to identify the signal peptide in order to know the protein state. Edman degradation is a very usable approach to sequence the N-terminus of proteins. It seems to be the most reliable methodology in such cases, but it uses picomols of protein. Moreover, this methodology can not be employed when the N-terminal amino acid is modified, or for protein mixtures. In this work, we used an easy proteomic approach to determine a protein state. An Escherichia coli cell lysate was separated by SDS-PAGE and some bands were digested with trypsin. The tryptic peptides were then analysed by mass spectrometry. Using the Maldi- TOF/TOF we identified the OmpA precursor (Outer Membrane Protein A) with a high score (279 in MASCOT), but no N-terminal fragment was found. When the N-terminus of the protein is not detected, the score of the precursor is higher than the active one. OmpA is a 35.2 Kda protein (325 amino acid residues) obtained from the cleavage of the N-terminus of the OmpA precursor containing 346 amino acid residues. In order to solve this problem, we digested "in silico” both sequences and we saw different patterns of N-terminal tryptic peptides amongst them. The four peptides for the precursor and one for the active protein were included in a peak list. Using a survey scan analysis in a CapLC-Q-TOF one ion was detected and fragmented giving a peptide sequence belonging to the active protein. If you are looking for a data base described protein, this is an easy and fast way to unambiguously identify a protein state POSTER SESSION

P-57 Towards the Phospho-Proteome of Corynebacterium glutamicum

A. K. Bendt1, A. Burkovski1, S. Schaffer2, M. Bott2, M. Farwick3 and T. Hermann3 1Institut für Biochemie, Universität zu Köln, 2Institut für Biotechnologie 1, Forschungszentrum Jülich, and 3Degussa AG, Halle-Künsebeck, Germany

We are interested in global regulatory networks in Corynebacterium glutamicum, a Gram- positive soil bacterium which serves as a model organism for mycolic acid-containing actinomycetes such as Mycobacterium tuberculosis and Corynebacterium diphtheriae [1]. To investigate the role of protein phosphorylation in this bacterium, we started an approach to analyze phosphoproteins by two-dimensional gel electrophoresis (2-DE) and two different detection methods: first, in vivo metabolic radio-labeling using [33P]-orthophosphate with subsequent autoradiography and second, immunostaining with phosphoamino acid-specific monoclonal antibodies. We showed the detection of at least 120 cytoplasmic protein spots containing phosphorylated proteins and identified many of them with MALDI-TOF-MS [2]. Most of the proteins identified are enzymes acting in the cells central metabolic pathways such as glycolysis, Krebs cycle and fatty-acid metabolism. Also chaperones as well as components of the protein synthesis machinery were identified. We were not able to identify regulatory proteins or signal transduction molecules, which are mainly low-abundance proteins and thus difficult to detect with the 2-DE methods applied. The challenge of future studies will be not only the visualization and identification of phosphorylated proteins, but also the localization of the corresponding phosphorylation sites as well as a quantification of protein phosphorylation.

References: [1] Nolden, L., Ngouoto-Nkili, C.-E., Bendt, A. K., Krämer, R., Burkovski, A. (2001): Sensing nitrogen limitation in Corynebacterium glutamicum: The role of glnK and glnD. Mol Microbiol 42: 1281-1295. [2] Bendt, A. K., Burkovski, A., Schaffer, S., Bott, M., Farwick, M., Herrmann, T. (2003): Towards a phosphoproteome map of Corynebacterium glutamicum. Proteomics 3, in press. POSTER SESSION

P-58 Identification of Phosphotyrosine-Proteins Regulating Epithelial Mesenchymal Transition During Acute Renal Failure

M. de Graauw1, R. van der Heijden2 and B. van de Water1 Divisions of 1Toxicology and 2Analytical Biosciences, Leiden/Amsterdam Center for Drug Research, Leiden University, The Netherlands

Acute renal failure (ARF) is still an important clinical problem caused either by ischemic episodes or exposure to nephrotoxic medicines, such as the anticancer drug cisplatin . Such renal injury is associated with detachment of viable cells from their extra-cellular matrix (cell- ECM) or loss of cell-cell interaction. These events may be reminiscent of a cellular stress- induced epithelial mesenchymal transition (EMT). MDCK cells are a good model to study EMT, a process that is most likely regulated by the tyrosine phosphorylation status of proteins. The non-receptor tyrosine kinase c-Src seems an important player in EMT. Indeed, EMT can be induced in temperature sensitive ts-v-Src MDCK cells by shifting them from a restrictive temperature (40°C) to a permissive temperature (35°C). This correlates with an increase in tyrosine phosphorylation (pTyr) status of several proteins. Importantly, herbimycin A, an inhibitor of phosphotyrosine kinases, blocks loss of cell-cell interaction as well as protein tyrosine phosphorylation during EMT. In order to identify the proteins with a change in tyrosine phosphorylation status we have set up 2D phosphotyrosine blotting. For this purpose w e used ts-v-Src MDCK cells held either at the permissive or non-permissive temperature. Changes in pTyr status of proteins were analysed with PDQUEST software. Selected pTyr protein spots were matched to Sypro Ruby protein spots on a 2D gel scanned with the Typhoon 9600. All proteins with a change in tyrosine phosphorylation during v-Src-induced EMT were picked and are awaiting identification by MALDI-TOF and QSTAR. POSTER SESSION

P-59 Phosphoproteomics of the Arabidopsis Plasma Membrane

T. S. Nühse1, A. Stensballe2, O. N. Jensen2, S. C. Peck1 1Sainsbury Laboratory, Norwich Research Park, Colney, Norwich, UK and 2Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark

The identification of phosphorylation sites is a major bottleneck in signal transduction research, and despite enormous advances in mass spectrometric technology, it is still not routine, especially for low-abundance proteins like plasma membrane receptors. However, by enriching phosphopeptides from complex digests it is possible to sequence large numbers of phosphorylation sites, and these data may help to understand the regulation of related proteins of interest. We are interested in signaling processes at the plant plasma membrane. Plasma membrane proteins are involved in the perception of microbial elicitors and regulation of early responses, and are targets of bacterial virulence factors. Proteomic approaches for membrane proteins based on 2D gels have been challenging, but recent "shotgun” approaches based on tryptic digestion of whole membranes and analysis of the peptides by LC-MS/MS have overcome the bias against large hydrophobic proteins. We have combined the "shaving” approach with the isolation of phosphopeptides by immobilized metal ion affinity chromatography (IMAC). The specificity of IMAC for the capture of unmodified phosphopeptides was between 75 and over 90%, far better than previously assumed. We have used a "three-dimensional” liquid chromatographic separation scheme (strong anion exchange – IMAC – reverse phase LC- MS/MS) to decrease the complexity of the sample, and at the same time obtained a far better yield of monophosphorylated peptides. In total, several hundred phosphorylation sites on Arabidopsis plasma membrane proteins have been identified. The most prominent groups of phosphoproteins include receptor-like kinases (over 30), ABC transporters, ion channels/pumps and metabolite transporters. One interesting observation was the abundance of phosphorylation sites on the unconserved juxtamembrane and C- terminal domains of receptor-like kinases- a finding that will have consequences for our understanding of signaling specificity and regulation of these proteins. For many of the identified phosphoprotein families, like mechanosensitive ion channels and cellulose synthases, this is the first evidence for regulation through phosphorylation. POSTER SESSION

P-60 Evaluation of the Potential of a Commercial Kit to Specifically Enrich and Analyse the Phosphoproteome

G. Rehg1, K. Hägele2, J. Volz2 and S. Dammeier1 Altana Pharma AG, 1Department of Functional Genomics & Proteomics, and 2Department of Physicochemical Research, Konstanz, Germany

The enrichment of phosphoproteins remains a central issue to analyse the phosphoproteome. Phosphoproteins were labeled in vivo in Jurkat cells with 33P-orthophosphoric acid and subsequently enriched directly by using commercial columns, without previous tryptic digestion. These columns are neither based on antibody affinity nor on the classical IMAC matrices with chelators complexing Fe3+ or Ga3+ ions. More than 80% of radioactivity were found reproducibly in the eluate. We analysed lysate, flow through and eluate by 2-DE. The gels were silver-stained and autoradiographed. Spots appearing on both, autoradiography and silver gel, refer to phosphorylated proteins. Several proteins were identified by mass spectrometry to confirm their occurrence as phosphorylated proteins. In order to estimate the benefit of the new columns for biological questions, Jurkat cells were stimulated with PMA, a PKC stimulans, and compared to non stimulated cells. Actually the changes in the spot pattern of the 2D gel go back to the stimulation by PMA since several proteins were identified known as substrates of PKCs. POSTER SESSION

P-61 Global Detection, Quantitation and Identification of Phosphorylated Proteins

M. Beaumont, C. Sheppard, F. Vega, N. Fouladpour, M. Oft, and C. Hannum DNAX Research, Palo Alto, CA, USA

Protein phosphorylation is a fundamental mechanism governing basic cell functions such as signal transduction and cell cycle events. Since most of these phosphorylation and dephosphorylation events occur as transient waves through pre-existing proteins they are beyond the scope of genomic studies. Consequently protein phosphorylation has become one of the most compelling areas for proteomics, whereby individual phosphorylations can be detected and completely characterized. In this regard we present our initial studies testing a fluorescent gel stain, Pro-Q Diamond™, that can detect proteins phosphorylated on tyrosines, serines, and threonines. Using model proteins we show that Pro-Q Diamond is specific, sensitive, and can be multiplexed with Cy5. We also present a single study investigating phosphorylation changes in a Ras-transformed mammary gland epithelial cell line, EP2, with and without TGF_ treatment at several time points. During such treatment EP2 cells switch from an epithelial to a mesenchymal morphology. Whole cell lysates were labeled with Cy5 and run on high resolution 2D gels that were post-stained with Pro-Q Diamond and scanned simultaneously for both total protein (Cy5) and phosphoprotein (Pro-Q Diamond) patterns. The resulting patterns were matched and analyzed using DeCyder™ software, and proteins showing changes in phosphorylation were picked robotically and analyzed by nanospray LC- MS/MS POSTER SESSION

P-62 Analysis of EGF Receptor Phosphorylation Sites by Tandem Mass Spectrometry

E. Boeri Erba1,2, E. Bergatto2, S. Cabodi2, G. Tarone2, P. Defilippi2, O. N. Jensen1 1Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odens,e Denmark; and 2Dipartimento di Genetica, Biologia e Biochimica, Università di Torino, Italy

Post-translational modifications are crucial for regulation and control of dynamic cellular processes. Protein phosphorylation on tyrosine, serine and threonine residues is one of the most common protein modification regulating signaling events. The EGF receptor is a 180 kDa protein tyrosine kinase which, in response to ligand binding, is phosphorylated and mediates many cellular responses in normal biological processes and in pathological states. Mutations of EGF receptor sites and its over-expression are implicated in a variety of cancers, including mammary carcinomas, squamous carcinomas, and glioblastomas, as well as other malignant diseases. In addition to the canonical activation by soluble ligands, we recently reported that EGF receptor can be phosphorylated by integrin-mediated adhesion in the absence of soluble ligands, leading to phosphorylation of a different subset of tyrosine residues (Moro et al., 2002). Here we present a mass spectrometry (MS) based experimental strategy for the identification and mapping of the major EGF receptor phosphorylation sites. The EGF receptor was isolated by immunoprecipitation and polyacrylamide gel electrophoresis from human epithelial ECV304 cells upon EGF treatment. Nanoscale immobilized Fe(III) affinity chromatography (Fe(III)-IMAC) columns were employed for enrichment of phosphorylated peptides from crude peptide mixtures prior to off-line analysis by matrix-assisted laser desorption/ionisation (MALDI) MS, nanoelectrospray tandem mass spectrometry (MS/MS) or MALDI quadrupole time-of-flight (QTOF) MS/MS. Alkaline phosphatase treatment of peptide mixtures in combination with nano-scale Fe(III)-IMAC and MALDI-MS allowed unambiguous identification of phosphopeptides by observation of 80 Da mass shifts. From the EGF-treated sample, several phosphorylation sites were determined in three different series of experiments. Similar experiments are also underway for the integrin-dependent activated EGF receptor. Preliminary results show that at least one tyrosine site, the 1148 residue, is not phosphorylated in this conditions, suggesting a differential phosphorylation by distinct stimuli. POSTER SESSION

P-63 Examination of EPO Receptor Functions using Different Proteomic Strategies

S. Körbel1, M. Schümann2, E.Krause2, H.Prietzsch1, T.Büchse1, J.Brock1 and T. Bittorf1 1Institute of Medical Biochemistry, Medical Faculty, University of Rostock, Germany, and 2Institute of Molecular Pharmacology, Berlin, Germany

The erythropoietin receptor induces a genetic program resulting in the proliferation, differentiation and survival of erythroid progenitor cells. The complex response of cells is characterized by multiple translational and transcriptional events. To study the role of distinct receptor structures within this process we established an EGF/EPO receptor hybrid and expressed it in murine Ba/F3 cells. We show that this receptor transmits EPO specific signals after stimulation by recombinant human EGF. Mutant receptors which were produced on the basis of this system differ in their ability to trigger distinct signalling pathways and cellular activities. The functional analysis of different receptor types was done by conventional techniques and the examination of phosphoproteomes. Tyrosine phosphorylated proteins were isolated from EGF stimulated cells by immunoprecipitation. Phosphoproteins were separated by two- dimensional electrophoresis and identified using the MALDI-TOF-technology. Although w e were able to demonstrate specific sets of phosphoproteins with different receptor types the identification of signalling proteins by mass spectrometry was hampered by their low expression rates. An alternative approach using 1D-electrophoretic separation and LC-MS/MS analysis, however, was sensitive enough to detect many phosphoproteins known to be involved in erythropoietin receptor signalling pathways. In addition, novel proteins interacting with this receptor system were identified. The complex data provided by this approach are discussed with respect to the cellular responses observed. POSTER SESSION

P-64 Proteomic Approach to the Identification of Glycosylated Prostasomal Proteins

S. Liberatori1, L. Bini1, B. Canas1, E. Carlini2, C. A. Palmerini3, G. Arienti2 and V. Pallini1 1Dipartimento di Biologia Molecolare, University of Siena, Siena; 2Dipartimento di Medicina Interna, University of Perugia, Perugia; and 3Dipartimento di Scienze Biochimiche e Biotecnologie Molecolari, University of Perugia, Perugia, Italy.

Prostasomes are membranous vesicles (200 nm diameter) secreted by prostate gland in human semen. Among a number of other biological functions, including antioxidant and antibacterial activities, prostasomes can bind to spermatozoa and fuse with them, exhibiting a favourable effects on their motility. Although the prostasomal global protein composition has been recently elucidated (Utleg et al. 2003), little is known about proteins involved in the interaction between prostasomes and spermatozoa. In the present study we exploited the resolution power of two dimensional gel electrophoresis to visualize and analyze prostasomal proteome, including primary and post-translationally modified protein products. Two- dimensional electrophoresis was performed using the Immobiline-polyacrylamide system. The second dimension was carried out on 9-16% polyacrylamide linear gradient gels (18cm x 20cm x1.5mm). Proteins were visualized by silver staining and computer-aided 2-D image analysis was carried out using the MELANIE 4 software (GeneBio, Geneva, Switzerland). 2-D separated proteins were electroblotted on PVDF membranes and tested with detection methods specific for glycosylated proteins. Data will be presented showing the glycosylation profiles of human prostasomes under physiological conditions.

References: Utleg AG, Yi EC, Xie T, Shannon P, White JT, Goodlett DR, Hood L, Lin B. (2003) "Proteomic analysis of human prostasomes” Prostate 56, 150-161. POSTER SESSION

P-65 Site-specific Characterisation of N-linked Glycosylation with a CapLC/Q-Tof LC-MS/MS System

M. Ritchie, N. Johnson, C. Jones, T. McKenna and J. Langridge Micromass MS Technologies Centre, Waters, Manchester, UK.

Defining the extent and type of post-translational modification of a protein is often key in understanding its structure and function. These modifications cannot be directly predicted from genomic information. Protein bound carbohydrate can play an important role in the structure and function of glycoproteins, affecting their stability and folding. It can also influence the pharmacokinetics and functional activity of a therapeutic protein. Conversely, as glycosylation is the product of a complex series of modifications, malfunction in the cell can be reported changes in protein glycosylation. The location of glycan is often as important as structural information, a factor not often accounted for in most standard glycosylation analyses. Here a method for detection, characterisation and location of glycosylation in a single HPLC–MS/MS experiment is described.

A proteolytic digest is analysed by reverse phase HPLC-ESI using a Q-Tof mass spectrometer. The instrument runs two MS surveys at different gas cell collision energies. The low energy survey (7eV) shows only the intact or parent ions. The high energy survey (35eV) shows the fragments of these ions. Upon detection of the carbohydrate oxonium ions at m/z 204 (HexNAc), 366 (HexHexNAc) and 274/292 (NeuAc) the instrument is switched into MS/MS mode and ions from the low energy spectra are selected by the quadrupole for fragmentation. The high mass of glycopeptides gives rise glycopeptide ions typically of 4 or more positive charges, a feature that is exploited for their preferential selection for MS/MS. The mass measurement of the parent ion is enhanced by the use of nano-lockspray. The glycosidic bonds tend to be more labile than the peptide bonds hence MS/MS spectra produce predominately glycopeptide Y-type fragment ions. Complex MS/MS spectra containing multiply charged ions are simplified by the use of MaxEnt3, and interpretation is facilitated by the use of Carbotools. POSTER SESSION

P-66 Trypsinolysis of Polyubiquitin-Chains Studied by MALDI-TOF MS Analysis

A. P. Døskeland Department of Biochemistry and Molecular Biology, University of Bergen, Norway

Ubiquitination is a widespread posttranslational protein modification of significance for cell signalling and protein degradation. Ubiquitin (Ub) is a 8 kDa peptide that is covalently linked to a lysine residue in a protein by the Ub-conjugating enzyme complex. The purpose of the present study was to know if the Ub-lysine isopeptide bond could be detected by matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI-TOF MS) analysis. This isopeptide bond has lower energy than the peptide bond in the protein backbone, and the risk of rupture had to be determined. Intactness of the isopeptide bond during MALDI-TOF MS analysis is required to identify Ub-protein conjugates and the ubiquitination site (Lys) of proteins. PolyUb-chains (mixture of Ub2-Ub7) were chosen as a model for ubiquitin-protein conjugates and subjected to trypsinolysis under non-denaturating conditions. MALDI-TOF analysis of the trypsinized mixture revealed that the covalent isopeptide bond between Lys of ubiquitinated proteins and the Gly76 from the Ub carboxylterminal Arg74-Gly75-Gly76 remained mainly intact. Some cleavage between Gly75 and Gly76 was also observed suggesting either that trypsin can have some carboxyendopeptidase activity or that this bond is particularly susceptible to laser induced breakage. No laser induced breakage was observed between Gly75 and Gly76 in monomeric ubiquitin. A model for the trypsinolysis, in solution, of Ub and polyUb-chains based on profiles obtained from SDS/PAGE and MALDI-TOF MS analysis is presented. POSTER SESSION

P-67 Identification of Protein-Protein Interactions by CyDye Analysis of Immune Complexes

F. Vega, M. Beaumont, M. Mattison, N. Fouladpour, Y. Jiang, W. Seghezzi, and C. Hannum DNAX Research, Palo Alto, CA, USA

Many biological processes are performed by complexes of proteins that are non-covalently associated. A complete understanding of the composition of such complexes has historically been very difficult to obtain, often involving prolonged boot-strapping efforts such as yeast two-hybrid analyses. However, if one has a precipitating antibody against a single component of such a complex it should be possible to pull down all of the proteins and identify them in a single experiment using proteomic methods. We have developed methods for the efficient recovery and CyDye labeling of proteins from immune precipitations for subsequent 2D gel separation and exquisite analysis using DIGE™ technology. The key to such an analysis is the ability to run both the negative control proteins and the specifically precipitated proteins on a single multiplexed gel. The specific proteins, even very poorly represented ones, rise clearly above the non-specific noise, giving a comprehensive picture of the complex. All of the protein species of interest can then be picked robotically and identified directly. The system described here is a complex of proteins co-precipitating (from Hela cells) with Aurora 2, a serine- threonine kinase involved in the control of mitosis. Comparison of the negative (Cy3) and positive (Cy5) patterns reveals 89 clear differences, most of which have been identified by nanospray LC-MS/MS POSTER SESSION

P-68 Identification of Proteins Interacting with SK3 by Coimmunoprecipitation

J. O. Thumfart1, M. Biniossek2, J. Andersen3 and U. Schulte4 1Physiologie II University Freiburg, Germany; 2Molecular Medicine and Cell Research University Freiburg, Germany; 3Protein Interaction Laboratory University of Southern Denmark, Odense, Denmark; and 4Complexio GmbH, Freiburg, Germany

Calcium-activated potassium channels with low conductance (SK) couple the intracellular calcium concentration and the membrane potential in several types of excitable cell. There, an increase in intracellular Ca2+ leads to activation of SK channels resulting in membrane hyperpolarization. This mechanism is physiologically important, for example in determining the tonus of smooth muscle or rhythmic release of hormones. It is known that Ca2+ activates SK channels by binding to calmodulin constitutively associated with the intracellular C-terminus of the channel. Hence, native channels consist of at least the pore-forming a-subunit and the calcium sensor calmodulin. Since these channels are activated in vivo by the second messenger Ca2+ within milliseconds, it is reasonable to assume, that they also interact with other proteins involved in Ca2+ signaling. Rat brain membrane preparations were solubilized and subjected to coimmunoprecipitation in order to identify additional proteins interacting with SK3. Eluted proteins were separated by SDS-PAGE and visualized by silver staining. Specific gel bands were then excised and the contained proteins identified by nanoflow HPLC MS/MS and consecutive database search (NCBInr). In a first round of experiments, some plasma membrane proteins were identified, among them plectin, bassoon and dynamin. These proteins have been reported to be synaptically localized and thus may indeed interact with SK3 channels and contribute to their physiological function. POSTER SESSION

P-69 Identification of New 14-3-3 Interacting Partners

N. Jaspert ZMBP, Universität Tübingen, Tübingen, Germany

Members of the eukaryotic 14-3-3 protein family have been implicated in the regulation of several cellular processes like signal transduction events, cell cycle control and apoptosis. In plants, 14-3-3 proteins play a significant role in the regulation of transcription factors, key enzymes of primary metabolism and the plasma membrane H+-ATPase. The common feature of 14-3-3 proteins seems to be the interaction with other polypeptides (Fu et al. 2000), the latter characterized by a phosphorylated sequence motif. The optimal interaction motif identified so far is RSXpSXP (Muslin et al., 1996), but 14-3-3 binding might also occur to unphosphorylated ligands (Petosa et al., 1998). 14-3-3 proteins form saddle-shaped homo-or heterodimers. Binding of the interaction motif takes place within the amphipatic groove of a 14-3-3 monomer. The major aim of our work is the identification of novel plant 14-3-3 target proteins. "Far- Western-Analysis” of crude protein extract obtained from Nicotiana tabaccum shows numerous interacting partners, enrichment of which can be achieved by means of 14-3-3 affinity-chromatography. 2 D-gel electrophoresis should result in further isolation of these polypeptides followed by their identification.

References: Fu B, Subramamanian RR, Masters SC (2000) 14-3-3 proteins: structure, function and regulation. Annual Review of Pharmacology and Toxology 40, 617-647 Muslin AJ, Tanner JW, Allen PM, Shaw AS (1996) Interaction of 14-3-3 with signalling proteins is mediated by recognition of phosphoserine. Cell 84, 889-897 Petosa C, Masters SC, Bankston LA, Pohl J, Wang B, Fu H, Liddington RC (1998) 14-3-3 zeta binds to phosphorylated Raf peptide and a high affinity unphosphorylated peptide via its conserved amphipatic groove. JBC 273, 16305-16310 POSTER SESSION

P-70 Mapping Protein Interfaces by Chemical Cross-Linking and FTICR Mass Spectrometry: Application to the Calmodulin/Melittin Complex

D. M. Schulz, C. Ihling, and A. Sinz Biotechnological-Biomedical Center, University of Leipzig, Leipzig, Germany

Chemical cross-linking, a conceptually simple approach, has gained renewed interest in combination with mass spectrometric analysis of the reaction products allowing for investigation of interacting sequences in protein complexes [1, 2]. However, the identification of intermolecularly cross-linked sites from the complex mixtures created by chemical cross- linking and subsequent enzymatic digestion remains a challenging task. We present a method for mapping protein interfaces employing chemical cross-linking combined with FTICR (Fourier Transform Ion-Cyclotron Resonance) mass spectrometry. The method was employed to define the interacting regions in the Ca2+-dependent calmodulin/melittin complex. The general procedure involves the conjugation of calmodulin and melittin with the zero-length cross-linking reagent EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) as well as the homobifunctional, amine-reactive cross-linkers BS3 (bis(sulfo-succinimidyl)suberate) and sulfo-DST (disulfosuccinimidyl tartarate) followed by enzymatic proteolysis, and analysis of cross-linking products by high-resolution FTICR mass spectrometry. MALDI-TOF mass spectrometric analysis of the intact complex was used for monitoring the extent of chemical cross-linking. Following chemical cross-linking, the reaction mixtures were separated by SDS-PAGE and subjected to enzymatic in-gel digestion with diverse proteases (trypsin, endoproteinase AspN, LysC) yielding a mixture of cross-linked and non-cross-linked peptides. Those highly complex mixtures were separated by nano-HPLC on reverse-phase columns applying water-acetonitrile-gradients with flow rates of 200 nl/min. The nano-HPLC system was directly coupled to an FTICR mass spectrometer equipped with a nano-electrospray ionization source. The obtained mass spectra were then screened for possible cross-linking products by employing customized software programs. Spatially proximal regions within the calmodulin/melittin complex were identified by pursuing this strategy allowing for the generation of a low-resolution structure model. Thus, the herein described analytical strategy represents a powerful application for structural proteomics studies.

References: [1] Bennett K.L., Kussmann M., Bjork P., Godzwon M., Mikkelsen M., Sorensen P., and Roepstorff, P. (2000) Protein Sci. 9, 1503-1518 [2] Sinz A. and Wang K. (2001) Biochemistry 40, 7903-7913 POSTER SESSION

P-71 Identification of Protein Ligands in Complex Biological Samples Using Intensity- Fading MALDI-TOF Mass Spectrometry

M. Ferrer-Navarro,‡, J. Villanueva,‡,† O. Yanes,‡,† E. Querol,‡ L. Serrano,*,§ and F. X. Aviles*,‡ ‡,† Institut de Biotecnologia i de Biomedicina, and Departament de Bioquímica, Universitat Autònoma de Barcelona, Spain, and *European Molecular Biology Laboratory (EMBL) Heidelberg, Germany † These authors contributed equally to this work. ‡ Universitat Autònoma de Barcelona; § EMBL.

The easy detection of biomolecular interactions in complex mixtures using a minimum amount of material is of prime interest in molecular and cellular biology research. In this work, a mass spectrometry MALDI-TOF based approach, which we call intensity-fading (IF MALDITOFMS), and which was designed for just such a purpose, is reported. This methodology is based on the use of the MALDI ion intensities to detect quickly the formation of complexes between nonimmobilized biomolecules in which a protein is one of the partners (protein-protein, proteinpeptide, protein-organic molecule, and protein-nucleic acid complexes). The complex is detected through the decrease (fading) of the molecular ion intensities of the partners as directly compared to the MALDI mass spectrum of the mixture (problem and control molecules) following the addition of the target molecule. The potential of the approach is examined in several examples of model interactions, mainly involving small nonprotein and protein inhibitors of proteases, at both the qualitative and semiquantitative levels. Using this method, different protein ligands of proteolytic enzymes in total extracts of invertebrate organisms have been identified in a simple way. The proposed procedure should be easily applied to the high- throughput screening of biomolecules, opening a new experimental strategy in functional proteomics. POSTER SESSION

P-72 A Unit of Cleavable Photophore as a Tool for Structural Analysis of Ligand-binding Region

Y. Sadakane and Y. Hatanaka Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama JAPAN

The large-scale study of the interactions between bioactive ligands and their receptor proteins is one of the goals of chemical proteomics. The technique of photoaffinity labeling cross-links the partners to each other through a covalent bond in an affinity-based manner. The photochemically introduced covalent bond leaves a tag for facilitating the determination of peptides forming the ligand-binding domain of receptor proteins. Thus, the photoaffinity labeling provides the structural information of the ligand-binding region within the receptor protein. In this study, we synthesized a unit of cleavable photophore, S-[4-(3-trifluoromethyl-3H-diazirin- 3-yl)-benzyl]-methane thiosulfonate1, to accelerate the structural elucidation of the ligand- binding region by photoaffinity labeling. Diazirine in the unit yields very reactive intermediate, carbene, and thiosulfonate group is well known for forming a disulfide linkage with free sulfhydryl group. To introduce the unit into a biotinylated 17-residue calmodulin-binding peptide (CBP, biotin-LKWKKLLKLLKKLLKLG), the Trp of the peptide was replaced to Cys, and then the unit was incorporated to the sulfhydryl group of Cys residue. Mass spectrometry analysis revealed that the photoaffinity CBP was cross-linked with calmodulin in a 1:1 molar ratio. The cross-linking was dependent on calcium ion, and was inhibited by calmodulin antagonist, trifluoperazine. These results indicate that the photoaffinity CBP recognizes the specific region within calmodulin. The calmodulin labeled with photoaffinity CBP was digested by V8 protease, and the cross-linked peptides were isolated by monomeric avidin beads. HPLC analysis revealed that retention times of the isolated peptides were shifted by a treatment of 50 mM dithiothreitol, indicating that these contained a disulfide linkage. An analysis of the isolated peptides by mass spectrometry is in progress.

References: Kaneda, M., Sadakane, Y., Hatanaka, Y. (2003) Bioconjugate Chem. in press. POSTER SESSION

P-73 Characterisation of Apoptotic Nuclear Signaling Pathways by a Novel Quantitative Proteome Approach

A. Tabbert1, A. Schmidt2, J. Kellermann2, F. Lottspeich2 and E. Ferrando-May1 1Dept. of Molecular Toxicology, University of Konstanz, Germany and 2Max-Planck Institute for Biochemistry, Martinsried, Germany

The ordered dismantling of the nucleus is a main feature of cell death by apoptosis. Several factors have been identified, which enact the cleavage and degradation of nuclear proteins and DNA, first of all the caspase family of proteases. However, little is known about the mechanisms responsible for the activation of apoptotic executioners inside the nucleus. The present project pursues a comprehensive characterisation of the protein alterations occurring in the nucleus in the early phase of apoptosis. This is achieved by comparative proteomics analysis of isolated liver nuclei exposed to a defined apoptotic environment under cell-free conditions. The quantitative proteome analysis is carried out using a new method termed Isotope Coded Protein Label (ICPL) which is based on isotopic labeling of all free amino groups. The complexity of the protein sample is reduced by 2D-electrophoretic separation followed by a high-throughput in-gel digestion. Final protein identification and quantification is performed using the AB 4700 Proteomic Analyzer. This approach results in an accurate and reproducible quantitation of changes in protein levels. An additional advantage of the technique is its high sensitivity resulting in the analysis of low amounts of biological material. In this study we aim to identify novel proteins or protein modifications involved in the transduction of early apoptotic signals in the nucleus. This will provide the basis for a molecular dissection of the mechanisms responsible for the initiation of nuclear apoptosis. The experimental setup will be presented in detail, including the procedures for the preparation of preapoptotic extracts, the conditions of in vitro reactions and the application of the ICPL technique. We will show preliminary results of the mass spectrometric analysis. POSTER SESSION

P-74 Signal-pathway Profiling of Cells Following Photodynamic Therapy with 5-Amino- Levulinic Acid-induced Protoporphyrin IX: Phototoxicity Goes Proteomics

R.C. Krieg1, K. Reher2, K. Schwamborn1, L.A. Liotta3, E.F. Petricoin III4, R. Knüchel1 1Institute of Pathology, Aachen University; 2Institute of Pathology, University of Regensburg, Germany; 3FDA-NCI Clinical Proteomics Program, Laboratory of Pathology, National Cancer Institute, NIH, Bethesda, MD, USA; and 4FDA-NCI Clinical Proteomics Program, Division of Therapeutic Proteins, CBER, Food and Drug Administration, Bethesda, MD, USA

Photodynamic therapy (PDT) utilizing 5-aminolevulinic acid (ALA) induced protoporphyrin IX (PPIX) as the photosensitizing source is a selective and versatile treatment modality for cancer and premalignant lesions in various organs. ALA-induced PPIX accumulates with high selectivity in tumors. Following irradiation with light the photosensitizer generates singlet oxygen, thus inducing submicroscopic photo-oxidation, finally leading to cellular death. This method is widely used nowadays. However the cellular aspects of PDT-induced cell death, leading to apoptosis or necrosis, are still poorly understood. This phototoxic detail has got great clinical impact, as the latter causes side effects in vivo, including inflammation and scares. Signal pathway profiling is a versatile approach for analyzing cellular death, as traditional assays (e.g. morphology, caspase activation, DNA-cleavage) might fail or check details at the very end of complex signal cascades. Factors influencing the cells’ decision of undergoing apoptosis vs. necrosis are pinpointed such as intracellular photosensitzer concentration, irradiation energy, irradiation wavelength, pulse vs. permanent irradiation mode or oxygen depletion. To extensively check these parameters in a kinetic mode, a great number of samples will occur automatically. To further analyze different checkpoints of cellular signal pathways one has to stress a sensitive as well as high-throughput method. Thus we analyze PDT treated in vitro samples with reverse phase liquid protein arrays, allowing to screen hundreds of sample for the expression of one protein at once on a miniaturized scale. Cell lines representing different in vitro-models for bladder (UROtsa, RT4, J82) and colon (CaCo2, HT29) respectively were cultured in 6 well plates (plateau phase), incubated with ALA and treated with different light doses (cell specific LD50 and LD90 values), simulating in vivo PDT. Cells were harvested 0, 5, 30, 60, 120 and 180 min after irradiation, respectively by lysing in protein lysis buffer containing a 1:1 mixture of 2x SDS electrophoresis buffer (125 mM Tris pH 6.8, 4% SDS, 10% glycerol, 2% ß-mercaptoethanol) and Tissue Protein Extraction Reagent (TPER, Pierce). Lysates were arrayed onto nitrocellulose covered glass slides (FAST-slides, Schleicher & Schuell) utilizing a conventional ring & pin technique (417 GMS arrayer, MWG-Biotech). This technique allows to spot 1152 different samples onto the area of a stamp. Following routine blocking against unspecific protein binding overnight the slides were immune histochemically tagged and stained on a DAKO Autostainer (Dako, Germany) using the CSA-System Peroxidase-Kit (DAKO) and following DAKO’s instructions. Antibodies were used in dilutions as checked in prior experiments. Ready stained slides were scanned on an UMAX PowerLook III flatbed scanner, using 600 dpi resolution and utilizing Adobe Photoshop 5.5. Images were densitometrically quantified using the software FluorChem (AlphaInnotech Corp., San Leandro, CA, USA). Analyzing the expression of caspases 3, 7, 9 (each total and cleaved) and AKT, ERK, BAD (each total and phosphorylated), clearly show a cell and dose specific signal response. These preliminary data strongly indicate activation of apoptotic and pro-apoptotic pathways for well differentiated cells in contrast to lower differentiated ones in an organ independent pattern. Reverse phase liquid protein arrays are a highly versatile tool for signal pathway profiling. In contrast to traditional western blots this method needs only small amounts of protein, is high- throughput capable and protein expressions observed can be quantified. Result of clear apoptotic signaling preferable in higher differentiated cells correlates with recent literature. POSTER SESSION

P-75 Differential Proteome Analysis and High Throughput RNA Interference: A Powerful Strategy for Gene Function Analysis in Mammalian Cells

T. Rudel, N. Machuy, B. Thiede and T.F. Meyer Max Planck Institute for Infection Biology, Dept. of Molecular Biology, Berlin, Germany

Apoptotic cell death is characterised by significant post-translational modifications. Processing of apoptosis regulators by caspases and the translocation of proteins between cellular compartments have been recognised as the most important signalling mechanisms of apoptosis regulators. We have used differential proteome analysis to identify proteins which were modified during apoptosis. Of the 100 proteins identified by this approach, about 70 proteins have not been associated to apoptosis before. In addition, only very few of the identified proteins have been functionally characterised so far. Gene knock down combined with phenotypic analyses are excellent approaches to determine the function of genes. Due to a high technical effort and time exposure classical knock down methods are, however, not well suited for high throughput analyses of gene function. Recently, the RNA interference (RNAi) technology has been developed for the down-regulation of gene expression in mammalian cells. The extraordinary efficiency and versatility of RNAi predestines it to be the ideal tool for analysing gene function in a global manner. We have established an automated high throughput platform for RNA-interference. The technique allows the automated transfection of siRNAs followed by the validation of gene specific RNAi and functional assays. The developed technology has been used for the functional analysis of candidate genes identified in the proteome analysis for apoptosis regulators. Our data demonstrate that automated RNAi is a suitable technology for the functional characterisation and validation of targets identified by proteome screens. POSTER SESSION

P-76 Comparisons of Human Endothelial Cell 2DE Patterns with Mass Spectrometry Identifications Between Quiescent, Apoptotic and Dedifferentiated States

A. Bruneel1, V. Labas3, J. Vinh3, A. Mailloux1, M. Vaubourdolle1, B. Baudin1,2 1Biochimie A - Hôpital Saint-Antoine, Paris ;2GRECAN-EA1772 –UFR Pharmacie, Caen; 3CNRS/UMR 7637 - ESPCI, Paris, France

The endothelium is a single layer of cells lining the inside face of all blood vessels; it constitutes an important metabolic organ allowing free blood circulation without activation of haemostasis, inflammation and immune response, ensuring survival of other vascular tissues. It also participates to vascular tone regulation and exchanges messages with both blood and sub-endothelium, particularly growth factors, peptides and nitrogen monoxide. Endothelial cells are involved in many pathological situations such as atherosclerosis, cancerogenesis with both neovascularization of tumours and metastasis dissemination, and in inflammation. We analyzed the proteomes of cultured human umbilical vein endothelial cells (HUVECs) in three states: 1- quiescent cell phenotype with, from 2DE patterns, the identification of more than 50 proteins by MALDI-TOF-MS and ESI-MS/MS, e.g. heat shock proteins, chaperons, proteins involved in cell motility, apoptosis and angiogenesis; 2- etoposide-induced apoptosis (etoposide is a pro-apoptotic anticancer drug) with at least 12 modifications in comparison to the quiescent state, including overexpression of glucose-related protein 94, proteinase inhibitor 9 and laminin binding protein; 3- PMA-dedifferentiated cells into smooth muscle-like cell morphotype with large discrepancies in comparison to both previous states. In summary, this proteomic approach appeared very instrumental for better understanding of the high metabolic capacities of the endothelium, the pathways involved in apoptosis as induced by chemotherapies, and the events related to dedifferentiation of endothelial cells as involved during neo-vascularization of tumours, i.e. angiogenesis with the expression of motility proteins, that can be related to protein-kinase C PMA-activation, and to inhibition of apoptosis. POSTER SESSION

P-77 Two Dimensional Gel Electrophoresis Applied to the Analysis of Differential Protein Expression Profiles in E2F1-/- and E2F2-/- T Lymphocytes

A Fullaondo1, M. Azkargorta2, A. Infante1, A. Iglesias1, A.M. Zubiaga1 and J.M Arizmendi2 1Dept. of Genetics, Physical Anthropology and Animal Physiology, Faculty of Sciences, University of the Basque Country, Bilbao, Spain; and 2Dept. of Biochemistry and Molecular Biology, Faculty of Sciences, University of the Basque Country, Bilbao, Spain

E2F transcription factors, critical targets of the Retinoblastoma family of proteins, are considered important regulators of proliferation, differentiation and apoptosis (Dyson, 1998). E2F is a family composed of at least six members (E2F1-6) that physically associate with a member of the DP family, and subsequently bind to the E2F site in target genes. Although the E2F proteins have been extensively studied, the specific function of each gene and their degree of functional overlap remain to be determined. The generation of mutant mouse strains for the E2F genes has been crucial to understand the role that each E2F member plays in vivo. During our characterization of E2F1- and E2F2- mutant mice, we have found that E2F1 and E2F2 play distinct roles in T lymphocyte function (Murga et al., 2001). E2F2-/- T lymphocytes hyperproliferate in response to antigenic signals, and E2F2-deficient animals develop autoimmunity. Conversely, E2F1-/- T cells show a reduced proliferative capacity. To determine the mechanistic basis for the specificity of E2F1 and E2F2 function in T lymphocyte proliferation, we are analyzing the gene expression patterns of E2F1- and E2F2- mutant T cells, both at the mRNA and the protein level. Protein expression analyses are being carried out by 2 dimensional gel electrophoresis (2DE). A comparison of the 2DE protein expression profiles obtained from E2F1-/-, E2F2-/- and wild-type controls has showed proteins that are differentially regulated by these E2F members, providing an insight for the specificity of E2F1 and E2F2 function.

References: Dyson, N. (1998). Genes Dev. 12, 2245-2262 Murga M., et al. (2001). Immunity 15, 959-970 POSTER SESSION

P-78 Analysis of Signal-dependent Changes in the Proteome of Drosophila Blood Cells During an Immune Response

O. Loseva and Y. Engström Stockholm University, Department of Molecular Biology and Functional Genomics, Stockholm, Sweden

The fruit fly, Drosophila, like other insects relies on innate immune reactions to protect against infections. The innate immune system is a universal form of host defense against pathogenic microbes. Invertebrates and vertebrates use very similar intracellular signaling pathways to regulate innate immune responses. Interactions of bacterial lipopolysaccharide (LPS), a component of the outer cell wall of Gram-negative bacteria, with Drosophila blood cells represent a model system for studying the activation and output of the innate immune system. The Drosophila mbn-2 cell line is of hemocyte origin and has retained its capacity to phagocytose and respond to microbial substances by Rel/NF-kB mediated activation of genes coding for antimicrobial peptides.

In order to obtain information about immune responses to LPS in Drosophila on the protein level we analyzed the proteome of mbn-2 cells (nuclear and cytoplasmic fractions) at two different time points following LPS challenge. We used a combination of two-dimensional polyacrylamide gel electrophoresis, computer-assisted image analysis by PDQuest 7.0 software and MALDI-TOF mass spectrometry to study the protein variation in a Drosophila cell line following treatment with LPS. We have characterized intracellular proteins that were up or down regulated after immune challenge, as well as post-translation modifications, which occurred as a result of activation of the immune system. The targets of the LPS response include proteins involved in mRNA processing, nuclear transport, antioxidant reactions, vesicle trafficking, cytoskeleton regulation, proteolysis and protein folding. POSTER SESSION

P-79 Dissecting the Role of WRKY Transcription Factors by Comparative Protein Profiling

J. Brümmer, B. Ülker, L. Jorda, H. Seki and I. Somssich Max-Planck-Insitute for Plant Breeding, Dept. of Molecular Phytopathology, Köln, Germany

The plant specific WRKY transcription factors are encoded by a large multigene family and are characterized by their DNA-binding WRKY domain, a peptide stretch of 60 amino acids containing the highly conserved W, R, K, Y amino acids and a Zn-finger motif. Present data indicate that WRKY proteins are translocated into the nucleus via a nuclear localisation sequence (NLS) where, upon binding to a cis regulatory element designated the W box (C/T TGAC T/C), they modulate the expression of target genes. Based on current knowledge, WRKY factors play a role in pathogen/stress induced signaling pathways but also in developmental processes i.e. senescence and trichome formation. In Arabidopsis thaliana 75 members where identified, 71 of which have been found to be expressed. Single WRKY gene loss of function mutants often do not show any obvious phenotypes indicating partial redundancy within this family of transcription factors. To overcome this problem we employ comparative protein profiling to identify WRKY-controlled targets and downstream pathways. We will analyse a range of knock-out lines under several defined stress-related conditions in comparison with wild type plants. POSTER SESSION

P-80 Alpha-Dystrobrevin and its Associated Proteins in Human Promyelocytic Cell Lines

R. Navakauskien, G. Treigyt and K.-E. Magnusson Department of Developmental Biology, Institute of Biochemistry, Lithuania and Division of Medical Microbiology, Department of Molecular and Clinical Medicine, Linkoping University, Sweden

Dystrobrevin is a dystrophin-related component of the dystrophin-associated protein complex (DPC) that is situated in the cytoplasm. The complex constitutes a scaffold for signaling molecules. Two different genes encode the dystrobrevin family of proteins. The alpha- dystrobrevin gene encodes six splice isoforms (designated α, β, γ, δ, ε and ζ). The precise role of dystrobrevins is unknown, but they are also proposed to play a role in intracellular signal transduction. Recently we have investigated the biochemical properties and spatial localization of the protein alpha-dystrobrevin and other isoforms in cells of the human promyelocytic leukemia line HL-60 granulocytic differentiation induced by retinoic acid. We have shown for the first time that alpha-dystrobrevin and its splice isoforms are present in human haematopoietic cell system, and biochemically it is found in two cell compartments - the cytosol and the nucleus. In our present studies we examine the alpha-dystrobrevin and its splice isoforms distribution in the cytoplasm and the nucleus of human promyelocytic leukaemia cell lines (HL-60, NB4) and the association of alpha-dystrobrevin with other proteins during apoptosis process. We have shown that spatial localization of alpha-dystrobrevin changes after induction of apoptosis. The level of cytosolic alpha-dystrobrevin isoforms increases and their nuclear level changes during apoptosis. Protein extracts from human promyelocytic leukaemia cell lines HL-60 and NB4 were used for immunoprecipitation with antibodies against alpha-dystrobrevin. Immunoprecipitates were resolved by two-dimensional electrophoresis. Proteins co-precipitating with alpha- dystrobrevin were analyzed by silver staining and supplied for MALDI-TOF MS analysis. We have identified e.g. tropomyosin, anexin, potassium voltage-gated channel proteins and myosin light chain. Our results provide new information about potential involvement of alpha-dystrobrevin and its splice isoforms in signal transduction during apoptosis of myeloid cells. POSTER SESSION

P-81 Translational Regulator rpL10/Grc5p Modulates the Protein Expression Pattern of the Cell

S. Lach1, M. Eder1, K. Pachler1, T. Karl1, A. Thuer1, K. Oender2, M. Loeffler3, N. Mehlmer1, B. Scheibe4, P. Eckl1, R. Crameri5 , M. Breitenbach1 and L. Koller1 1Dept. of Genetics, University of Salzburg, Austria; 2SJS/Dept. of Dermatology, Salzburg, Austria; 3AKH/I.M.III Vienna, Austria; 4Amersham Biosiences, Freiburg, Germany; 5SIAF, Davos, Switzerland

In yeast, ribosomal protein rpL10p/Grc5p/Qsr1p/Rix5p is a multifunctional protein with successive activities in ribosome biogenesis in the nucleus, ribosomal subunit export into the cytoplasm, subunit joining during translation initiation and possibly linking the biogenesis of two major cellular compartments, the ribosomes and the cell surface (1, 2). Ribosome structure analysis show that rpL10p/Grc5p is located close to the functional centre of the fully assembled ribosome, i.e. the peptidyl transferase centre, the GTPase centre of the small subunit and the decoding centre of the small subunit (3, 4). These structural determinants suggest a participation of rpL10p in kinetic control of ribosome function and/or differential translation. rpL10p/Grc5p interacts with ribosome associated protein Nmd3, which is involved in 60S ribosomal subunit export and with cell surface glycoprotein Sed1p, for distinct isoforms of which we have show a physical interaction with translating ribosomes. To investigate how rpL10/Grc5p might contribute to modulation of translation we are establishing 2-D gel electrophoresis for our experimental system. We wished to first obtain reproducible 2-D gels from wild type and RPL10/GRC5 mutant cells before subjecting the crude protein exctract to differential separation techniques with subsequent 2-D Gel analysis. So far, we observed several differentially expressed proteins when comparing wild type and mutant RPL10/GRC5 cells. We extend these studies to cells harbouring mutant alleles of the ribosome associated protein / ribosome subunit export adapter protein Nmd3p and the cell surface protein Sed1p. We hypothesize, that modulation of translation associated functions of rpL10p/Grc5p may result from structural changes of the protein, also in the context of binding to a regulatory ribosome associated protein.

References: 1) Gadal et al., Mol Cell Biol. 2001, 21:3405-15 2) Oender et al., Yeast 2003, 20: 281-294 3) Ban et al., Science 2000, 289: 905 – 920 4) Spahn et al., Cell 2001, 107:373-386 POSTER SESSION

P-82 Proteomic Analyses of Ribozyme Induced bcl2-Down Regulation in MCF7 Cell Line

L. Bianchi1, S. Liberatori1, L. Poliseno2, L. Citti2, L. Mariani2, A. Salvetti2, L. Bini1, G. Rainaldi2 and V. Pallini1 1Dipartimento di Biologia Molecolare, Università degli Studi di Siena, Siena, Italy; 2Laboratorio di Bioterapia Molecolare, Istituto di Mutagenesi e Differenziamento, CNR, Pisa, Italy

Ribozymes are catalytic RNA molecules able to cleave themselves or other specific RNA target molecules. They provide a really useful tool for functional analysis in items of what happens to the cell in which the gene of interest is down-regulated by ribozyme activity. In this work we chose bcl2 as target for such an application. Bcl2 is a mitochondrial protein with crucial function in cellular homeostasis having cytostatic and antiapoptotic proprieties. The anti-bcl2 hammerhead ribozyme Rz-bcl2 was stably transfected in MCF7 cells and the cleavage of bcl2 mRNA was demonstrated by cleavage assay and wester blot analysis. The depletion of Bcl2 endogenous levels was accompanied by a decrease in cytostatic and antiapoptotic activities as the increment of growing rate and of the sensitivity to the apoptotic drug staurosporine demonstrated. In order to investigate how the down-regulation of bcl2 affects the global state of the cell we proceeded with a proteomic approach. Two reference electropherograms of Rz-bcl2 transfected cells, one with the ribozyma catalytically active form and the other with the ribozyma catalytically inactive form, were obtained. Comparing the 2D maps we found 53 spots differentially expressed and some of them have been identified by MALDI-TOF mass spectrometry. These data may be considered a further step toward the understanding of the ribozyma effects on cytostaticity and cell survival, and, consequently, of bcl2 cellular functions. POSTER SESSION

P-83 An Efficient Proteomics Method to Identify the Cellular Targets of the p38 MAP Kinase Inhihibitor SB 203580

K. Godl, J. Wissing, A. Kurtenbach, P. Habenberger, S. Blencke, H. Gutbrod, K. Salassidis, M. Stein-Gerlach, A. Missio, M. Cotten and H. Daub Axxima Pharmaceuticals AG, München, Germany

During the last two decades the development of small-molecule inhibitors of protein kinases became important for signal transduction research as well as for the treatment of a variety of diseases. However, the selectivity of these compounds is a critical parameter for the interpretation of biological results. Conventional methods to determine the cellular targets of protein kinase inhibitors cover only a subset of the protein kinases present in a human cell. Therefore, efficient methods for proteome-wide assessment of compound selectivity are needed. Direct affinity purification methods based on immobilised protein kinase inhibitors might address this important issue, but only if the efficiency of this approach is significantly improved compared to earlier reported procedures. Here we establish an efficient method for purification of protein kinases affected by the widely used p38 kinase inhibitor SB 203580. Immobilisation of a suitable SB 203580 analogue and optimised adsorption and elution conditions for affinity chromatography permitted the dramatic enrichment of a small subset of cellular proteins. Subsequent gel electrophoresis and mass spectrometry analysis led to the identification of several protein kinases, which had not been described as cellular targets of SB 203580 previously. In vitro kinase assays showed that cyclin G-associated kinase (GAK) and CK1 were almost as potently inhibited as p38_, whereas receptor-interacting kinase (RICK) was even more sensitive to inhibition by SB 203580. Cellular kinase activity of RICK, a known signal transducer of inflammatory responses, was already affected by submicromolar concentrations of SB 203580 in intact cells. Therefore, our results warrants a re-evaluation of the vast amount of data obtained with SB 203580 and might have significant implications on the development of p38 inhibitors as anti-inflammatory drugs. Based on the procedure described here, efficient affinity purification techniques can be developed for various protein kinase inhibitors and thus provide crucial information about their cellular modes of action. POSTER SESSION

P-84 How Selective are Potent Protein Kinase C Inhibitors? A Powerful Proteomics Method to Address This Question

D. Brehmer, K. Godl, B. Zech, J. Wissing and H. Daub Axxima Pharmaceuticals AG, München, Germany

Small ATP-competitive protein kinase inhibitors have become a major, and promising research area in medicinal chemistry within the last decade. One of the most important criteria for an inhibitor, which has to reach clinical phases, is its selectivity against a specific protein kinase target. By advancing the method of affinity chromatography employing immobilised kinase inhibitors, we established a proteomic method for the identification of alternative cellular targets. Here we were used the compound class of bisindolymaleimides, which were originally described as potent and selective protein kinase C blockers and have been widely used in the field of signal transduction. Affinity chromatography, 16-BAC/SDS-PAGE electrophoresis and subsequent mass spectrometry analysis led to the identification of various previously unknown cellular targets, including both protein kinases and other types of nucleotide-binding enzymes. Moreover, this method can be adapted so that only the activated form of known cellular targets, for example PKCα or Rsk1 can specifically bind to the immobilized bisindolymaleimides. To prove the reliability of our target identification procedure, we performed in vitro binding studies of several identified kinases and further determined the IC50 values for bisindolylmaleimide inhibition in activity assays for four novel targets: The Ste20-related kinase (SLK), the cyclin dependent kinase 2 (CDK2), the adenosine kinase (AdK) and the Quinone- Reductase Type 2 (NQO2). Furthermore, the binding pattern of cellular proteins changed conspicuously in the presence of minor chemical modifications within the immoblilised bisindolylmaleimide compounds. These results revealed a good structure-activity relationship of bisindolymaleimides, which enabled us to identify BisX as a more potent CDK2 inhibitor than its close derivatives (BisIII and BisVIII). Molecular docking studies of these bisindolymaleimide derivatives onto the CDK2/Staurosporin X-ray structure provided results, that were consistent with binding studies and IC50 value determinations. The higher potency of BisX to CDK2 can be explained by a distinct orientation within the ATP binding pocket compared to BisIII and BisVIII. POSTER SESSION

P-85 A Proteomics Approach for the Identification of Cellular Targets Affected by Fused Pyrimidine Protein Kinase Inhibitors

J. Wissing, K. Godl, M. Cotten, P. Habenberger, M. Stein-Gerlach, A. Missio, and H. Daub Axxima Pharmaceuticals AG, München, Germany

Small molecules belonging to the fused pyrimidine class of compounds are known to inhibit several protein-tyrosine kinases which have been implicated in cancer progression. Importantly, derivatives of this compound class retain effectiveness against most STI571/Gleevec-resistant BCR-ABL mutants isolated from advanced chronic myelogenous leukaemia patients. After immobilization of a suitable fused pyrimidine, we employed a combination of affinity chromatography, 1D-and 2D-electrophoretic methods and mass spectrometry for the identification of its cellular targets. To have a chance to detect protein kinases of very low abundance, we started with total cell extract prepared from 40g or 1 x 1010 HeLa cells. Within this amount of starting material, a 100 kD protein expressed with only 100 copies per cell corresponded to 164 ng or 1 pmol of protein. The use of Bruker´s LIFT technology resulted in a success rate of more than 80% with respect to fluorescence stained proteins. The combination of methods mentioned above led to the identification of more than 25 protein kinases as well as several other cellular proteins with more or less high affinity for the immobilised fused pyrimidine inhibitor. Interestingly, we identified not only protein-tyrosine kinases but also serine/threonine kinases such as RICK, GAK and p38α, which were potently inhibited by the fused pyrimidine in vitro. Our results demonstrate that fused pyrimidines are not specific inhibitors of tyrosine kinases as assumed previously, but instead seem to be rather selective for protein kinases containing a small amino acid residue in a conserved position controlling the binding of this inhibitor. Furthermore, we show efficient inhibition of p38 and RICK kinase activities in intact cells upon fused pyrimidine treatment. As these two protein kinases have established functions as signal transducers of inflammatory responses, our data demonstrates the utility of proteomics methods employing immobilised kinase inhibitors for identifying novel targets linked to previously unrecognised potential therapeutic applications.

We thank Robert Brehm for excellent technical assistance. POSTER SESSION

P-86 Proteomic Analysis of Breast Cancer "Matched Pair” Cell Lines Hs578T and Hs578Bst: Comparison of Subproteomes Derived from Affinity Chromatography using Immobilized Kinase-Inhibitor Purvalanol B

J.E. Ehlert, B. Mutschler, A. Lingnau and M.H.G. Kubbutat ProQinase/KTB Tumorforschungs GmbH, Freiburg, Germany

Overexpression of several protein kinases has been shown to be involved in cancer formation. Many different approaches aim at the identification of tumor-relevant kinases, most of which are based on genomic screens. Only few proteomic approaches have been taken, most likely due to the low abundance of kinase proteins. To overcome this restriction, we aim at the enrichment of protein kinase subproteomes from crude lysates of tumor cells in order to compare them by 2D-gel analyses to the respective subproteomes from untransformed cells, and to identify kinases upregulated in tumor cells by subsequent MS analyses. In the present study, we use the immobilized form of the tri-substituted purine-derivative Purvalanol B (PurvB) as affinity matrix to generate protein kinase subproteomes. Although Purvalanol B is known as highly specific inhibitor of cyclin-dependent kinases (CDKs) 1 and 2, its immobilized form has previously been shown to bind a variety of other kinases. We now show analyses of the PurvB-binding subproteome of the breast cancer cell line Hs578T, in which we find about 70% of the detectable spots to represent protein kinases. This set of kinases is composed of CDK5, ERK1, ERK2 and CaMKII delta. Moreover, we identified differentially phosphorylated forms of protein kinases. We compare these results with those obtained from 2D gel analyses of the PurvB-binding subproteome of the untransformed "matched pair” cell line Hs578Bst. Our data demonstrate for example that normal and tumor cells show similar levels of CaMKII delta, whereas those for CDK5 were found elevated in tumor cells. Data obtained from this proteomic approach will be compared with cDNA micro array data aquired from the same cell types. In summary, our data indicate that immobilized kinase-inhibitors might be useful to enrich and compare protein kinase subproteomes of tumor and normal cells. POSTER SESSION

P-87 Investigating the Resistance to Tamoxifen in a Human Breast Tumor Xenograft Line: A Proteomic Approach

V. Besada1, I. Fichtner2, M. Diaz1, M. Becker2 , L. Castellanos-Serra1 1Department of Proteomics, Center for Genetic Engineering and Biotechnology, Habana, Cuba; 2Experimentelle Pharmakologie and Onkologie, Max Delbrueck Centrum für Molekulare Medizin, Berlin-Buch, Germany

Breast cancer is the most common cancer in women and the leading cause of cancer death in women worldwide. Estrogen receptor-positive tumors are commonly treated with estrogen antagonists, the first line treatment being the non-steroidal drug Tamoxifen (TAM). TAM is indicated for prolonged treatments (generally during five years); nevertheless, there are overwhelming clinical evidences indicating that a non-negligible number of patients with positive estrogen receptors finally onset resistance to this drug. Fichtner et al (1, 2) developed a xenografted mouse line bearing a TAM resistant human breast tumor, that was obtained from an originally TAM-sensitive line as a consequence of its repetitive passages in the presence of the drug. In this work, we compared both the sensitive and the resistant tumor line obtained thereof through two-dimensional gel electrophoresis. The proteins (3) differentially expressed were identified by mass spectrometry analysis. These proteins seem to be involved in (or are differentially expressed as a consequence of) the onset of resistance to this agent.

References: [1] Naundorf et al.: J. Cancer Res. Clin,.Oncol. 119: 35-40 (1992). [2] Naundorf et al. Brit. J. Cancer 82:1844-1850 (2000). [3] Patent: DE 103 10 198.5 (2002) POSTER SESSION

P-88 SEL1L-Induced Proteome Modulation in MCF-7 Mammary Carcinoma Cell Line

C. Canton1*, L. Bianchi2*, L. Bini2, S. Liberatori2, V. Pallini2, S. Cristoni1, M. Cattaneo3, A. Albertini3, L. Rossi Bernardi1, I. Biunno3 1Centre for Biomolecular Interdisciplinary Studies and Industrial application (CISI), University of Milan, Segrate (MI), Italy; 2Department of Molecular Biology, University of Siena, Italy; 3Institute for Biomedical Technologies (National Research Council), Segrate, Milan, Italy *These authors have contributed equally to this work

The expression of SEL1L, the human orthologue of the Ceanorhabditis elegans sel-1 gene, has been recently associated with a reduction of proliferative activity and aggressive behaviour in the mammary carcinoma cells line MCF-7. Analysis of breast carcinoma patients indicated a statistically significant correlation between SEL1L down-modulation and poor prognosis suggesting a role of SEL1L in breast tumor development. The biological mechanisms responsible for this anti-proliferative effect remain still unclear. To clarify the complex intracellular pathways modulated by SEL1L expression and identify proteins interacting with it, we analysed MCF-7 human breast carcinoma cells expressing the entire SEL1L cDNA under a dexamethasone (DEX) inducible promoter. Cellular extracts from DEX-induced SEL1L- transfected (MCF7-SEL1L) and mock transfected cells (MCF7-pDEX) were separated by high- resolution 2D electrophoresis and two reference electropherograms were obtained. The subsequent Melanie 3 software analysis of the 2D maps highlighted the presence of qualitative and quantitative differences between gels. MALDI-TOF Mass Spectrometry was carried out to identify the protein spots differentially present in SEL1L-transfected and mock transfected cells. POSTER SESSION

P-89 Analysis of Biomarkers for Cervical Cancer

N.I. Govorukhina1, I. Keizer1, S. Roelfsema1, A.G.J. van der Zee2, S. de Jong3, H.W.A de Bruijn2 and R. Bischoff1 1Centre for Pharmacy, University of Groningen; 2Department of Gynecology, University Hospital Groningen; 3Department of Medical Oncology, University Hospital Groningen, Groningen, The Netherlands

Cervical carcinoma is the second most frequent carcinoma in women world-wide, while in the developing countries cervical carcinoma is the most frequent carcinoma in women. Squamous cell carcinoma antigen (SCCA) is a well-known tumor marker for patients with (adeno)squamous cervical cancer. Studies, including those from our own institute, have shown that 1) pretreatment serum SCCA levels can be used to identify patients with a poor prognosis, and 2) that the course of serum SCCA during and after therapy adequately predicts disease recurrence. However, we also demonstrated that in early stage cervical SCC only a minority of patients will have increased pretreatment serum SCCA levels, prohibiting the use of serum SCCA for early diagnosis [1]. SCCA is produced from two genes resulting in SCCA-1 and SCCA-2 and furthermore each form can be alternatively spliced [2,3]. Since measuring overall amounts of SCCA by immunological techniques does not allow early diagnosis of malignant cervical lesions, we have developed a new approach based on LC-MS to detect SCCA in human serum after tryptic digestion. To this end we have depleted the serum of HSA and IgG prior to digestion to enhance the useful protein load on the analytical system. Initially the method was developed by spiking recombinant human SCCA-1 [4] into serum. We were able to detect 6 of the expected tryptic peptide fragments from SCCA-1 based on their molecular mass. The identity of these peptides was confirmed by MS-MS. Initial results with serum from a cervical cancer patient showed the same peptide fragments, while control serum from a healthy volunteer did not. We are presently confirming these initial results by MS-MS of selected peptide fragments. Besides SCCA proteins we are currently searching for more biomarkers in cancer patients. In future studies we will extend the number of patient samples and also miniaturize the capillary LC-MS system to enhance sensitivity.

References: [1] Esajas, M.D., Duk, J.M., de Bruin, H.W., Aalders, J.G., Willemse, P.H., Sluiter, W., Pras, B., ten Hoor, K., Hollema, H., van der Zee, A.G. J. J.Clin.Oncol. 2001, 19: 3960-3966. [2] Schneider SS, Schick C, Fish KE, Miller E, Pena JC, Treter SD, Hui SM, Silverman GA. A serine protease inhibitor locus at 18q21.3 contains a tandem duplication of the human squamous cell carci-noma antigen gene. Proc Natl Acad Sci USA 1995; 92:3147-3151. [3] Suminami Y, Kishi F, Murakami A, Sakaguchi Y, Nawata S, Numa F et al. Novel forms of squamous cell carcinoma antigen transcripts produced by alternative splicing. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression 2001; 1519: 122-126. [4] courtesy of Prof. Gary Silverman, Harvard Medical School, Cambridge, MA, USA. POSTER SESSION

P-90 Proteomic Analysis of a Human Prostate Cancer Cell Line

Z. Liu1, M. Márguez1, S. Nilsson1, A.R. Holmberg1 and A. Alaiya2 1Div. of Chemistry and 2Div. of Cancer Proteomics, Karolinska Institute, CCK, Stockholm, Sweden

Somatostatin (sms) is sometimes used in the treatment of hormone refractory prostatic cancer in spite of modest results in controlled clinical studies. Consequently, its optimal use in this context remains to be determined. The human prostatic cancer cell line LNCap has been used in several previous studies. There it has been confirmed that sms can indeed affect the protein expression of this tumour cell line. An important tool to increase the understanding about how sms changes the tumour cell protein expression is proteomic analysis. In this study a new sms derivative (smsdx), currently undergoing clinical phase1 studies, was used as an additive to the LNCap cell culture. Proteomics analysis using rapid mini two-dimensional electrophoresis was performed to determine effects on the cells protein expression. Marked quantitative differences were observed in the protein expression profiles in smsdx treated LNCap cells compared with normal control cells. Sets of 63 (yet unidentified) protein spots were differentially expressed. This difference is statistically significant using Mann Whitney analysis. The 63 dataset was used to accurately discriminate control cells from smsdx treated cells using hierarchical cluster analysis. Smsdx is a new sms derivative with long in vivo half-life and pan receptor affinity. These results convey new important information about this potentially useful substance. Further studies are now pending to identify the affected proteins POSTER SESSION

P-91 Proteomics-based Characterization of Micrometastatic Tumour Cells

B. Willipinski-Stapelfeldt and K.Pantel Institute of Tumor Biology, University Hospital Hamburg-Eppendorf, Hamburg, Germany

Introduction: Malignant tumours derived from epithelial tissues (e.g. breast, prostate and lung carcinomas) are the most common form of cancer and their distant metastases are the main cause of cancer-related death. The bone marrow is a clinical relevant site of micrometastatic disease in patients with solid epithelial tumours. Clinical studies have indicated that the presence of occult carcinoma cells in bone marrow is an independent risk factor (Pantel et al: Lancet 347(9002):649-53, 1996; Braun, Pantel et al. NEJM 342: 525-533, 2000). For this reason, the direct identification of bone marrow micrometastases is of particular importance. The conditions necessary for the growth of epithelial cells that must be present in mesenchymal organs such as bone marrow are extensively unknown. Disseminated tumour cells were not been amenable to detailed molecular and biochemical analyses, because they occur at low frequencies of 10-6 nucleated bone marrow cells. Recently, we have therefore established tumour cell lines generated from bone marrow of cancer patients by immortalisation with a SV-40 oncogene cDNA. (Putz E et al., Cancer Res. 59:241-248, 1999) and therefore established a model that allow the characterization of disseminated tumour cells. Material and Methods: Four micrometastatic cell lines generated from breast cancer patients, as described above, were compared by 2-dimensional (2D) gel electrophoresis to MTSV-1.7 cells which are SV-40 immortalized mammary duct cells with a phenotype similar to luminal cells in the terminal duct lobular unit of the normal breast. Differential expressed spots were identified with MALDI-ToF and verified via 1D and 2D Western-Blot . Results and Conclusions: Most striking was a strong vimentin expression in all micrometastatic cell lines, while it is nearly not detectable in the MTSV-1.7 cells. In contrast, cytokeratins 19 and 8 are downregulated in the micrometastatic cells compared to MTSV-1.7 cells. The ectopic expression of the mesenchymal cytoskeleton protein vimentin and the downregulation of cytokeratins as the typical epithelial cytoskeleton components, indicate a partial or complete epithelial-mesenchymal transition (EMT) of micrometastatic cells. From the literature, there is evidence that EMT is involved in dedifferentiation programme of tumour cells and enables tumour cells to become mobile and disseminate to secondary sites by increasing their cellular plasticity (J.P: Thiery: Nature Rev 2:442-453, 2002). Our results show that the proteomics-based characterization of micrometastatic cells is able to identify putative metastasis-associated proteins in micrometastatic cells. POSTER SESSION

P-92 Proteomic Analysis of Human Colon Carcinoma Cell Line with Acquired Resistance to Oxaliplatin

L. Molina, M. Laplanche, I. Gourdier, L. Crabbé, N. Vié, P. Martineau, M. Del Rio and N. Chapal CNRS UMR 5160, Faculté de Pharmacie, Montpellier, France

Colorectal cancer is a common human malignancy in developed nations with increasing incidence. The main therapy against cancer is the surgical resection and according to the tumour progression chemotherapy will be recommended as complementary treatment. In the case of colorectal cancer therapy, oxaliplatin in association with 5-fluorouracile is widely used and shows clear clinical benefits. However, many patients escape to the therapy because of intrinsic or acquired chemoresistance. The molecular mechanisms involved in oxaliplatin resistance are poorly understood and any mechanism is clearly defined. We have recently shown that mitochondrial-mediated apoptosis could play an important role in the resistance to oxaliplatin and could be mediated by Bax-dependent and Bax-independent mechanisms [1]. In order to further investigate the molecular mechanisms of oxaliplatin resistance in colorectal cancer, we have analysed the global protein expression profile of cancer cell lines using the proteomic approach. Thus, we have generated in vitro cellular models of resistance by isolating resistant variants from the HCT116 colon carcinoma cell line [1]. We have analysed clones with progressive resistance: a sensitive clone HCT116/S, two resistant clones HCT116/R1 and HCT116/R2 (28 and 68-fold resistant respectively). Then, we have compared the protein profiles of the parental cell line and their chemoresistant counterparts. Different protein expression patterns were observed and 10 proteins were identified by mass spectrometry and data searches.

Identification of new biomarkers of drug resistance will be extremely helpful in the selection of the appropriate chemotherapeutic drug in the colon cancer. Furthermore, the understanding of drug resistance mechanisms should lead to the identification of news therapeutic targets as well as the improvement of existing drugs.

Reference: [1] Gourdier I, Del Rio M, Crabbe L, Candeil L, Copois V, Ychou M, Auffray C, Martineau P, Mechti N, Pommier Y, Pau B.: Drug specific resistance to oxaliplatin is associated with apoptosis defect in a cellular model of colon carcinoma. FEBS Lett. 2002, 529, 232-6. POSTER SESSION

P-93 Cox-2 and Cancer: A Proteomics Approach

N.M. Griffin, C.M. Cox, G. Scully, P.B. Maguire and D.J. Fitzgerald Clinical Pharmacology, Royal College of Surgeons, Dublin, Ireland

Cyclooxygenase (Cox) is the enzyme that catalyses the first step in the formation of prostaglandins and thromboxanes from arachidonic acid. The enzyme exists in two isoforms, Cox-1, important in the initiation of an inflammatory response and Cox-2, prominent in both the initiation and the resolution of the inflammatory response. Evidence is emerging that the Cox-2 isoform plays a central role in colorectal cancer and regular users of aspirin, which inhibit Cox activity, have a decreased risk of the disease. To provide a more in-depth understanding of Cox-2 effects in a cellular system, especially in relation to Cox-2 specific cancers, we used a proteomics approach to elucidate those proteins regulated by Cox-2 in a gastric epithelial cell line. Stably transfected cell lines were created in the gastric AGS cell line, where the Cox-2 construct was under the control of a tetracycline (tet) promoter. The expression of Cox-2 in this system was regulated by the antibiotic doxycyclin, a non-toxic derivative of tetracycline. Following the establishment of a characteristic proteome from the gastric AGS cells using two-dimensional electrophoresis, we compared 2-D gels of Cox-2 overexpressing cells with Cox-2 negative cells. We identified the differential proteins using MALDI-TOF mass spectrometry and proteins of interest identified include the ras oncogene and the structural components of cell-cell adhesive junctions cadherins and desmoplakin 1. This tet-regulated Cox-2 system was also used in gene expression experiments where we showed that Cox-2 increased the expression of Bcl-X, an anti-apoptotic gene, as well as decreased the expression of Bax-α, a pro-apoptotic gene. Thus, identification of Cox-2 regulated proteins will aid in understanding the mechanisms of action of Cox-2 and may identify therapeutic targets for the treatment of Cox-2 expressing cancers, such as colorectal. POSTER SESSION

P-94 Proteome Analysis of Radiation-induced Human Thyroid Carcinomas

E. Zeindl-Eberhart1, S. Liebmann1, J. Mattow2, P. Jungblut2 and H.M. Rabes1 1Institute of Pathology, Ludwig Maximilians University, Munich, Germany and 2Max-Planck Institute of Infection Biology, Berlin, Germany

A high incidence of papillary thyroid carcinomas (PTC) was observed in children and young adults who had been exposed to radioactive fallout after the Chernobyl reactor accident. Genome analysis revealed a high prevalence of rearrangements of the proto-oncogene cRET: H4/RET (PTC-1) and ELE/RET (PTC-3). Recent results disclosed that the type of genetic alterations determines the tumor phenotype: PTC-1 rearrangements were found in papillary and follicular variants, while in tumors with PTC-3 rearrangements the solid variant of PTC was found. The tumor phenotype might be reflected in a peculiar pattern of tumor-associated proteins. Comparative proteome analysis was performed with normal thyroid tissue, thyroid tumor tissue and lymph node metastasis expressing the PTC-1 or PTC-3 rearrangement-type, or expressing no rearrangement (PTC-0). Proteins were extracted and fractionated in soluble and insoluble proteins and were separated by 2-DE. Tumor-associated variant proteins, identified by MALDI-MS, showed characteristic pattern for PTC-1 and/or PTC-3 tumors. The identified variants could be assigned to different protein families. Here we focus on variants whose function could be attributed to cell mobility/motility, stress response and signal transduction. The results suggest that the combination of both molecular and proteome analysis is a valuable tool for studying pathogenetic mechanisms as in the case of these human thyroid carcinomas. POSTER SESSION

P-95 A Proteomics Investigation into Neuroblastoma Chemoresistance to Etoposide

A. Urbani1, J. Poland5, A. Biroccio2,3, L. Bellincampi3, P. Sinha5, M. Schnölzer6, S. Bernardini3,4 and G. Federici2,3,4 1Proteomics and Analitical Biochemistry Laboratory, Centro Studi sull’Invecchiamento (Ce.S.I.), University of Chieti and Pescara, Chieti, Italy; 2Clinical Biochemistry Laboratory, Children Hospital Bambin Gesu’ – IRCCS, Rome, Vatican State; , 3Clinical Biochemistry Laboratory, Department of Laboratory Medicine, University Hospital of Rome "Tor Vergata”, Rome, Italy; 4Clinical Biochemistry Laboratory, Department of Internal Medicine, Faculty of Medicine University of Rome "Tor Vergata”, Rome, Italy; 5Clinical Biochemistry Laboratory, Institut für Medizinische und Chemische Labordiagnostik LKH-Klagenfurt, Austria; 6Zentrale Proteinanalytik, Deutsche Krebsforschungs Zentrum (DKFZ), Heidelberg, Germany

Neuroblastoma is one of the most common paediatric cancers, the 96% of cases occur before age 10. This tumour is an embryonal malignancy of the postganglionic sympathetic nervous system, therefore it develops anywhere along the sympathetic nervous system chain. Therapeutic protocols often implies the use of etoposide as a drug of choice for the DNA damaging in combination with other cytotoxic compounds, such as the platinum- based drugs. Unfortunately in order to eradicate the tumour high doses of chemotherapeutics are often needed to minimize the development of drug-resistant clones. Molecular markers of such a chemoresistance onset are not presently available. We have investigated the changes in protein profiles levels in the model cell line of human neuroblastoma SH-SY5Y upon exposure to 1 uM concentration of etoposide. The SH-SY5Y (ATCC CRL-2266) is a thrice cloned subline of the neuroblastoma cell line SK-N-SH established from a metastatic bone tumour. Moreover a comparative proteomics analysis between the parental cell line and an etoposide resistant clone was pursued. Total protein extracts were fractionated at high resolution using the 2D-PAGE technique on broad pH gradient 3-10. Comparison of the silver stained image datasets of the different treatment shows few changes in protein profiles level upon onset of chemoresistance. The dUTP pyrophosphatase is the only detectable protein, which is down regulated, while most of the changes highlight polypeptides overexpression. POSTER SESSION

P-96 Landscaping Activity of the Tumor Suppressor Smad4 as Revealed by a "Secretome” Analysis

M. Volmer1,2, Y.Radacz1, K. Stühler2, S. Klein-Scory1, W. Schmiegel1, H. Meyer2 and I. Schwarte-Waldhoff1 1IMBL, Medizinische Universitätsklinik, Knappschaftskrankenhaus and 2Medizinisches Proteom Center, Zentrum für Klinische Forschung, Ruhr-Universität Bochum, Germany

Smad4 is a central mediator in the TGF-beta signal cascade and also a pivotal point in the cross-talk with other signal cascades. It functions both as a signal transmitter and as a transcriptional comodulator which influences the expression of a variety of genes by interaction with other transcription factors. Functional inactivation of Smad4 is a common event in the process of carcinogenesis of gastrointestinal tumors. At a significant rate loss of Smad4 occurs in one half of pancreatic and one third of metastatic colon carcinomas. To analyse the functions of Smad4 and its role in tumor suppression we use restoration of smad4 expression in deficient human carcinoma cell lines (e.g. in the colon carcinoma cell line SW480). By this means we could show that the reexpression of Smad4 is adequate to suppress tumor growth but does not restore TGF-beta mediated groth inhibition. We found that reexpression of Smad4 in SW480 cells leads to a morphological reversion from spindle- cell shape to an epithelioid morphology associated with reduced migratory activity in an in vitro invasion assay. Further lines of evidence indicate an impact of Smad4 on the interaction of the tumor cells with their environment: For example, using an adhesion assay we found that Smad4 positive and negative cells deposit functionally different (extracellular) matrices on culture dishes. Here we present a proteomic approach to decipher differences in the composition of conditioned media. The secreted soluble proteins in serum-free supernatant were concentrated and loaded on 2D-gels. After silver staining the expression patterns were compared using ProteomWeaver™ software. Differentially secreted proteins were excised, trypsin digested and identified using mass-spectrometry. This approach led to the identification of novel Smad4 targets, among them SPARC (Secreted Protein Acidic and Rich in Cysteins). SPARC is a very prominent protein in the supernatants of Smad4 deficient SW480 clones, only. Northern blot analysis revealed that Smad4 suppressed Sparc expression at the level of transcription. SPARC is a glycoprotein of the extracellular matrix. It has been characterized as an anti-adhesive protein and is known to play an important role in remodeling in a variety of tissues during development and repair processes. Very strong SPARC expression has been found in invading tumors at the tumor-host tissue border and invasion promoting effects were proven in in vitro assays. Thus, a role of Smad4 as a negative regulator of SPARC expression is consistent with its tumor suppressor function. POSTER SESSION

P-97 Tobacco Smoke-induced Mutations and the Development of New Biomarkers for the Identification of High-risk Persons

J. Rauch1,*, M. Ahlemann1,*, O. Gires1,2, S. Lang1 and R. Zeidler3 1Department of Head and Neck Surgery, Ludwig-Maximilians University of Munich, Munich, Germany; 2Clinical Cooperation Group Molecular Oncology, GSF-Research Center for Health and Environment and Department of Head and Neck Surgery, Ludwig-Maximilians University, Munich, Germany; 3Vaecgene Biotech Inc., Munich, Germany * These authors contributed equally

Today, it is widely accepted that cigarette smoking causes cancer. It is estimated that up to 20% of all tumors are attributable to smoking. As yet, the molecular basis underlying tobacco- induced carcinogenesis is not sufficiently clear. Therefore, the principal aims of this project are the analysis of tobacco-induced changes, and, furthermore, the identification of differentially expressed genes and proteins. Subsequently, sensitive biomarkers for Head and Neck tumors shall be established to allow an early and reliable detection of cancer. In order to analyse tobacco-induced mutations, a suitable cellular system was established: HEK293 and A549 cells were treated with carcinogens such as Benzo(a)pyrene, BPDE-2, and NNK. Subsequently, differential gene expression was analysed using 2D-electrophoresis. Additionally, mRNA expression profiling will be performed using Atlas cDNA gene arrays. The frequency of antigens and antibodies specific for these proteins will be examined in blood samples using the Bioplex technology. In parallel, a PCR-based system was established to screen biomarkers using head and neck cancer cell lines (RT-PCR). In order to test known and new biomarkers in vivo, we compiled a tissue library composed of tumor and healthy samples, which will be tested for the expression of selected biomarkers at the mRNA level. Specific and sensitive biomarkers shall allow for the early prognosis and diagnosis of malignancies, especially in individuals at high risk such as tobacco and alcohol abusus. POSTER SESSION

P-98 The Multivariate Analysis of Serum Thermostable Fraction from Patients with Different Oncological States

E.I. Goufman and A.I. Archakov Institute of Biomedical Chemistry, Moscow, Russia

Serum is most suitable body fluid for diagnostics. In order to reveal the possibilities of serum application for oncology disorders diagnosis the proteomic study was performed. The serum termostable fraction from patients with ovarian cancer, uterus cancer, breast cancer and benign tumor of the uterus were analyzed using 2D-PAGE combined with MALDI-TOF/PMF. 38 protein spots corresponding to haptoglobin, transthyretin, apoprotein A-I, apoprotein A-IV, apoprotein D, apoprotein J, Zn α glycoprotein, α2 HS glycoprotein, antitrypsin, and fibrinogen on the proteomic map reflect presence the oncological process in patients. The different oncological states were clearly classified using multivariate (multiprotein) analysis based on self-organizing map (SOM) applied to the protein optical densities of these proteins. The nonspecific changes, which correspond to all tumors, and tumor-type specific changes in protein expressions were revealed by principal component analysis (PCA) applied to self- organized maps. The research results allow considering the 2D proteomic map as multiprotein diagnostic panel for differential diagnosis of some oncological states. POSTER SESSION

P-99 Functional Proteomics to Study Genetic Predisposition to Environmentally Related Cancer

E.H.C Dirksen1 J. Cloos2, B.J.M. Braakhuis2, R.H. Brakenhoff2, A.J.R. Heck1 and M. Slijper2 1Department of Biomolecular Mass Spectrometry, Utrecht University, Utrecht, and 2Section Tumorbiology, Department of Otolaryngology, VUMC, Amsterdam, The Netherlands

Convincing evidence has been provided that the development of head and neck squamous cell carcinoma (HNSCC) is not only related to environmental factors such as smoking and alcohol abuse, but also to an individual cancer susceptibility. It has been shown that patients with environmentally related tumours have an impaired DNA damage processing capability. This intrinsic sensitivity for DNA damage ("mutagen sensitivity”) is measured as mean number of chromatid breaks in peripheral blood lymphocytes after challenging the cells with DNA damaging agents, such as bleomycin. In a combined family and twin study it has been shown that mutagen sensitivity is a heritable factor, thus hypersensitive phenotype is associated with a high risk to develop environmentally related cancer. To investigate which signalling pathways are involved in this inherited mutagen sensitivity, two major routes are pursued, namely at the levels of the transcriptome and the proteome. B-cells of groups of both hypersensitive and insensitive individuals were treated with bleomycin in a time-course and the induction of protein expression was followed using 2D gel electrophoresis. For a comprehensive analysis, the method of visualizing the proteins is very important. Silver staining of the proteins limits the linear dynamic range of detection extremely, although we could already spot some significant differences between the hypersensitive and insensitive groups at two levels, namely that of protein expression and of protein post- translational modification. However for fine-tuning, i.e. to detect significant differences at a wide range of protein expression levels, we needed a significant increase of the quantification potentials. For this purpose we used the DIGE approach (2D difference gel electrophoresis), and labelled the protein samples prior to electrophoresis with the spectrally resolvable fluorescent dyes Cy2, Cy3 and Cy5. A further advantage of this approach was, that it allows for an experimental design in which biological variation can be distinguished from experimental variation. This resulted in a comprehensive and statistically reliable analysis of expression induction by bleomycin at the level of the proteome. Importantly, we established that proteome analysis using 2D gel electrophoresis uniquely reveals different post-translationally modified forms of proteins. Moreover, these differences cannot be distinguished through differential analysis at the level of the transcriptome. POSTER SESSION

P-100 Combined Effects of γ-radiation and Arsenite on Tk6 Cell Proteome

S. Tapio1, I. Danescu-Mayer1, M. Asmuss1, F. Bunk1, A. Posch2, M. Gomolka1* and S. Hornhardt1* 1Federal Office for Radiation Protection, Institute of Radiation Hygiene, Neuherberg, Germany; 2Tecan Munich GmbH, Kirchheim, Germany *the authors contribute equally to the investigation

Cohort studies on uranium miners indicate that the inhalation of arsenic present in most mines together with g-radiation increases the risk of lung cancer. In spite of the epidemiological evidence, arsenic has failed to induce cancer in animal models. For a better understanding of the interaction between radiation and arsenic, we investigated the differences on the proteome level of human lymphoblastoid Tk6 cells, exposed to a single dose of 1Gy of 137Cs-γ-rays, to 1µM arsenite (As[III]) or both agents in combination, using 2D electrophoreses and MALDI-TOF for screening and identification. We were able to identify nine proteins, the levels of which were influenced by at least one of the exposure conditions. After radiation or arsenite treatment a decrease compared to that of the control level was observed for ubiquinol-cytochrome C reductase complex, succinate dehydrogenase [ubiquinone] flavoprotein subunit, isocitrate dehydrogenase [NAD] subunit alpha and serine/threonine protein phosphatase. After the combined treatment the protein amounts stayed lower than the control levels for the first two proteins. Interestingly, for isocitrate dehydrogenase and phosphatase they were near or higher than the control levels. On the contrary, after the combined treatment the levels of protein subunits for 26S proteasome and electron transfer flavoprotein were decreased, although the treatment with radiation or arsenite alone showed an increase in comparison to the control level. The amounts of adenine phosphoribosyl transferase, glutathione transferase omega 1 and endoplasmic reticulum protein Erp29 did not change after irradiation, whereas after treatment with arsenite an increase was observed corresponding to the level of the combined treatment. We conclude that both arsenite and g-radiation influence the levels of several proteins involved in main metabolic and regulatory pathways, either directly or by triggering the defence mechanisms of the cell. The combined effect of both exposures on the level of some essential proteins like the proteasome or serine/threonine protein phosphatase may contribute to the cocarcinogenic effect of arsenic. POSTER SESSION

P-101 A Lung Adenocarcinoma Proteome Map – Search for Early Biomarkers

P. Zürbig1, H. Rütters1, R. Halter1 and Jürgen Borlak1,2 1Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover and 2Department of Pharmacology and Toxicology, Medical School of Hannover, Hannover, Germany

Lung cancer is the most common cancer in the world and smoking is the major risk factor, accounting for about 90% of the cases. In 1996, an estimated 1.3 million new cases were diagnosed worldwide (World Cancer Research Fund, 1997). Prevention and early detection are the main tools to reduce lung cancer morbidity. In human lung adenocarcinomas, the c-raf and c-myc protooncogenes are frequently overexpressed. Two transgenic mouse models were developed to further our knowledge on the specific roles of these protooncogenes. The transgene were targeted to alveolar type II cells through utilisation of the surfactant protein C promoter. Protein expression profiles in lung tumors of SP-C/c-myc and SP-C/c-raf transgenics were then investigated by 2D-SDS-PAGE, image analysis and MALDI-TOF/TOF mass spectrometry. So far more than 3500 gel spots were analyzed for proteome mapping. Lung tumor proteome maps were compared to appropriate controls. Expression levels of key proteins involved in cell growth and metabolism differed significantly between tumor and control tissues. For instance, enhanced expression of known lung tumor markers, such as the murine stress- inducible phosphoprotein, was observed in all tumors investigated. The generation of a lung proteome map for adenocarcinomas is well on its way and holds promise for an identification of novel biomarkers at early stages of tumor development and for identifying novel targets for therapy.

This work was supported by a grant from the Ministry of Science and Culture of the State of Lower Saxony of Germany (J. Borlak). POSTER SESSION

P-102 COFRADIC™-based Non-gel Proteomics of a Sputum Sample from a COPD-patient Reveals More than 200 Different Proteins

P. Van Damme1, A. Staes1, J. Van Damme1, K. Hugelier1, L. Martens1, L. Dupont2, K. Kas3, J. Vandekerckhove1 and Kris Gevaert1 1Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University and Flanders Interuniversity Institute for Biotechnology (VIB09), Ghent, Belgium; 2Department of Respiratory Medicine, University Hospital Gasthuisberg, Leuven University, Leuven, Belgium; 3VIB Headquarters, Zwijnaarde, Belgium

Human chronic obstructive pulmonary disease (COPD) is characterized by the progressive development of airflow limitation and includes pulmonary emphysema, chronic bronchitis and airflow obstruction. The identification of biomarkers in sputum which allow early diagnosis, monitoring and optimisation of therapies for lung diseases is one of the most ambitious goals in respiratory medicine. The induced sputum technique allows sampling of the airways in a non- invasive manner and thereby offers a unique opportunity to specifically isolate potential biomarkers that can be used in respiratory medicine. Recently, our lab has developed a gel-free proteomics approach based on a chromatographic procedure to isolate representative peptides out of complex mixtures (e.g., proteome digests). This procedure is called combined fractional diagonal chromatography (COFRADIC™, Gevaert et al., 2002 & 2003). Sets of peptides that are routinely isolated for further LC-MS/MS analysis include methionine-containing peptides and amino terminal peptides. Here, we report on the identification of more than 200 different proteins from a human COPD-sputum sample using COFRADIC™. The clinical sample was first digested in solution with trypsin and the generated peptide mixture was fractionated on a reverse phase (RP) HPLC column. Following a series of secondary, identical RP-HPLC separations, methionine-peptides were specifically isolated as their sulfoxide derivatives and were used to identify their precursor proteins by automated LC- tandem mass spectrometric (MS/MS) analysis. The MASCOT database search algorithm (http://www.matrixscience.com) unambiguously identified 748 methionine-containing peptides, of which 482 carried unique sequences. In total, 216 different human proteins were identified. In contrast, using a traditional, gel-based approach, only 49 different proteins were identified following LC-MS/MS analysis. Next to a large number of novel proteins, the list of identified proteins contained a significant number of biomarkers which have been associated with the pathogenesis of COPD. These biomarkers amongst others, include the 92 kDa type IV collagenase precursor (P14780), the serine proteinase inhibitor a-2-macroglobulin precursor (P01023), the cysteine proteinases cathepsin D (P07339 ) and cathepsin H (P09668) as well as the α1-antitrypsin precursor (P01009). These findings clearly indicate a high possibility that our list contains hitherto undiscovered biomarkers for COPD or related lung diseases. Next to secreted proteins, w e identified a fairly high number of intracellular proteins that can originate from cellular lysis, which might be a marked biological phenomenon in the lungs of COPD patients.

References: Gevaert, K., Van Damme, J., Goethals, M., Thomas, G.R., Hoorelbeke, B., Demol, H., Martens, L., Puype, M., Staes, A. and Vandekerckhove, J. (2002) Mol. Cell. Proteomics 1, 896-903. Gevaert, K., Goethals, M., Martens, L., Van Damme, J., Staes, A., Thomas, G.R. and Vandekerckhove, J. (2003) Nat. Biotechnol. 21, 566-569. POSTER SESSION

P-103 Differences in the Protein Expression Pattern of Murine Fibroblasts after Oncogenic Transformation with Various K-ras Mutations

C. Recktenwald1, S. Mendler1, R. Lichtenfels1, R. Kellner2 and B. Seliger1 1Johannes Gutenberg University, III. Department of Internal Medicine, Mainz, Germany and 2Merck KGaA, Darmstadt, Germany

Mutated Ki-ras appears to be involved in the carcinogenesis of pancreatic, lung and colorectal carcinoma. Three "hot spots” of Ki-ras mutations occurring at codon 12, 13 and 61 might differentially affect the biology of tumors as well as the clinical outcome of Ki-ras-associated malignancies. In order to define potential mutation-specific therapeutic targets, we first generated stable transfectants of NIH3T3 cells carrying Ki-ras mutations at codon 12, 13 and 61. Transfectants with the Ki-ras wild type gene, mock transfectants and parental cells served as controls. These in vitro model systems were then analyzed for the protein expression pattern using two dimensional gel electrophoresis followed by mass spectrometry and / or protein sequencing. Employing the proteome approach a series of differentially expressed proteins were identified. Some of the proteins showed a common regulation by ras transformation, whereas others were dependent on the mode of Ki-ras mutation. The differentially expressed proteins identified belong predominantly to the families of cytoskeletal proteins, heat shock proteins, annexins, metabolic enzymes and oxidoreductases. Evaluation of differentially expressed proteins was performed by RT-PCR and/ or Western blot analysis of different Ki-ras mutant and respective control cells. Our results suggest that the Ki-ras mutants are a powerful tool to study Ki-ras mutation-mediated protein expression which will lead to the identification of novel target structures for the treatment of Ki-ras-associated tumors. POSTER SESSION

P-104 A Rat Liver Proteome Analysis for an Identification of Tumor Markers

G. Gazzana1, H. Rütters1, M. Niehof1 and J. Borlak 1,2 1Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover and 2Department of Pharmacology and Toxicology, Medical School of Hannover, Hannover, Germany

Polychlorinated biphenyls (PCB) are persistent environmental chemicals that accumulate at the apex of food chains. PCB are well-known for their ability to promote tumor growth in laboratory animals and are classified as class A carcinogens. Nonetheless the evidence for their tumor-initiating potential remains controversial. It is known that PCB have the ability to bind to the aryl hydrocarbon receptor, a transcription factor that induces expression of a wide range of different genes including Cyp monooxygenases. To gain further insights into the molecular mechanisms of PCB’s mode of action and to identify biomarkers for tumor growth, we initiated the proteome analysis of rat liver following treatment of rats with Aroclor 1254, a commercial mixture of PCB. For proteome mapping, liver extracts of control and treated animals were separated by two-dimensional SDS-PAGE employing different protein loads and lysis buffers for extraction of cytosolic and nuclear/membrane proteins. pH ranges for isoelectric focusing were selected according to a "virtual gel” constructed from gene expression data. Protein spots were visualized using Colloidal Coomassie Brilliant Blue or Ruthenium (II) tris (bathophenanthroline disulfonate). Approximately 800 spots per gel were excised and analysed by MALDI-TOF/TOF mass spectrometry after tryptic in gel digestion. The majority of identified proteins in control and treated samples belongs to the following categories: blood proteins, mitochondrial proteins, ribosomal proteins, metal-binding proteins, and proteins involved in different metabolic pathways like glycolysis and fatty acid biosynthesis. By image analysis more than 20 proteins were identified as being differentially expressed. This was, in part, dependent on the type of lysis buffer employed. Comparison of gene and protein expression data revealed significant differences that may deepen our understanding of the PCB-related carcinogenesis at a molecular level.

This work was supported by a grant from the Ministry of Science and Culture of the State of Lower Saxony of Germany to J. Borlak. POSTER SESSION

P-105 Differential Proteome Analysis OF Rat Liver Following TCDD Exposure

R. Pastorelli1, D. Carpi1, S. Tavazzi1, E. Fattore1, C. Chiabrando1, L. Airoldi1, H. Hakansson2 and R. Fanelli1 1Department of Environmental Health Sciences, Istituto "Mario Negri", Milano, Italy and 1Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden.

This preliminary study was undertaken to investigate the extent to which 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) exposure alters liver protein expression and to determine which, if any, protein markers might be indicative of TCDD toxicity mechanisms. We applied proteomic technologies to study changes in the levels of liver proteins of male rats treated with a single oral dose of TCDD (10 µg/kg) on postnatal day 21. Liver tissue was taken 22 days after the treatment. Total liver proteins were separated by two-dimensional gel electrophoresis and visualized using the Coomassie colloidal staining method. Gels were run in triplicate. Using ImageMaster software analysis, approximately 370 spots in each gel image were matched and quantitated. Only protein spots that changed more than 2-fold in magnitude, in the same direction (i.e. up or down) and were observed in all the triplicate gels were considered for gel excision and further analysis. These differential expression studies revealed the presence of significant derangements in 10 proteins, following administration of TCDD. After in-gel trypsin digestion, the proteins of interest were analysed by matrix-assisted laser desorption/ionization time-of- flight (MALDI-TOF) mass spectrometry. TCDD treatment appears to modulate the expression of several enzymes related to oxidative stress, including an aldehyde dehydrogenase isoform with putative retinoid-related activity. For the first time, we observed a significantly increased expression in the presence of two isoforms of the Selenium-Binding Protein 2 after TCDD treatment. The physiological role of this protein is still unknown. Interestingly, we observed an up-regulation of a protein related to the androgen metabolism, suggesting a putative new molecular target of endocrine disruption by TCDD. In summary, these preliminary data suggest that this approach will allow us to have a global perspective, at the protein expression level, of the many different known effects of TCDD. Though most of the protein expression changes observed are consistent with the extensive literature on TCDD effects, some of these changes may lead to new insights into the mechanism of TCDD toxicity. POSTER SESSION

P-106 Difference Gel Electrophoresis (DIGE) is a Powerful Tool to Detect TIMP-1 Induced Proteins in Human Hepatoma Cells

C. Henkel1, S. Poetsch2, B. Bacher2, S. Matern1 and E. Roeb1 1Medical Clinic III University Hospital, RWTH Aachen, Germany and 2Amersham Biosciences Europe GmbH, Freiburg, Germany

Introduction: Matrix metalloproteinases (MMP) are involved in the remodeling process of extracellular matrix and the basement membrane. Increased expression of MMP inhibitors such as the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) promotes progression of fibrosis in liver. Although several stimulators of TIMP-1 expression were described, only little is known about the influence of TIMP-1 on the cellular protein expression pattern in liver cells (E. Roeb et. al. FASEB J 2000). In this study protein maps of two different human hepatoma cell lines, one of it is a TIMP-1 over-expressing cell line, were investigated by Difference Gel Electrophoresis (DIGE) and MALDI mass spectrometry in order to determine proteins that could explain the function of TIMP-1, e.g. in liver fibrosis. Methods and Instrumentation: Two different cell lines were compared: HepG2 infected with Ad5-CMV-mTIMP-1 and mock transfected HepG2. The adherent hepatoma cells were harvested and washed in saccharose buffer (10 mM Tris, 250mM Saccharose) and proteins were extracted in DIGE lysis buffer (8 M urea, 4% CHAPS and 30mM Tris pH 8.5). Interfering substances of the protein samples were removed using the PlusOne 2-D Clean-Up Kit. The Protein concentrations in the lysates were determined by using 2-D Quant Kit. Two samples and a pooled standard were labeled with Cy3, Cy5 and Cy2 CyDye DIGE fluors, respectively. A pooled internal standard containing equal amounts of both samples and allows spot specific normalization of the data and statistical analysis. The first and second dimension electrophoresis was done under standard conditions (see Ettan DIGE User Manual) using Immobiline drystripspH 3-10 NL, 18cm, IPGphor, and Ettan Dalt six. The gels were scanned on a Typhoon 9410 and analysed fully automatically by DeCyderTM software. Spots showing significant differences (student's t-test, p<0.05) were chosen for picking and further analysis on an Ettan Maldi ToF mass spectrometer. Results: The experiment to determine interacting proteins of TIMP-1, which may regulate the function of TIMP-1, was divided into two steps. First we compared the protein pattern of TIMP- 1 and HepG2 (control) by using Difference Gel electrophoresis (DIGE). Both samples were run in 5 replicate where at least two samples can be processed in the same gel. The use of an internal standard allows normalization of the data and the detection of significant differences with high confidence. Protein mixtures were labeled with different CyDye™ DIGE fluors and separated using two-dimensional gel electrophoresis. Five gels were run with 150 µg protein/gel and as the second step two gels were run as preparative gels 500 µg protein/gel. Image analysis revealed approximately 2300 spots, which matched on all gels. Among these spots, 160 spots showed significant differences. In detail, 60 spots decreased by a factor > 1.5 and 100 spots increased by a factor >1.5. The MALDI ToF mass spectrometry showed several proteins (e.g. Heat shock Protein 70, Enolase 1, MYC promotor binding Protein), which need further investigations with respect to the influences of these proteins in the development of fibrosis. POSTER SESSION

P-107 Serum Proteomic Signatures by SELDI-TOF MS Analysis for Diagnosis of Primary Biliary Cirrhosis and Other Chronic Cholestatic Diseases

B. Baudin1,2, A. Bruneel1, M. Cubizolles3, O. Chazouillères4, R. Poupon4 and M. Vaubourdolle1 1Biochimie A and 4Hépatologie, Hôpital Saint-Antoine, Paris, France; 2GRECAN-EA1772 - UFR Pharmacie, Caen, France ; 3Ciphergen France, Cergy-Saint-Christophe, France

Primary biliary cirrhosis (PBC) is a chronic and cholestatic hepatic disease with progressive destruction of biliary tracts leading to cirrhosis; the main treatment is ursodeoxycholic acid (URSO) and, in some patients, liver transplantation will be necessary. The best diagnostic marker is the presence of antimitochondria antibodies; and sequential increases in serum bilirubin are of poor prognosis. Sometimes a differential diagnosis must be made with primary sclerotic cholangitis (PSC) because the response to URSO is worse and some of the patients with PSC will be transplanted. We used SELDI ProteinChip® biomarker system from Ciphergen, based on protein array technology and SELDI-TOF MS, for the comprehensive proteomic profiling of sera from patients with PBC or PSC in the goal to identify serum signatures that could improve both diagnosis and therapeutic care of these chronic cholestatic diseases. Four groups, of six patients each, were studied: 1- non active PBC under URSO; 2- serious PBC before transplantation; 3- PSC; 4- controls matched in sex and age with PBC patients. Each serum fraction was analysed on four ProteinChip® Arrays with two different binding conditions for each retention chromatographic array (CM10 array for cationic exchange at pH 5 and 7, LSAX30 array for anionic exchange at pH 7 and 9, IMAC40 array for immobilized metal ion capture affinity using nickel and zinc, and H50 array for reversed phase using 5 % and 10 % acetonitrile), i.e. 24 samples x 8 conditions = 192 experiments. After specific removal of proteins not bound onto the arrays, sinapinic acid matrix was loaded and bound proteins were detected with ProteinChip® Reader (mass spectrometry in time-of-flight separating proteins on the basis of their molecular mass); the ProteinChip® Software automates biomarker analysis and gives statistics. All these biomarkers were chosen after visual peak inspection; they correspond to either increase or decrease of the peak intensity for the considered group. We found more than 40 proteomic features among the serum samples; in particular a biomarker of severity in PBC (decreased in serious PBC samples) was detected at 95.2 kDa on anion exchanger, pH 9 (6 versus 6, p=0.004); two biomarkers of PBC (increased in PBC samples compared to controls) were detected at 8.6 and 9.0 kDa (low scale), and two of PSC (increased in PSC samples compared to PBC samples) at 34.5 and 101.8 kDa (high scale) by profiling on cation exchanger, pH 7 (at least 4 versus 4, p<0.05); two other markers of PBC were detected at 5.9 and 8.9 kDa (low scale) by profiling on IMAC40 loaded with Zn2+; profiling on H50 mainly detected markers of high molecular mass. Several other potential markers were also found with p<0.05. These preliminary results show that serum proteomic signatures based on SELDI ProteinChip® technology can discriminate PBC from PSC, and both groups of patients with chronic cholestatic diseases from controls; moreover markers of severity and low response to URSO will be of particular interest for the assessment of PBC under therapy. The identification of the most representative protein markers will not only improve the diagnosis and prognosis of these hepatic diseases but also will enhance our knowledge on their physiopathology. From these pathological mechanisms we can expect to identify new targets for their specific therapy, in particular using drug design. POSTER SESSION

P-108 Proteomics Analysis of Brain Gliomas

F. Odreman1, M. Vindigni2, M. Skrap2, G. Stanta1,3, M. Lujardo1 and A. Vindigni1 1International Centre for Genetic Engineering and Biotechnology, Area Science Park, Trieste, Italy; 2Neurosurgery Unit, Hospital of Udine, Udine, Italy; 3Department of Clinical, Morphological and Technical Sciences, University of Trieste, Italy

Gliomas are the most common type of malignant brain tumors accounting for about 45% of all intracranial tumors. On the basis of histological observations, brain gliomas are divided in two main groups defined as low- and high-grade gliomas. Previous studies on the molecular genetic events associated with glioma formation and subsequent progression have identified a number of specific genetic alterations, including amplification or mutation of the epidermal and platelet-derived growth factor receptors, and loss of cell cycle growth regulators such as p53 and p16. In addition to these known genetic changes, it is likely that there are other alterations in gene expression associated with the pathogenesis of gliomas, which may yield novel insights into potential new targets for therapeutic drug design. In this study, we investigated the differences in the protein expression profiles between low- and high-grade gliomas by two-dimensional gel electrophoresis. Changes in the protein expression patterns between the gels were highlighted with the assistance of the PDQuest software. Successively, most of the protein spots that were uniquely present in either low- or high-grade gliomas were identified by mass spectrometry. In particular, by means of western blot analysis we have analyzed the eventual presence of Ku and RPA, two proteins that play a key role in DNA repair. Our results showed that most high-grade gliomas were consistently positive for Ku86 while the signal for Ku70 was dramatically reduced or totally absent. On the other hand, both Ku86 and Ku70 were equally present in low-grade gliomas. POSTER SESSION

P-109 Proteome Study of Human Cerebrospinal Fluid Following Traumatic Brain Injury Indicates Fibrin(ogen) Degradation Products as Trauma Associated Markers

A. Conti1, Y. Sanchez-Ruiz2, A. Bachi2, L. Beretta3, E.Grandi3 and M. Alessio1 1Proteomics Unit, 2Mass Spectrometry, 3Intensive Care Neurosurgery Department, San Raffaele Scientific Institute, Italy

Introduction: During a traumatic brain injury (TBI) event the expression of many proteins increases in the damaged regions. These proteins belong to different groups such as proinflammatory cytokines, cytoskeletal proteins and cytoskeletal-associated proteins. In addition, alterations of intracellular protein assembly and transport, proteolysis and changes in protein phosphorylation, have also been described. The proteomics technologies allows the analysis of a large number of differently modified proteins. The cerebrospinal fluid (CSF) is the reference "tissue” for proteomic studies of central nervous system (CNS) pathologies for the following reasons: first, it is of great diagnostic importance, being in contact with the brain it contains proteins released directly from the CNS; second, CSF samples from patients and controls are routinely available as a consequence of therapeutic interventions and patients monitoring; third, the most abundant proteins in the CSF are already known and their identification by comparison with digitalized reference maps is feasible. This work aim was to identify specific markers for TBI pathological condition by applying a proteomics analysis of patients CSF. Material and Methods: Following ethic approval by hospital’s institutional review board, CSF samples were collected from six brain trauma patients. Control CSF was obtained from patients hospitalised for minor urologic, gynecologic or orthopedic surgeries. Samples were centrifuged in order to eliminate cells and supernatants immediately used or stored at –80° C. This experimental approach includes the generation of two-dimensional gel electrophoresis (2D-E) maps, that resolve proteins on the basis of their isoelectric point and relative mass. Interesting proteins were then identified either by computer assisted comparison with CSF reference map, or by mass- spectrometry (MS) analysis and database search. Results: Image analysis identified four proteins spots showing a significant expression increase in TBIs . Comparison with human CSF 2-DE reference maps allowed the unambiguous identification of these proteins. Spot #1 corresponded to α1 antitrypsin (A1AT), spots #2, 3 and 4 corresponded to haptoglobin 1β, α2 and α1 (HPT1β and α2/1) respectively. Proteins expression increase was quantified by image analysis, comparing TBI and control normalized spot volume . Three protein spots (# 5, 6 and 7) were present only in TBIs . Theoretical relative mass and pI assigned to them were: spot #5, pI 5.56 and Mr 41227; spot #6, pI 5.74 and Mr 40840; spot #7, pI 5.73 and Mr 24276. These spots were excised from preparative 2D-E gels and investigated by mass spectrometry. Due to the low amount of protein recovered from the gel was impossible to identify spot #7 protein. Spots # 5 and 6, resulted to be the same protein, corresponding to the carboxyl- terminal portion of the fibrinogen β_(Fbβ). The measured Mr and the sequence overlapping suggest that these are products of proteolytic degradation. Since our CSF samples were from patients with tissue damage, there was a risk of blood contamination, even though the inclusion criteria excluded patients in which CSF appeared contaminated by erythrocytes. To confirm that this was not the case we performed 2D-E from both peripheral and jugular vein bulb reflous blood and compared them to CSF 2D-E. Fibrinogen β and γ_ that have been reported to be present also in CSF, were observed in all kinds of sample. Interestingly, the spots #5, 6 and 7, found specifically in TBI patient CSF, were not found in the corresponding patients plasma. Specific molecular marker analysis, confirmed that no blood contamination occured in our CSF samples and that the presence of spot #5,6 and 7, is a specific feature of TBI patient CSF. In conclusion, in TBI, proteins of the acute phase that undergo an important expression level modulation, were identified. We also identified two β-fibrinogen polypeptides specifically generated in CSF during brain trauma. These proteins are probably linked to the protective pathways activated by the organism after nervous tissue injury. POSTER SESSION

P-110 Study of Proteins Pattern in Human Cerebrospinal Fluid Obtained from Patients Affected by Peripheral Neuropathies

A. Conti1, A. Cattaneo2, A. Bachi2, S. Iannaccone3 and M. Alessio1 1Proteomics Unit, 2Mass Spectrometry, 3Neurology Dept., San Raffaele Scientific Inst., Italy

Introduction: Peripheral neuropathies syndrome is usually characterized by asymmetrical, slowly progressive weakness, most often beginning distally in the arms with no upper motor neuron signs. The disease can occur in presence or absence of pain and its identification is important to decide the best therapeutic approach. This syndrome pattern of weakness symptoms are not character- istic and unique and therefore additional laboratory evaluation, including electrodiagnostic studies and measurement of serum autoantibodies, is usually needed to help in diagnose this disease in individual patients. We propose the use of cerebrospinal fluid (CSF) as reference biological fluid for the research of markers for this pathology by using the Proteomics approach. In fact, being in tight contact with central nervous system (CNS) CSF contains proteins released directly from the CNS. Moreover samples from patients are routinely available as a consequence of patients monitoring. Another reason for this choice is that many proteins of the CSF are already known and their identification by comparison with digitalized reference maps is feasible. Material and Methods: Following the approval of ethic procedures by hospital’s institutional review board we started collecting liquor samples. We analysed CSF from three main groups: pain in neuropathic patients (PNn..), no pain in neuropathic patients (NPNn..) and healthy controls (CNn..). The respectively samples (8 PN, 8 NPN and 6 CN) have been treated to allow 2-D electrophoresis (2-DE) and analytical highly sensitive silver staining. One hundred ug of proteins from each CSF sample were used for the generation of analytical 2-DE. The proteins were precipitated with aceton, resuspended and run on the first dimension by using a pH 3-10 non linear strips, followed by second dimension (9-16% acrylamide gradient gels). The slab gels were silver stained, scanned at high resolution and the resulting 2D protein patterns analyzed by Image Master 4.1 software (Amersham Bioscience). Results: Control (CN) were 3 male and 5 female with age ranging between 25 and 62 years (average 41.25 sd 13.61); neuropathic patients with pain (PN) were 4 male and 4 female with age ranging between 31 and 69 years ( average 48.8 sd 12.3); neuropathic patients without pain (NPN) were 5 male and 3 female with age ranging between 38 and 74 years (average 55.2 sd 14.5). No statistical differences have been determined among patient groups sex and age features, as calculated by student T test. On the contrary, CSF total proteins concentration showed statistical differences between patients groups. In fact, CSF proteins concentration was lower in controls (average 0.28 mg/ml, sd 0.12) compared to PN (average 0.48 mg/ml, sd 0.21) and NPN (average 0.53 mg/ml, sd 0.27). Despite a greater proteins concentration in PN and NPN patients, the number of protein spots detectable in the 2-DE maps was lower in these two groups (average 390 sd 126, and 426 sd 80, respectively) compared to control (average 548 sd 116). This may suggests that a proteins degradation occurred in the PN and NPN patients and that the small degradation peptides are quantified by protein detection system but not resolved by 2-DE. The protein profile image analysis showed a spot matching ranging between 50-60% of the total spots number (about 200-300 proteins were comparable among the different group gels), and about 25- 40% of the total spots are shared by the CN, PN and NPN groups. Image analysis showed 27 protein spots differentially expressed in the 3 groups. An experimental molecular weight (MW) and an isoelectric point (pI) have been assigned to these 27 protein spots by map calibration. These protein spots can be classified as groups with different expression pattern: proteins expressed only or at higher level in PN patients (4 spots); proteins that are absent or expressed at lower level only in PN patients (6 spots); proteins that are expressed at higher level in both PN and NPN (1 spot); proteins that are absent or with lower expression level in both PN and NPN patients compared to control (16 spots). None of the interesting protein spots were directly identified comparing with the CSF 2-DE reference map available on-line (www.expasy.org). For this reason preparative 2-DE will be generated and protein spots of interest analysed by mass spectrometry after partial tryptic digestion. POSTER SESSION

P-111 Proteomic Analysis of Cerebrospinal Fluid of Patients with Multiple Sclerosis: Effects of T Cell Vaccination

D. Dumont, J.-P. Noben, J. Raus, P. Stinissen and J. Robben Biomedisch Onderzoeksinstituut (BIOMED), Limburgs Universitair Centrum and School for Life Sciences, Transnational University Limburg, Diepenbeek, Belgium

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system that is generally believed to be of autoimmune origin. Although the exact mechanism of disease initiation remains elusive, it is postulated that autoreactive T cells play an essential role in the pathogenesis of MS by targeting components of myelin sheaths. Based on the potential pathogenicity of myelin reactive T cells several immunotherapeutic strategies have been designed to specifically inactivate these T lymphocytes. One possible therapeutic approach is T cell vaccination (TCV) which involves immunization with attenuated autologous myelin reactive T cells. In a previous study, we conducted a pilot clinical trial of TCV with cerebrospinal fluid (CSF)-derived activated CD4+ T cells in five MS patients. In all patients, anti- vaccine responses were observed and myelin reactive T cell responses remained low or were reduced. A proteomic approach is currently used to further analyse the effects of TCV. CSF proteins from five MS patients before and after TCV were separated by two-dimensional gel electrophoresis, visualized by silver staining and were evaluated using quantitative electrophoresis image analysis software. Differentially expressed proteins are currently analysed by mass spectrometry. This proteomic approach may lead to an improved understanding of the effects of TCV on protein expression in CSF. POSTER SESSION

P-112 Analysis of Cerebrospinal Fluid of Depressed Patients

M. Rosenhagen, D. Milfay, G. Maccarrone, M. Uhr, F. Holsboer and C. Turck Max-Planck Institute of Psychiatry, Munich, Germany

Objectives: Comparative analysis of the proteome of cerebrospinal fluid (CSF) of depressed and healthy subjects with the aim to identify disease specific proteins that may be targets for novel treatments in psychiatric and neurological diseases. Background: Analysis of protein patterns of cerebrospinal fluid (CSF) is of great diagnostic importance because it reflects the metabolic state of the brain in several diseases as well as under healthy conditions. The majority of CSF proteins are serum proteins, while only a small number are derived from the CNS. The identification of CSF proteins is also important for the development of new, clinically useful neuronal markers and for studying the pathogenesis of mental disorders. Method: Cases were in-patients with the diagnosis of a severe major depressive episode. Controls consisted of patients without a history of psychiatric illness that were admitted to the neurology ward for routine diagnosis and the exclusion of severe neurological disorders. CSF proteins were separated by two dimensional (2D) gel electrophoresis. They are separated first by charge and then by size. Differences in spot patterns were analyzed by the computer assisted program PDQUEST. Identification of proteins were done by mass spectrometry. Results: Preliminary evaluation of the data of our 2D analysis has shown both modest qualitative and quantitative differences in the protein patterns between cases and controls. Several proteins were identified by mass spectrometry. Further analysis is on-going, focussing on the evaluation of the results.

References: A. Sickmann et al. (2000): Identification of proteins from human cerebrospinale fluid, separated by two-dimensional polyacrylamide gel electrophoresis. Electrophoresis 21, 2721-2728 C. Rohlff (2001): Proteomics in neuropsychiatric disorders. Int J Neuropsychopharm 4, 93-102 T. Rabilloud (2002): Two-dimensional gel electrophoresis in proteomics: Old, old fashioned, but still climbs up the mountains. Proteomics 2, 3-10. POSTER SESSION

P-113 Proteomics of Lipid Rafts from Brain Microvascular Endothelial Cells

M. Jäger, S. Märten, T. Oppolzer, T. Bangsow, B. Pelzer and S. Wolf Esplora GmbH, c/o Institute of Biochemistry, Darmstadt, Germany

Blood-brain barrier (BBB) cells have cellular tight junctions and express special transport systems which serve to actively transport nutrients into the brain. On the other hand there is also an active efflux of toxic metabolites and xenobiotics from brain to blood. The main component of the BBB are the microvascular endothelial cells (BMEC) which stnd in close contact with two other cell types, pericytes and astrocytes. Many diseases of the central nervous system like Alzheimer or Parkinson remain difficult to treat because many potential therapeutic cannot reach the brain or are discharged. Therefor the understanding of transport processes taking place at the BBB is an important step for the development of effective drug delivery systems. Lipid rafts (LR) are plasma membrane microdomains which are enriched in glycosyl- phosphatidylinositol (GPI)-anchored proteins, as well as proteins involved in signal transduction and intracellular transport. Rafts are rich in cholesterol and sphingolipids, which provide a particularly ordered lipid enviroment. As brain microvascular endothelial cells loose there barrier characteristics e isolated lipid rafts from cultured BMEC passage P0 and P2 and separated them by SDS-Page. Bands were excised and a tryptic digest was performed. The resulting peptides were eluted, separated by nano-HPLC and analyzed by ESI-MS/MS-spectrometry. The amount of lipid rafts isolated of fraction P0 was higher than of P2. This was also shown by Anti-Caveolin western blot and alkaline phosphatase (AP)-activity. The pattern between both LR fractions were similar and only slight differences were observed. By LC-ESI-MS/MS and subsequent database searching some 40 proteins were identified in both fractions. But there were also some unique proteins found in P0 and P2. Importantly we could also show that the newly described ABCG2 transport protein (Eisenblatter & Galla, 2002) belonging to the multi drug resistance family is located in lipid rafts at the blood brain barrier.

Reference: Eisenblatter T, Galla HJ (2002), Biochem Biophys Res Commun. 293, 1273-1278. POSTER SESSION

P-114 Neuroanatomical Comparison of the Ventral and Dorsal Striatum in the Non-human Primate by 2-Dimensional Difference Electrophoresis

W.M. Freeman and S.E. Hemby Yerkes National Primate Research Center, Emory University School of Medicine, Atlanta, GA, USA

The dopaminergic terminal fields of the mesolimbic (nucleus accumbens, NAcc) and nigrostriatal (striatum) are key substrates of motivated behavior and movement. These brain regions are critical in diseases such as drug abuse and Parkinson’s disease, respectively. Development of drugs to effectively treat either the etiology or symptomatology of these diseases with a minimum of side effects will require drug targets that are specific to either of these brain regions. To find proteins specifically or preferentially expressed in either of these brain regions, the nucleus accumbens (NAc) and putamen were dissected from rhesus macaques (Macca mullatta, 1.5-3 years, n=3) were compared by 2-dimensional difference electrophoresis (2-DIGE). Initially, proteins were fractionated into cytosolic, membrane and nuclear fractions by ultracentrifugation. Cytosolic and membrane fractions were labeled using minimal labeling Cy dyes, separated by 3-10NL isoelectric focusing and SDS-PAGE electrophoresis and compared using DeCyder software. Differentially expressed proteins, as determined by DeCyder analysis of normalized spot volumes (p<0.05) were excised by an Ettan Spot Picker and peptides from in-gel trypsin digestion were analyzed by either MS or MS/MS. Protein identifications were made using public, human databases. Results of this study represent the first proteomic profile of the NAcc and striatum, and one of the first uses of the 2-DIGE methodology in non-human primate neurobiology. These results will be compared to the results of ongoing studies into changes in protein expression with cocaine abuse and antipsychotic action in the NAcc and striatum of non-human primates for overlap. POSTER SESSION

P-115 Profiling of Urinary Tryptophan Metabolites by On-line Solid-phase Extraction – liquid Chromatography – Tandem Mass Spectrometry for the Assessment of Metabolic Dysfunctions Associated with Autism

K. Dettmer, J.W. Newman, C.E. Wheelock and B.D. Hammock University of California, Dept of Entomology and Cancer Research Center, Davis, CA USA

Autistic spectrum disorder (ASD) is a neurological disease characterized by dysfunctions in social interaction and communication. While the syndrome is of unknown etiology, various hypotheses have been presented suggesting both genetic and environmental (i.e. nutrition, chemical exposure) factors. Analyses of single biomarkers have identified apparent changes in tryptophan metabolism in relation to ASD, such as elevated levels of the neurotransmitter serotonin in platelets or abnormally high quantities of indolyl-3-acryloylglycine in urine. To provide a more comprehensive coverage of tryptophan metabolism, we are quantitatively analyzing over 20 compounds within the serotonin, kynurenine, indole pyruvate, and indole pathways which hold information about critical metabolic branch points. The quantitative analysis of tryptophan metabolites is performed using on-line solid-phase extraction (SPE) coupled to high-performance liquid chromatography (HPLC) with MS/MS detection. On-line SPE–HPLC will increase the method automation and laboratory throughput. Moreover, analyte losses are minimized and the amount of sample required per analysis is reduced, since the entire sample is analyzed. Between 50 and 200 µL of the filtered and acidified urine sample is injected on a pre-column (Oasis HLB), followed by a washing step. Subsequently, the solvent flow through the pre-column will be reversed with solvent flow being directed to an analytical HPLC column. The chromatographic separation is followed by electrospray ionization in both positive and negative mode and multi-reaction monitoring (MRM) mass spectral data are acquired. Analyte pre-column breakthrough volumes will be presented as well as the precision, accuracy, and sensitivity of the analytical technique. POSTER SESSION

P-116 Identification of ANA Targets by Serologic Proteome Analysis in Autoimmune Hepatitis Type 1

S. Huguet1, V. Labas2, J.C. Duclos-Vallee3, A. Bruneel4, J. Vinh2, C. Johanet1 and E. Ballot1 1Service d’Immunologie, Hopital Saint-Antoine AP-HP, Paris; 2Laboratoire de neurobiologie CNRS UMR 7637 ESPCI, Paris; 3Centre hépatobiliaire Hopital Paul Brousse, EPI 99-41, UPRES 1595, Villejuif; 4Service de Biochimie, Hopital Saint-Antoine AP-HP, Paris; France

The classification of antinuclear antibodies (ANA) is important for diagnosis and prognosis, and for understanding the molecular pathology of autoimmune disease. ANA are particularly important serological markers for autoimmune hepatitis (AIH) type 1. However, they are f e w studies investigating their molecular targets in this disease. We have undertaken to characterize antinuclear targets in AIH type 1 using the proteomic tool. Patient sera were screened for ANA by indirect immunofluorescence on Hep-2 cells and rat liver. 29 sera from patients with AIH type 1 and ANA positive and 14 negative controls were analyzed by one dimensional (D) immunoblot performed with nuclear fraction of rat liver obtained by centrifugation on sucrose density gradient. Among the stained band, we focus especially to a 36 kDa protein that was recognized by 52% of patients’sera vs 7% of controls (p<0.001). On 2D immunoblot performed with 5 sera, this protein correspond to 5 spots located between pI 8.0 and 9.0. After in gel tryptic digestion, their identification was performed using peptide mass fingerprint analysis obtained by MALDI-TOF MS (matrix-assisted laser desorption/ionisation-time of flight mass spectrometry) with 50% sequence coverage, and was unambiguously confirmed by 14 different amino-acid sequences obtained by nanoscale LC-ESI-MS/MS (nanoscale liquid chromatography electrospray tandem mass spectrometry). This lead to the clear identification of heterogeneous nuclear ribonucleoprotein (hn RNP) A2/B1. Autoantibodies to proteins of the hnRNP complex (A1,A2,B1,B2,C1,C2) have now been described in some rheumatic diseases but it is the first report in AIH type 1. This study identify the hnRNP A2/B1 as ANA targets in the sera of AIH patients and shows a concrete example of 2D electrophoresis and mass spectrometry association as a versatile and powerful tool for identification of novel autoantigen specificities. POSTER SESSION

P-117 Diagnosis by Complex Autoantibody-Repertoires and Protein Profiles in Autoimmune Diseases using Bio-Chips

F.H. Grus, S.C. Joachim and N. Pfeiffer Dept. of Ophthalmology, University of Mainz, Germany

Normal sera contain a large number of naturally occurring auto-antibodies which can mask important disease-associated ones. Western Blotting has evolved as the most important tool to demonstrate autoantibodies in autoimmune diseases, because its possibility of simultaneous screening for a wide spectrum of different antigens. In previous studies we could show the diagnostic potential of the analysis of autoantibodies in autoimmune diseases by means of multivariate statistics and artificial neural networks. However, the Western Blotting procedure remains very time-consuming and is also limited in sensitivity. Therefore, we used an on-chip approach for the analysis of autoantibodies. This ProteinChip system uses ProteinChip arrays and SELDI-TOF (surface-enhanced laser desorption/ionisation in time-of-flight mass spectrometry) technology (Ciphergen, Fremont, USA) for capturing, detection, and analysis of proteins without labeling or without the need of chemical modification. The micro-scale design of the arrays allows the analysis of very small quantities of proteins. In the present study, we used arrays with biological activated surfaces that permit antibody capture studies. Protein-A-Chips were incubated with sera of glaucoma patients and healthy subjects (n=22). After washing, the chips were incubated with a complex solution of auto-antigens and subsequently washed again. If the Protein-A bound autoantibodies recognized their antigens, these proteins could be separated by their molecular masses and were be detected by mass spectrometry. Previous studies using monoclonal antibodies could demonstrate that the detection limit is in the attomole level. Furthermore all sera were analyzed by conventional Western Blotting for direct comparison. In the present study, we could show complex on-chip antibody-antigen reactions. At higher molecular weights (>30kDa) the detection sensitivity of this on-chip method was comparable to conventional Western Blotting. At lower molecular weights, the Western Blot technique is easily exceeded by the on-chip method. Considering that this on-chip procedure is quite easy to use, is much less time consuming than Western Blotting, and is much more sensitive at least in the low molecular weight range, the SELDI-TOF technology is a very promising approach for the screening of autoantibodies in autoimmune diseases. Due to its versatility, this on-chip technology could allow the large-scale screening for complex autoantibody and protein distributions for diagnostic purposes and early detection of autoimmune diseases could be made possible, e.g. in glaucoma. POSTER SESSION

P-118 Multiple Sclerosis: A Proteomics Approach to Identify New Markers of Disease

A. M. Jacob1, T. Ziemssen2, F. Weber1 and C. W. Turck1 2MPI of Psychiatry, 2MPI of Neurobiology, Munich, Germany

Multiple sclerosis (MS) is a chronic inflammatory, demyelinating disorder of the CNS white matter with heterogeneous pathology. Extensive studies have been done on the pathogenesis of MS using animal models. The most widely used MS autoimmunity model, Experimental Autoimmune Encephalomyelitis (EAE), shares many features with human MS. EAE is induced in animals by active or passive immunization with myelin antigens. Our goal is to identify new disease markers that are relevant in the pathogenesis of MS employing proteomics methods, namely two dimensional gel electrophoresis and mass spectrometry. Presently, we are comparing protein expression in brain and spinal cord tissues from different mouse EAE models and appropriate controls. In the future, we plan to extent these studies to different stages of the disease. In addition, we will analyze human specimen such as cerebrospinal fluid, brain and spinal cord tissue (post mortem) in order to characterize the different types and stages of the disease. The data presented are from a preliminary proteomic analysis of brain and spinal cord tissue from mice with passive EAE induced by the administration of Th1 cells reactive to myelin oligodendrocyte glycoprotein (MOG). POSTER SESSION

P-119 Proteome Analysis of Regulatory T Cells – Promising Targets for Innovative Immunotherapeutical Approaches

P. Lutter1, H. Jonuleit2, E. Schmitt3, S. Bailey1, H.E. Meyer1,2, C. Huels1 and P. Weingarten1

1Protagen AG, Dortmund, Germany; 2Department of Dermatology, University of Mainz, Germany; 3Institute of Immunology, University of Mainz, Germany; 4Medical Proteom-Center, Ruhr-University Bochum, Germany

Several mechanisms control the discrimination between self and non-self, including the thymic deletion of auto-reactive T cells and the induction of anergy in the periphery. In addition to these passive mechanisms, evidence has accumulated for the active suppression of auto- reactivity by a population of regulatory T cells that co-express CD4 and CD25. Regulatory T cells play a central role in the maintenance of the immunological balance and are powerful inhibitors of T cell activation both in vivo and in vitro [1]. The enhancement of suppressor-cell function might be a target for immunotherapeutic approaches for the treatment of immune- mediated diseases like multiple sclerosis and Crohn's disease. By contrast, the elimination of regulatory T cells or the inhibition of their functions might prove to be beneficial for the induction of tumour immunity or the treatment of chronic infectious diseases. To elucidate the still unclear effector functions of regulatory T cells we performed differential proteome analyses with diverse human and murine T cell populations. The whole protein extracts of conventional and regulatory T cells of four individual human donors and BALB/c mouse pools were separated by high resolution two dimensional gel electrophoresis according to Klose [2]. The proteomes of resting as well as anti-CD3/CD28 stimulated CD4+ and CD4+CD25+ T cells were compared. The identification of candidate proteins which are of functional relevance for both human and murine regulatory T cells contributes to the understanding of the causes and mechanisms involved in autoimmune diseases, allergy, and cancer and will lead to the development of new drugs to manipulate the activity of regulatory T cells.

References: [1] Jonuleit H., Schmitt E., Kakirman H., Stassen M., Knop J., Enk A.H. (2002): Infectious Tolerance: Human CD25+ Regulatory T Cells Convey Suppressor Activity to Conventional CD4+ T Helper Cells. J. Exp. Med. 196: 255-260 [2] Klose, J. (1999): Large-gel 2-D electrophoresis. Methods Mol. Biol. 112: 147-172 POSTER SESSION

P-120 Identification of the Profile of Disease-Associated Humoral Antigens

M. Schaffrik1*, J. Rauch1*, M. Ahlemann1*, M. Münz2, D. Eberle1, B. Mack1, S. Lang1, R. Zeidler3 and O. Gires1,2 1Department of Head and Neck Surgery, Ludwig-Maximilians University of Munich, Munich, Germany; 2Clinical Cooperation Group Molecular Oncology, GSF-Research Center for Health and Environment and Department of Head and Neck Surgery, Ludwig-Maximilians University, Munich, Germany; 3Vaecgene Biotech Inc, Munich, Germany * These authors contributed equally

Pathogens such as bacteria and viruses as well as malignant cells trigger the immune system to select for and amplify disease-specific antibodies to counteract the developing condition. Here, we describe an innovative technology, which takes advantage of the presence of disease-specific antibodies in the serum of patients. The technology termed AMIDA (Autoantibody-Mediated IDentification of Antigens) is based on the immunoprecipitation of disease-specific antigens by autologous serum antibodies. Separation of antigens by two- dimensional gel electrophoresis is followed by their identification via mass spectrometry. We have successfully applied AMIDA on patients suffering from head and neck squamous cell carcinomas. Among 32 antigens identified, we found 15 known autoantigens and tumor- associated proteins, and 17 novel candidate antigens, which had not been isolated by alternative techniques such as SEREX, antibody repertoire fingerprinting or other methods. The novel antigens include seven proteins of unknown function, which had not been recognized as tumor-associated antigens before. Validation of one of the antigens identified, cytokeratin 8 (CK8), revealed its ectopic expression pattern on the surface of primary carcinomas and the strong prevalence of CK8-specific antibodies in the serum of carcinoma patients already at early disease stages. As a promising new technology, AMIDA can foster significant improvements with respect to diagnosis and therapy of human diseases, which elicit a humoral immune response. POSTER SESSION

P-121 Subproteomic Analysis in Nickel Allergy: New Metal-protein Interactions in Human Antigen-presenting Cells

C. Junkes1, K. Heiss1, N. Guerreiro1, D. Wild1, H.U. Weltzien1 and H.-J. Thierse1 1Max-Planck Institute for Immunobiology, Freiburg, Germany and 2Novartis Pharma AG, Muttenz, Switzerland

Objective: In humans, nickel (Ni) is the most common metal allergen causing allergic reactions in about 10-15% of the human population, but the molecular events underlying this disease still have to be elucidated. Low-molecular-weight agents, such as Ni, cobalt and copper, which by definition are non-antigenic in the free state (hapten), have the potential to interact with serum or cellular proteins in a way which renders them antigenic. We have previously demonstrated that such nickel-protein-interactions, e.g. with human serum albumin (HSA), which occurs in enormous concentrations in human skin, might lead to functional metal-protein complexes. These extracellular Ni-protein complexes are able to induce and activate Ni-specific CD4+ and CD8+ human T cell clones of Ni-allergic patients. T cell receptor (TCR) - transfected cell lines are also activated in a Ni-dependent and HLA-restricted manner. With the aim of identifying so far unknown cellular nickel-protein interactions and/or nickel-binding proteins in human antigen presenting cells (APC), we used a human B cell line as a model system for a subproteomic approach. Methods: After cell lysis, Ni-interaction molecules on/in human antigen presenting cells were detected via nickel-NTA-enrichment, 2D electrophoresis, mass spectrometry and database analysis. If possible, data were confirmed by Western blotting, graphite furnace atomic absortion spectometry and/or Biacore analysis. Results and conclusion: More then 20 new potential nickel-interaction and/or nickel-binding proteins were identified in human B cells. Among others, some chaperones/heat shock proteins were detected. Possible consequences of those Ni-protein interactions will be discussed with respect to the development and pathophysiology of T cell mediated human nickel allergy.

Supported by the BMBF, Klinische Forschergruppe, FKZ 01GC9701/7 POSTER SESSION

P-122 Investigations into the Processing of Phl p 6, a Major Grass Allergen on the Mucosal Interface

C. Blume1, B. Lindner2, W.M. Becker1 and A. Petersen1 Research Center Borstel, 1Biochemical and Molecular Allergology and 2Biophysics, Borstel, Germany

Allergic disorders e.g. hayfever and asthma result from sensitisation by specific molecules from allergen sources. Up to 40% of patients with type 1 allergy are sensitive towards grass pollen allergens. Whilst pollen extracts contain over 1000 different components, only a f e w result in allergic responses and are thus allergens. Timothy grass pollen (Phleum pratense) is one of the major allergen sources in Europe, containing 13 different groups of allergens. Each allergen group displays structural microheterogeneity resulting from single amino acid residue exchanges and/ or glycosylation. Here we wished to study the degradation of allergenic molecules, in order to establish whether there are differences between the processing of these molecules between allergic and non allergic individuals. The major allergen Phl p 6 is used here as a model allergen. The initial work was to optimize the conditions and sensitivity of detection of the cleavage products. Natural Ph1 p 6 consists of at least seven isoforms as identified by ESI-MS and was compared with a single recombinant isoform of Phl p 6. In order to confirm that the recombinant material behaves in the same manner as the naturally occuring material both were subjected to digestion by trypsin and separated by RP-HPLC. The resulting chromatograms where similar and the MALDI-TOF analysis resulted in the detection of most of the theoretically predicted 12 tryptic fragments. We are in progress to examine the digestion of recombinant Phl p 6 by different body fluids e.g. nasal secretion, bronchial lavage fluid, gastric fluid and duodenal fluid utilising RP-HPLC and ESI-MS in order to establish which fragments are generated. This will allow us to identify if there are differences in the enzyme activity between allergic and non allergic individuals as well as which of these fragments cause mediator release and thus an allergenic response. POSTER SESSION

P-123 Partial Characterization of Inflammation Modified MRP8 and MRP14 Proteins in Human Plasma

A. Sünter University of Tartu, Institute of General and Molecular Pathology, Department of Human Biology and Genetics, Estonia

The S100 myeloid-related proteins MRP8, MRP14 and their heterocomplex MRP8/14 (calprotectin) are Ca-binding proteins that have been shown to have a role in inflammatory responses. The molecular structure of MRP8 and MRP14 proteins as well as the gene organization is now well known. Although there are a number of hypotheses, the exact functions of these proteins remain unknown. Several authors have suggested the clinical relevance of calprotectin in a number of inflammatory diseases. MRP proteins reflect the granulocyte activation and hence might serve for disease activity measurement. Conventional markers (CRP level etc.) lack the required sensitivity and specificity due to the fact that these parameters reflect systemic rather than local activity in the inflammatory process. Immunoblotting with our own monoclonal antibodies (13CFP 8F7 and 13CFP 12D3) demonstrated the variety of complexes in plasmas of patients with inflammation. This variety in complex formation is caused probably by heterogeneity of monomers modified in inflammatory area. The 2D electrophoresis demonstrates the heterogeneity of MRP monomers modified by inflammation. These two IgG1 type monoclonal antibodies 13CFP 8F7 and 13CFP 12D3 bind to protein complexes of MRP8 and MRP14. Both antibodies react only with modified proteins in inflammatory area, not with unmodified MRP8, MRP14 and their complexes (MRP proteins). I compared the commercially available MRP complex specific monoclonal antibodies (MAC 387, 27E10) with the monoclonal antibodies developed in our laboratory (13CFP 8F7 and 13CFP 12D3). The antigenic determinant recognized by 13CFP 12D3 is overlapped by antigenic determinant recognized by MAC 387 and different from determinants recognized by monoclonal antibody 27E10. The antigenic determinant recognized by monoclonal antibody 13CFP 8F7 is unique. From all the antibodies used (Mac 387, 27E10, 13CFP 12D3, 8-5C2, S36.48) only one - monoclonal antibody 13CFP 8F7 appears to be a good marker for monitoring disease in acute pancreatitis patients with severe inflammation. This antibody binds to unique complexes present in plasma of pancreatitis patients with severe inflammation. I suppose that MRP proteins in plasma carry a lot of information about the processes in inflammation area. Using proteomic methods we can read the information saved in MRP protein molecules. POSTER SESSION

P-124 Proteomic Profiling of Platelet Membrane Rafts

M. Foy, A. Treumann, D. Kenny, D.J. Fitzgerald and P.B. Maguire Department of Clinical Pharmacology, Royal College of Surgeons, Dublin, Ireland

Lipid rafts are compositionally distinct membrane microdomains consisting of dynamic assemblies of cholesterol, sphingolipids and a specific subset of proteins. They are more ordered in structure than the remainder of the plasma membrane. Functionally, they are believed to act as platforms for signal transduction by selectively attracting certain proteins while excluding others in response to intra- or extracellular stimuli. Evidence exists that indicates that lipid rafts govern the general processes that contribute to platelet activation mechanisms, however, the exact role of platelet lipid rafts remains to be clarified. In this study, we have employed a comprehensive proteomic profiling of lipid rafts from both control and activated platelets in order to attain a more functional understanding of these domains. Lipid rafts were isolated from control and VWF/ristocetin activated platelets using sucrose gradient ultracentrifugation. Fractions were separated using one-dimensional SDS-PAGE and then probed for the presence of flotillin-1, a known lipid raft marker. Coomassie stained protein bands from control and activated lipid raft fractions were then excised, digested with trypsin, and proteins identified using liquid chromatography-tandem mass spectrometry. Using this approach, 142 proteins were identified in control and activated raft fractions. Hallmark membrane raft proteins identified include flotillin-1, lyn tyrosine kinase and stomatin. In addition, proteins common to both control and activated platelet lipid rafts included members of the src-family kinases, G proteins, tetraspannin proteolipids, aIIbb3 and CD36. Moreover, a number of proteins were found to be unique in the lipid rafts of VWF/ristocetin activated platelets. These included CD109, the glucose transporter GLUT 4, phospholipase C, 14-3-3 zeta and glycoprotein Ib-alpha, the receptor for VWF. Changes in the proteome of platelet lipid rafts upon activation suggest that these dynamic membrane domains may act as concentrating platforms that co-cluster receptors and signalling molecules, thus ensuring efficient, co- ordinated platelet activation processes. POSTER SESSION

P-125 Functional Proteomics of the Platelet; Identifying Novel Pathways of Atherothrombosis

P.B. Maguire, J. Coppinger, G. Cagney, D. Harney, M. Foy, A. Treumann, K. Wynne and D.J. Fitzgerald Inst. of Biopharmaceutical Sciences, Royal College of Surgeon’s in Ireland, Dublin 2 Ireland

Platelets play a key role in the initiation of arterial thrombosis, and therefore in the occurrence of myocardial infarction and stroke. As anucleate cell particles however, they do not lend themselves to analysis by traditional cell and molecular biology. In contrast, proteomic analysis offers unprecedented opportunities to investigate platelet biology and there have been many studies globally analysing the platelet proteome. However, the number of platelet proteins identified to date is relatively low, as these global expression maps preclude the detection of low abundance proteins of biological interest. We have focused on specific platelet subproteomes in an effort to generate biologically meaningful datasets. These include the phosphotyrosine proteome (over a time course of activation), the membrane lipid raft signaling domains and the proteins that are secreted in response to thrombin activation. We used immunoprecipitation to capture the dynamic phosphotyrosine signaling events occurring in the platelet over a time course of thrombin-activation and differences in the resulting phosphoproteomes were identified by MALDI-TOF and Q-TOF mass spectrometry. Several previously unknown tyrosine phosphorylated proteins were discovered, including the non-muscle myosin heavy chain type IIA. The phosphorylation site was partially characterized by immunoblotting for tyrosine phosphorylation of peptides seen on cyanogen bromide digestion. To further enrich for signaling proteins we isolated the membrane lipid-raft domains, which are believed to act as platforms for signal transduction by selectively attracting certain proteins while excluding others. Using Q-TOF MS, we achieved a proteomic profile of over 80 proteins in the lipid rafts of control and over 100 proteins in the lipid rafts of ristocetin- activated platelets and have demonstrated that the platelet glycoprotein Ib-IX-V complex and a host of signaling proteins including several hypothetical proteins are recruited to lipid rafts upon platelet activation. Additionally, we characterised the secreteome of thrombin-activated platelets, as platelet secreted proteins can adhere to the arterial wall and promote the development of atherosclerosis and thrombosis. We used a multi-layered proteomics approach of 2D electrophoresis and multi-dimensional chromatography both coupled to MS and identified over 300 proteins secreted by human platelets, many mapping to EST’s of unknown function. Several of the previously unknown secreted proteins were localized in platelets on immunofluorescence and released upon activation, and while absent in normal vasculature, were identified in human atherosclerotic lesions. These proteomics approaches to understanding the signaling and secretion events leading to and following platelet activation have revealed a wealth of platelet proteins, which in the future, may prove suitable as therapeutic agents or novel targets for the treatment of atherothrombosis. POSTER SESSION

P-126 Proteomic Analysis of Platelet Derived Microvesicles and Exosomes

N. O’Donoghue, K. Culligan, J. Coppinger, N. Moran, D.J. Fitzgerald and P.B. Maguire Dept. of Clinical Pharmacology, Royal College of Surgeons In Ireland, Dublin, Ireland

In addition to the release of soluble proteins and mediators from internal granules, activated platelets can also release two distinct membrane vesicle populations into the external environment. Cell surface-derived microvesicles have a protein content similar to the activated plasma membrane and exhibit procoagulant and inflammatory functions. In contrast, exosomes of endocytic origin are released by the fusion of subclasses of α-granules and multivesicular bodies (MVBs) and contain the tetraspan protein CD63, however their function remains unknown. Interestingly, exosomes are also secreted by a multitude of other cells including those of hemopoietic lineage, where they are thought to play an immunoregulatory intracellular role. Exosomes have an internal diameter of 40 - 100nm whereas microvesicles have an internal diameter of 100nm – 1µm. This difference in size was exploited in this study to separate and isolate intact microvesicles and exosomes from thrombin-activated platelets using sucrose density ultracentrifugation. Western blot analysis for the plasma membrane marker GP1bα and the tetraspan CD63 confirmed the presence of isolated microvesicles and exosomes respectively. Microvesicle and exosomal fractions were separated using one-dimensional SDS-PAGE and the resulting coomassie-stained protein bands were trypsin digested and analysed by both MALDI-TOF and Q-TOF mass spectrometry.

Platelet specific proteins such as GPIbα and αIIbβ3 were identified in platelet microvesicles. Proteins identified in platelet exosomes include thrombospondin, multimerin, latent transforming growth factor beta binding protein and entactin. In addition, several of the proteins identified in platelet exosomes are prothrombotic e.g., Factor V, while others are immune modulators e.g., platelet factor 4 (PF4) and platelet basic protein. Thus, platelet microvesicles and exosomes contain factors of major significance in the development of atherosclerosis and thrombosis. In the future, we intend to characterize the membrane proteomes of platelet-derived microvesicles and exosomes using quantitative mass spectrometry techniques including ICAT. POSTER SESSION

P-127 Platelet Proteomics: a Tool to Analyse Age-related Changes in the Protein Expression Profile

M. Zellner, W. Winkler, M. Wei-fen Chang, J. Grillari and R. Oehler Surgical Research Laboratories, University of Vienna, Austria

Rationale: Increased reactivity of blood platelets can be observed in a number of different disease states including arteriosclerosis, diabetes, and obesity as well as in aged persons. The balance between reactive oxygen species (ROS) and the anti-oxidative capacity has a regulatory role in the activation of platelets. In addition, this balance has strong effects on the expression and modification of cellular proteins. The present study determines whether platelet proteomics is suited to analyse the effect of age-related changes in protein expression and modification. Methods: Proteomics is the analysis of the entire protein complement expressed by a cell. To analyse the proteome of platelets we isolated platelets from peripheral venous blood of 15 aged (mean age = 85) and 15 young (mean age = 25) subjects. The cellular proteins were separated by 2D flurescence difference gel electrophoresis system. The 2D-patteren was analysed by the specialised software DeCyder. Results: The two-dimensional gel electrophoresis showed up to 1800 protein spots. About 1400 spots were detected repeatedly in every gel of the same group. The comparison of the two groups revealed several distinct changes. The list of the changes will be presented. Conclusion: The present study shows that platelets from aged subjects have a different protein expression and modification profile in comparison to young control. Thus, platelet proteomics is a suitable tool to analyse age related changes in proteins. The identity of the proteins and their potential relationship to the oxidative balance within platelets will be determined in future studies. POSTER SESSION

P-128 A Proteomic Approach to the Study of Human Intra-Abdominal Adipose Tissue: Implications for Pathologies Associated to Insulin Resistance

M. Cortón1, G. Villuendas2, J.I. Botella2, J.L. San Millán2, H.F. Escobar-Morreale2 and B. Peral1 1Instituto de Investigaciones Biomédicas, CSIC-UAM, Madrid, Spain, and 2Departments of Molecular Genetics and Endocrinology, Hospital Ramón y Cajal, Madrid, Spain

The polycystic ovary syndrome (PCOS) is one of the most common pathologies in women of fertile age, with a 6’5% prevalence in Spain. PCOS is defined by oligo-ovulation and hyperandrogenism, and is considered not only a reproductive endocrinopathy but also a metabolic disorder. It is associated with insulin resistance, hyperinsulinemia, glucose intolerance, obesity, and an altered lipid profile. The reasons for metabolic disorders in PCOS have not been completely elucidated and there are not explanation to the fact that only some obese insulin resistant women develop this endocrinopathy. One of our objectives is the application of a proteomic approach, combining two-dimensional polyacrylamide gel electrophoresis (2-DE) and mass spectrometry (MALDI-TOF MS), in order to address whether in the adipose tissue of obese women with and without PCOS there are differentially expressed proteins. Here we present a novel protocol for preparation and solubilization of human intra-abdominal adipose tissue in order to separate larger amounts of proteins in 2-DE gels by using different range immobilized pH gradient (IPG) strips. Proteins from this tissue show some difficulties for solubilization due to the presence of great amounts (often >50% of the tissue volume) of triglycerides. Samples were obtained from morbidly obese women submitted to surgical treatment for obesity. The homogenisation is performed using a Polytron PT-1200C directly in the lysis buffer: 8.4 M urea, 2.4 M thiourea, 5% CHAPS, 50mM DTT and 1% IPG buffer 3-10 (total volume 2 mL/g fat pad). The resolution of 2-DE has been significantly improved when adding hydroxyethyl disulfide (DeStreakTM ) to the rehydration solution at alkaline and wide pH interval 3-10, together with application of sample by cup-loading close to the anode and by in-gel sample rehydration, respectively. The inclusion of HED instead of DTT was shown to eliminate the horizontal streaking and to simplify the spot patterns. Following these strategies we have optimized the sample preparation procedure for the proteomic analysis of human intra-abdominal adipose tissue and we have identified several proteins by MALDI-TOF MS. POSTER SESSION

P-129 Effect of Obesity on Adipose Tissue Peptide Secretion: A Proteomics Approach to Diabetes Type II

H. Roelofsen, S. Zaaijer and R.J. Vonk Department of Pediatrics, Section Nutrition and Metabolism, University Hospital Groningen, Groningen, The Netherlands.

The world wide prevalence of type II diabetes mellitus is increasing rapidly. Dietary fats and rapid digestible carbohydrates are believed to play an important role in the development of this type of diabetes. In patients with type II diabetes, insulin is less able to promote the uptake of glucose into muscle and liver and to inhibit the production of glucose by the liver. The underlying mechanisms by which diet components induce a state of insulin resistance are largely unclear. Adipose tissue is thought to play a central role in the development of the insulin resistant phenotype. During obesity adipocytes maturate (enlarge) by storing triglycerides. Besides its role in energy storage, these differentiated adipocytes function as an endocrine organ that secretes numerous peptide hormones such as leptin, resistin and TNF_. These proteins have been shown to play a role in the regulation of food intake (leptin) and the development of insulin resistance (TNF_, resistin). It is anticipated that differentiated adipocytes produce yet unknown peptide hormones that may play regulatory roles in glucose and fatty acid metabolism. The aim of the study is to determine the effects of obesity on the adipose tissue secretome using proteomics technology. Methods: Adipose tissue, isolated from control (black 6) and obese, insulin resistant ob/ob mice, was cultured for 4 days in M199 media supplemented with insulin (120 nM) and penicillin/ streptomycin. Subsequently protein profiles of the conditioned media were generated using SELDI proteinchip technology (Ciphergen biosystems). The media, diluted 2-fold in binding buffer (NH4Ac pH 4.0/0.05% Triton x-100) were applied to weak cation exchange (WCX) proteinchips and, after washing, they were analyzed by time of flight mass spectrometry. Results: Data from the 2-100 kDa range show 48 proteins that are clearly increased in the ob/ob media while 28 proteins were found decreased in the ob/ob media compared to control media. Conclusion: Results show that in total 76 proteins in the range of 2 to 100 kDa have altered secretion patterns when comparing adipose tissue from obese and lean mice indicating that major changes take place in the adipose tissue secretome during obesity. The discovered proteins have to be further identified to determine their exact function and role in the development of diabetes type II. POSTER SESSION

P-130 Covalent Fluorescent Inhibitors for Isolation of Lipolytic Enzymes of Mouse Adipose Tissue

R. Birner1, H. Susani-Etzerodt1, G. Rechberge2, M. Kollroser3, R. Zimmermann2 and A. Hermetter1 1Institut für Biochemie, Technische Universität Graz, Austria; 2Institut für Molekularbiologie, Biochemie und Mikrobiologie and 3Institut für Gerichtsmedizin, Karl-Franzens-Universität Graz, Austria

In western Europe, more than 50% of the population is overweight, and throughout the world about twice as many people (approx. 15 million per year) die from cardiovascular diseases such as heart attack and stroke as die from cancer. Dysregulation of the vascular and cellular metabolism of lipids and the excessive deposition of neutral lipids in adipocytes and the arterial wall are causally involved in the pathogenesis of atherosclerosis and obesity. The goal of our joint project is to discover novel genes, processes and pathways that regulate lipid homeostasis in humans, mice and yeast being a prototype model organism in lipid metabolism. The contribution of our group in the joint project is proteome screening with specific suicide lipase inhibitors which are fluorescently labelled. Since the inhibitors covalently bind to lipases which are serine hydrolases, the enzyme-inhibitor complexes can be isolated by 1D- or 2D-gelelectrophoresis, detected with a laser scanner and analysed by mass spectrometry (nano-HPLC-iontrap) after tryptic in gel digestion. Screening of the proteome of mouse adipose tissue with O-((6-(7-nitrobenz-2-oxa-1,3-diazol- 4-yl)amino)hexanoyl)aminoethyl-O-(p-nitrophenyl) n-hexylphosphonate (NBD-HE-HP) led to the detection of lipase-, esterase- and (lyso-)phospholipase-type enzymes. Next to known enzymes, also yet uncharacterised proteins were identified. Our results indicate that the fluorescent suicide inhibitor NBD-HE-HP specifically binds to lipolytic enzymes and can therefore be used as a powerful tool for screening for lipolytic activities in complex biological samples.

This work is financed by the GOLD (Genomics of Lipid-associated Disorders) project (http://gold.uni-graz.at/) which is one of four joint GEN-AU (GENome research in AUstria) projects (http://www.gen-au.at/) funded by the Austrian Federal Ministry for Education, Science and Culture. POSTER SESSION

P-131 A Comparison of IgG Serum Autoantibodies Patterns Against Retinal, Optic Nerve, and Optic Nerve Head Antigen in Patients with Glaucoma

S.C. Joachim, F.H. Grus and N. Pfeiffer Dept. of Ophthalmology, University of Mainz, Germany

Glaucoma is one of the leading causes of blindness worldwide. There is evidence that an autoimmune mechanism is involved in the development of glaucoma in several patients. The aim of this study was to compare the autoantibody repertoires against different antigens (retina, optic nerve, and optic nerve head) in sera of glaucoma patients and healthy subjects. A total of 66 patients were divided into three groups: healthy volunteers without any ocular disorders (CO, n=30), patients with primary open-angle glaucoma (POAG, n=19), and normal tension glaucoma (NTG, n=17). The sera of patients were tested against Western Blots of retinal, optic nerve, and optic nerve head antigens. Immunodetection was performed by using 4-chloro-1-naphthol staining. The IgG autoantibody patterns were digitized and subsequently analyzed by multivariate statistical techniques. All patients showed complex patterns of autoantibodies against retinal, optic nerve, and optic nerve head antigens. The analysis of discriminance revealed a statistical significant difference between the patterns of all groups. Our multivariate approach could quantify the differences in immunoreactivities of patient sera against the different antigens. The POAG group had the most significant difference against retinal antigen (P=0.0021) compared to the other antigens. In the NTG group the highest reactivity appeared against optic nerve head (P=0.00053) and optic nerve (P=0.0025). In this study we could demonstrate that all groups show different autoantibody patterns against the three ocular antigens. These autoantibodies are highly specific for each patient group. Thus, further analysis of autoantibody pattern could provide important information to learn more about the role of autoimmune mechanisms in the pathogenesis of both POAG and NTG. POSTER SESSION

P-132 Investigating Rat Retinal Proteins using a MALDI TOF MS and MALDI QIT TOF MS

R. Martin1, S. Wallenborg2, H. Matsumoto3, S. Kurono3, N. Komori3 and H. Klement4 1Shimadzu Biotech, Manchester, UK; 2Gyros AB, Uppsala, Sweden; 3Dept Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA; 4Shimadzu-Biotech, Duisburg, Germany

Introduction: Several years ago an "ocular proteomics” project was initiated, the intention of which was to catalogue all of the proteins expressed in bovine retinas [Nishizawa et al , Exp.Eye Res., 1999, 69, 195-212]. Recently a cataloguing effort on retinal proteins from mice and rats has been attempted. These rodents have been used extensively in laboratories as a model system and their genome projects are nearly completed, thus serving as excellent models for ocular proteomics studies. Increasingly, large numbers of protein samples are being investigated and this large volume of samples in turn introduces a number of limiting steps in the work flow. A novel CD based system has recently been introduced that is capable of performing sample clean-up, concentration and elution onto an incorporated area specifically designed for MALDI acquisition. Advantages of this methodology include high reproducibility, increased sensitivity and reduced contamination. We have applied this new technology in combination with two MALDI based mass spectrometers for the identification of proteins from rat retinal extracts. The first series of analyses aimed to identify as many proteins as possible by peptide mass fingerprinting and database searching using both a curved field reflectron MALDI mass spectrometer and a MALDI QIT TOF MS. Additionally, identification of the protein samples was aided or confirmed by analysis on a novel MALDI- quadrupole ion trap-time of flight system providing important MS/MS information used to identify proteins by database searching of fragmentation data. Methods: Protein samples extracted from rat retina were processed by isoelectric focusing (IEF) gel and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as previously described [Matsumoto and Komori, Methods Enzymol. 2000, 316, 492-511]. The protein spots then underwent in-gel digestion with trypsin. The resultant digests were divided into two aliquots, dried down and stored at –20°C. One set of samples was then reconstituted in 5 µl of 0.1% TFA and processed on the GyroLab Workstation (Gyros AB, Uppsala, Sweden), resulting in a CD containing desalted tryptic digests deposited with CHCA matrix (1mg/ml in 0.1% TFA/50% acetonitrile with 0.1 % octyl glucoside and 1mg/ml fucose). The CD was introduced into the mass spectrometers using a specially designed MALDI target adaptor. The second set of samples were reconstituted in 5 ul 0.1% TFA, desalted and a 0.5 ul aliquot of each sample spotted directly onto a 384 well stainless steel MALDI target with an equal volume of dihydroxybenzoic acid matrix (12.5 mg/ml in 0.1% TFA/50% acetonitrile). To generate peptide mass fingerprints, an Axima CFRplus MALDI TOF mass spectrometer (Shimadzu Biotech) incorporating a curved field reflectron was used in positive ion reflectron mode. In addition, a MALDI QIT TOF MS [Martin and Brancia, Rapid Commun. Mass Spectrom., 2003, 17,1358-1365], the Axima QIT (Shimadzu Biotech) was used to generate peptide mass fingerprints and MS/MS data for protein identity confirmation. Both instruments can accommodate the same target format and therefore samples could be analysed on one system and subsequently on the alternative system with out the need to reprepare samples. For all database searches, Mascot (Matrix Science) was employed on an internal server. Results: A total of 30 rat retinal tryptic digests were analysed using a combination of MALDI TOF MS and MALDI QIT TOF MS using both the CD based technology and more conventional MALDI preparation techniques. All but one of these samples were identified via database searching both PMF and MS/MS data. Comparable PMF data was acquired on both MS systems. In the cases where the PMF approach was not successful, (due to a number of possible factors including few peptides in the sample or low protein concentration), MS/MS experiments proved to be extremely useful in aiding identification. This is mainly a consequence of the high resolution and mass accuracy of the fragmentation spectra obtained on the MALDI QIT TOF MS. Additionally, it is possible to select ions for MS/MS experiments with very high resolution, allowing one to gain sequence information about individual peptides that may be extremely close in nominal mass. The combination of the MALDI ionization source and quadrupole ion trap also permit the use of higher mass peptide ions (up to 5000 Da) in MS/MS experiments. POSTER SESSION

P-133 Two-dimensional Difference Gel Electrophoresis and Mass Spectrometry Identify Proteins Involved in Adult Retinotopic Map Reorganization

G. Van den Bergh, S. Clerens and L. Arckens Lab. for Neuroplasticity and Neuroproteomics, K.U.Leuven, Belgium

The induction of small but overlapping central retinal lesions produces a visually inactive zone in the retinotopic targets in cat primary visual cortex. In ensuing weeks, neurons within this so-called ‘cortical scotoma’ regain visual responsiveness, but receive input from the perilesional retina. In this study, the molecular mechanisms underlying this form of cortical retinotopic map plasticity were investigated by two-dimensional difference gel electrophoresis (2-D DIGE) and mass spectrometry. Cortical grey matter was collected from the ‘cortical scotoma’ and from more peripheral parts of area 17, and a 2-D DIGE comparison was made at post-lesion survival times of 3 days, 14 days and 1 month. Surprisingly, no differences in protein expression were observed at any of the survival times investigated. A comparison of central and peripheral regions of area 17 in normal control animals also did not result in the detection of protein expression differences in function of visual eccentricity. Because at the cortical level the border surrounding the scotoma is believed to play a principal role in the reorganization process, we then compared the protein patterns from this small rim around the cortical scotoma with the corresponding cortical region of normal, control animals. Two protein spots showed a clear difference in protein expression and were subsequently identified by Q-Tof MS. One was more abundantly expressed in the normal cortex and may present a plasticity suppressor protein. The second protein showed an increased expression in the scotomal border region and points towards a change in the regulation of axonal transport and the stability and formation of new synapses during retinotopic map plasticity. POSTER SESSION

P-134 Proteome Analysis of the Regenerating Retinas in Marmosets (Callithrix jacchus)

Karin Rose1, Martin Zeller2, Simone König2 and Solon Thanos1 1University Eye Hospital Münster, Dept. of Experimental Ophthalmology; and 2Integrated Functional Genomics, Interdisciplinary Clinical Research Facility, Münster, Germany

The adult retina serves as a model to study neuronal survival and axonal regeneration after injuries. In order to examine the posttraumatic ability to regenerate axons and form new connections, an organ culture system of monkey retina was established. Fresh retinas obtained from monkey cadavers of various ages were explanted with the ganglion cell layer facing the laminin-1 (α1β1γ1) coated surface (regenerative group), whereas explants with no contact to laminin-1 and non-explanted retinas served as controls (non-regenerative group). The present study intends to investigate whether the initial axon growth in vitro is associated with the synthesis of new or modulation of pre-existing proteins that can be detected by 2D- gel electrophoresis and proteome analysis. To elucidate the mechanisms involved in regeneration, a mapping for basic information and a comparative proteome analysis of the regenerating retina was carried out to screen for differentially expressed proteins. For this, total protein extracts from both groups were separated by 2D-PAGE, the protein patterns were analysed, and spots of interest identified by Maldi-MS. Up to one thousand spots could be separated by 2D electrophoresis and 25 of them were identified by Maldi-MS, generating landmarks for ongoing differential proteome analysis. So far as analysed, several proteins were differentially expressed depending on the age of the animal or regeneration status. Among others, crystallins such as alpha-crystallin seem to be discernible and selectively upregulated after axonal growth. Ongoing analysis aims at characterizing further candidates associated with axonal growth. The relevance of these candidates for the regeneration process and growth cone formation remains to be discussed and may provide a deeper understanding of neuronal regeneration.

Supported by the Deutsche Forschungsgemeinschaft and the IZFK (Project F5) POSTER SESSION

P-135 Proteomics of the Thyroid: Protein Identification and Expression Profiling

K. Berger1, J.-D. Wissmann2 C. Ihling3, A. G. Beck-Sickinger2, A. Sinz3, and D. Führer1 1Department of Internal Medicine III, University of Leipzig; 2Institute of Biochemistry, University of Leipzig; 3Biotechnological-Biomedical Center, University of Leipzig, Germany

Nodular or multinodular goiter is the most neoplastic thyroid disease and can be found in up to 47 % in people living in iodine-deficient areas; 5 % turn to malignancy and represent cancer. To date, preoperative diagnosis is often difficult as thyroid adenomas and carcinomas share certain cytological features. Since malignancy is associated with quantitative and qualitative alterations in proteins, e.g. change of expression levels or post-translational modifications, proteomic studies of thyroid tissue has the immense endowment to produce diagnostic fingerprints of the protein expression levels of benign and malignant thyroid nodules. Hence, the power and flexibility of proteomic approaches should hasten our understanding of specific processes at the protein level. This work reports on the establishment of quantitative proteomics of cytosolic proteins of the human thyroid. Cytosolic proteins from nodular and healthy thyroid tissue were extracted and separated by pH 4-7 and pH 3-10 gradient strips and 8-16 % SDS gradient gels. For quantification of differently expressed proteins we show that the fluorescent dye Ruthenium II ( tris bathophenanthroline disulfonate) (RuBPs) is an attractive method for protein expression profiling in proteomics. First, because of its high sensitivity, which is comparable to that of Sypro Ruby. Second, because of its broad linear range, which is orders of magnitudes higher than that of silver stain, thus allowing detection of low-abundant proteins, and third, because of its good compatibility with mass spectrometry. Prominent spots were picked from 2-D gels, in-gel digested with trypsin, and the proteins were identified by nano-HPLC/nano-ESI FTICRMS and MALDI-TOFMS. In this work we show that proteome analysis, the study of quantitative and qualitative alterations of protein expression, may benefit to our understanding of the aetiology of thyroid diseases. Thus, our future work will concentrate on delineation of protein expression in different thyroid pathologies. The proteomics approach described herein permits a direct assessment of the biologically as well as pathophysiologically relevant specific protein expression in thyroid pathology. POSTER SESSION

P-136 Exploring Nuclear Gene Defects in Mitochondrial Disorders Using Comparative Proteomics

D. Molina1, M. Jaksch2, A. Lamanda3, S. Gallati1 and A. Schaller1 1Division of Human Genetics, University Children’s Hospital, Bern, Switzerland; 2Metabolic Disease Centre Munich, Germany; 3Department of Chemistry and Biochemistry, University of Bern, Switzerland

Leigh Syndrome (LS) is a neurodegenerative disorder mostly based on neuroradiological findings of bilateral symmetric lesions in basal ganglia and in the brainstem, reflecting focal necrotic areas on pathological examination. However, the genetic causes of LS are extremely heterogeneous. The syndrome has been mainly attributed to deficiencies of the mitochondrial energy metabolism such as respiratory chain (RC) defects, ATPase or pyruvate dehydrogenase malfunctions. Among these, failure of cytochrome c oxidase (RC complex IV; COX) is relatively frequent. RC deficiencies are not only caused by mutations in genes coding for the structural subunits of the respiratory chain themselves, but also in nuclear genes coding for proteins involved in assembly and biogenesis of the complexes. Here, a proteomic approach was used to investigate whether such mutations would affect the pattern of mitochondrial proteins at a broader level. Purified mitochondria from control cell lines and from patients with known mutations in SURF1 (COX assembly gene with unknown function) and SCO2 (encodes a copper transporter to COX) were analysed by two- dimensional electrophoresis for up- and downregulated proteins. We present a comparison of pathologic and normal results and assignments by mass spectrometry. This approach opens new insights into potential protein-protein interactions, as well as into genotype-phenotype correlation of mitochondrial disorders. POSTER SESSION

P-137 Establishment of a 2D-Electrophoresis Map of Mitochondria from Human Lymphoblastoid Cells: On a New Path to Diagnose Mitochondrial Disorders

J. Xie1, S. Techritz1, S. Haebel3, H. Neitzel2, J. Klose2 and M. Schuelke1 1Department of Neuropediatrics and of 2Human Genetics, Charité University Hospital, Berlin, Germany; 3Interdisciplinary Research Center for Biopolymers, University Potsdam-Golm, Germany

Mitochondriopathies are multisystem diseases that can be caused by any defect in the energy (ATP)-generating pathways of the mitochondria. They are difficult to diagnose and only 20% can be solved on molecular level. This warrants new diagnostic strategies. In order to be able to diagnose mitochondrial diseases by analysing deviant protein patterns, we first established a 2D-electrophoresis map of normal human mitochondria, isolated from EBV-transformed lymphoblastoid cells. Maps of mitochondrial proteins already exist from placenta and cultured neuroblastoma cells. For diagnostic purposes, however, these materials are generally not available. Cultured lymphoblastoid cells, in contrast, are suited much better for diagnostic purposes since they can be easily obtained from any patient at any age. In addition, they allow investigation of the genetic variability in a larger number of control patients. Identification of proteins was achieved by high-resolution 2D-electrophoresis coupled with peptide mass fingerprinting via MALDI-TOF mass spectrometry. After database search, we could identify a total of 95 different proteins, 74 of which were of confirmed mitochondrial origin. The identified proteins are components of the main biological pathways located in the mitochondrion. 16 of the identified proteins belonged to the respiratory chain. Despite the fact, that 18 proteins were annotated in SWISS-PROT as ”membrane associated proteins”, only four of them had putative transmembrane domains. None of the 13 proteins encoded by the mitochondrial DNA could be identified. These proteins are highly hydrophobic membrane proteins, which also escaped detection by other research groups. Their identification on a 2D-electrophoresis gel poses a challenge for the mitochondrial research community. POSTER SESSION

P-138 Interaction of the Slit Diaphragm Proteins Densin and Nephrin in the Kidney Glomerular Filtration Barrier

E. Heikkilä, H. Ahola, E. Åström, I. Izawa, M. Inagaki and H. Holthöfer Biomedicum Helsinki, Molecular Medicine, University of Helsinki, Finland

The ultrafiltration barrier in the kidney glomerulus consists of three distinct layers including the fenestrated endothelium, the glomerular basement membrane (GBM) and the visceral epithelial cells, podocytes. Podocytes cover the entire urinary space aspect of the GBM with secondary cellular processes called foot processes. They are connected to each other by specialized cell-cell contact, slit diaphragm (SD). The SD is believed to form the crucial single part of the glomerular filtration barrier in preventing loss of circulating plasma proteins into urine. We recently reported of a novel slit diaphragm associated protein, densin, wich was discovered as a co-precipitate of nephrin, the major structural backbone of the SD. Densin had previously been found only in the postsynaptic densities in the forebrain. The localization of densin to the slit diaphragm was verified by electron microscopy. To extend the study of the in vivo complex formation of densin with nephrin the appropriate co-precipitations of human glomeruli were performed. To demonstrate a direct physical association and map the domains required for the interaction, two sets of GST-protein-protein interaction assays were performed. Both endogenous nephrin of human glomeruli and in vitro -translated intracellular part of nephrin could be successfully precipitated with a recombinant GST-fusion protein containing the intracellular part of densin. The identification of the specific motifs involved in the binding will be reported. These data demonstrate a novel in vivo interaction of densin with nephrin in the slit diaphragm of human kidney glomeruli. Further, this interaction is a direct one and involves distinct intracellular motifs of both proteins. POSTER SESSION

P-139 Mass Spectrometric Analysis of Glomerular and Pancreatic Nephrin

J. Rinta-Valkama1, A. Pätäri1, L. Valmu2, N. Kalkkinen2, H. Holthöfer1 1Department of Bacteriology and Immunology, University and University Central Hospital of Helsinki, Finland, 2Protein Chemistry Research Group and Core Facility, Institute of Biotechnology, University of Helsinki, Finland

Nephrin is a transmembrane protein of the immunoglobulin superfamily and has a crucial role in kidney filtration function. It was first found in the kidney as a 185 kDa protein, but later w e found nephrin expression also in the pancreas. Interestingly, the molecular weight of nephrin in pancreas is 165 kDa. The aim of this study was to verify by MALDI-TOF and LC-MS/MS the identity of the 165 kDa band in pancreas. Immunoprecipitation was performed from normal human pancreatic tissue lysate and normal human glomeruli lysate with anti-nephrin antibody. The appropriate protein bands were cut from the silver stained SDS-PAGE gel followed by trypsin digestion. One half of the sample was analysed with MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) and the other half with LC-MS/MS tandem mass spectrometry. The peptides obtained from glomerulus were measured with MALDI-TOF-mass spectrometer (Biflex) and used for database searches. The mass of one peptide matched with nephrin peptide spectrum, and sequencing of this peptide with LC/MS-MS revealed an amino acid sequence identical to extracellular part of nephrin. We succeeded to precipitate and identify nephrin from the human glomeruli. Verification of pancreatic nephrin is still ongoing. POSTER SESSION

P-140 Mononuclear Cell Proteins Involved in Idiopathic Nephrotic Syndrome of Childhood: A Proteomic Approach

E. Gonzalez, M. Kemper, T. Neuhaus and E. Girardin Geneva University, Genève, Switzerland

Clinical and laboratory data suggest the implication of T-lymphocytes in the pathogenesis of idiopathic nephrotic syndrome (INS) but the proteins involved in the link between T- lymphocytes and glomerular cells are still unknown. The aim of this study was to identify differences in protein expression in mononuclear cells from patients with INS during relapse and healthy patients. Method: Peripheral blood mononuclear cells were taken from 7 children with INS during relapse and 7 healthy children. Mononuclear cell proteins were solubilized and denaturated using a lysis buffer [7M urea, 2M thiourea, non-ionic detergents (2% CHAPS and 2% Zwittergen) and a reducing agent (65mM dithiothreitol)]. Isoelectrofocusing was performed using non-linear immobilized pH gradient from 3 to 10 (Immobiline DryStrips 3-10NL, Amersham Biosciences). In order to increase the definition of our gels, we performed zoom-gels using narrow pH gradients: pH 4.5 to 5.5 and 5.5 to 6.7. The second dimension was performed using precasts 12-14% gradient polyacrylamide gels (ExcelGel XL SDS 12-14, Amersham Biosciences). Analytic gels were silver stained. After scanning with a laser densitometer, gels were analysed and matched with the Melanie® Software (GeneBio, Switzerland) to highlight significant differences in protein patterns. After automatic matching of 8 to 12 gels, each group of spots was statistically analysed using the spots relative volume and manual editing was performed. For protein identification, preparative gels were stained using SYPRO Ruby. Spots with an upregulated expression in relapse were picked, destained and trypsinised. Sequencing was performed using TOFTOF-MALDI-MS/MS mass spectrometer. Results: Mass Spectrometry results: Identified proteins: L-plastin, Annexin3, Glutathione peroxidase, α-Tropomyosin, α-Enolase, Albumin, Myosin heavy chain, Heat shock cognate 71kDa protein, GAPDH, Lamin B2). L-plastin, a-tropomyosin, annexin III are involved in cytoskeleton rearrangement and increased adhesion. α-enolase, a key glycolytic enzyme, was found to be expressed on the cell surface and to bind plasminogen. It was recently found to be involved in a variety of systemic and autoimmune disorders. Transcripts of L-plastin and elongation factor 1-α2 were also found to be upregulated in PBMC in relapse (D. Sahali, J am soc nephrol 13:1238- 1247, 2002). Conclusion: (i) A recognizable pattern of proteins characterizes samples from INS patients in relapse. (ii) These data show an increase expression of mononuclear cell proteins involved in cytoskeleton rearrangement and increased adhesion in INS patients in relapse. (iiI) A proteomic approach may contribute to specify the role of mononuclear cells in the pathogenesis of INS.

(Abstract was truncated to one page) POSTER SESSION

P-141 Saliva Protein Identification Using MALDI-TOF-TOF

R. Vitorino1, M.J.C. Lobo2, J.A.R. Duarte3, A.J. Ferrer-Correia1, J. Dubin4, K.B. Tomer4, P.M. Domingues1, F.M.L. Amado1 1Department of Chemistry, University of Aveiro, Portugal, 2Instituto Superior de Saúde - Norte, Portugal, 3Sport Biology Department, FCDEF, University of Porto, Portugal, 4NIEHS, RTP, NC, USA

Saliva is a heterogeneous fluid comprising proteins, glycoproteins, electrolytes and small organic compounds. Many of these compounds are constantly being transported from blood. The determination of "salivary biomarkers” as a means of monitoring general health and for the early diagnosis of disease is of increasing interest in clinical research. Based on the linkage between salivary proteins and systemic diseases, the aim of this work was the identification of saliva proteins using proteomics. Salivary proteins were separated using 2-D gel electrophoresis over a pH range between 3-10, digested, and then analyzed by a MALDI-TOF- TOF mass spectrometer. Of the identified proteins, 20 were separated and identified in saliva for the first time using proteomics tools. Moreover, three proteins that have not been previously identified in saliva, PLUNC, Cystatin A and, Cystatin B were identified. Also of interest are the identifications of the presence of seven cystatins, lipocalin-1, prolactin and several other proteins associated with inflammatory response. POSTER SESSION

P-142 α Canine α1-Antitrypsin Isoforms

I. Miller1, A. Preßlmayer1, R. Wait2 and M. Gemeiner1 1Institut für Medizinische Chemie, Veterinärmedizinische Universität Wien, Austria; and 2Imperial College of Science, Technology and Medicine, London, UK

α1-Antitrypsin (Pi) is an important protease inhibitor, present in considerable amounts in serum / plasma of most animal species. For some species (e.g. human, rat) it is known as a positive acute phase protein. In our studies of canine serum samples (including acute and chronic inflammation, e.g. in kidney diseases or gammopathies), no upregulation was noticed, compared to healthy individuals.

In addition, α1-antitrypsin is a protein known for its polymorphism, and - especially in humans - attempts have been undertaken to find out details about isoforms and correlation to diseases. In humans, hereditary deficiency of serum α1-antitrypsin (e.g. the Z-variant) is typically associated with chronic pulmonary and hepatic diseases. Also for the dog, isoforms have been described, but only with native isoelectric focusing. The present approach shows that there are also different banding patterns in SDS-PAGE and in IEF with reducing / denaturing additives. We studied canine α1-antitrypsin isoforms in Western blots and 2-DE patterns, using the same samples. After identification by a cross-reactive anti-human antibody, the respective spots / spot groups could be clearly discerned in silverstained protein patterns (pH range 4-9 or 4-6). Three forms of α1-antitrypsin could be detected, two homozygous and the mixed form. In general, there is a clear correlation between banding patterns in SDS-PAGE (1-2 bands) and 2-DE (one or two spot groups / chains). When more α1-antitrypsin is present, 2-DE gives a clearer picture: the number of spots in the single chains increases, but the additional spots usually display either higher or lower Mr than average. Effects like this may easily obscure SDS-PAGE patterns and influence isotyping results. Thus, 2-DE produces more accurate results and may help to find out about possible correlation of isoforms and diseases. In addition, if using pH 4-9 strips, the same 2-DE separation allows studying overall protein pattern of the respective animal. POSTER SESSION

P-143 Effects of Caffeine and Low Temperature on the Entire Protein Expression Profile of BHK Cells Expressing wt- or F508del-Cystic Fibrosis Transmembrane Conductance Regulator

A. Simas1, A. Dragomir2, M. Roxo Rosa1, M. DAmaral1,3, G.M. Roomans2, D. Penque1 1Centro de Genética Humana, Instituto Nacional de Saúde Dr Ricardo Jorge, Lisboa, Portugal; 2Department of Medical Cell Biology, University of Uppsala, Uppsala, Sweden; 3Department de Química e Bioquímica, FCUL, Universidade de Lisboa, Portugal

Cystic Fibrosis (CF), the most common genetic disorder in Caucasian, is a misfolding disease since most individuals with CF carry at least one F508del-CFTR allele whose the product fails to reach the epithelial cell surface where it works as cAMP-regulated Cl- channel. Previous studies have shown that low temperature and some compounds namely xanthine derivatives can overcome the processing defect of F508del-CFTR and activate cAMP-regulated Cl- channel in CF cells. Although some explanations have been proposed for these observations, the mechanisms involved in the restoration of F508del-CFTR defect by those treatments remain to be elucidated. We have studied the effect of caffeine, a xanthine derivative, on wild type and F508del-CFTR processing and function. Using a proteomics approach, we have also investigated and compared the effects of caffeine and low temperature on the entire protein expression profile of BHK cells stably expressing wt- or F508del-CFTR. Our objective is to search for a common group of proteins that is differentially expressed under these treatments suggesting their direct involvement in the restoration process of the F508del-CFTR trafficking defect. Western blot analysis of cells under caffeine treatment, using anti-CFTR Lis-1 antibody, revealed that, similar to low temperature, 1mM and 5mM caffeine promote the rescue of F508del-CFTR by causing appearance of the complex-glycosylation form (band C) of this mutant. Caffeine has no significant effect on the processing efficiency of wt-CFTR. Immunocytochemistry confirmed this result by showing the presence of F508del-CFTR in the cell membrane of some cells under 1mM and 5 mM caffeine. However, increasing the concentration of caffeine to 10 mM seems to be toxic to the cells since it causes accumulation of wt- and F508del-CFTR into vesicles dispersed in the cytoplasm of the cells. Functional analysis by MQAE efflux showed that treatment of BHK cells expressing F508del-CFTR with 1mM caffeine for 24 hrs significantly increases the forskolin and IBMX-stimulated chloride efflux in comparison with the same cells without caffeine treatment. However, the values for chloride efflux observed under stimulation are still lower in comparison with BHK cells expressing wt-CFTR indicating that similarly to low temperature as well as other compounds, caffeine can only partially revert the trafficking defect of F508del-CFTR. We also analysed by 2D-SDS-PAGE the total protein extracts of BHK cells expressing F508del- CFTR after caffeine treatment (37ºC for 24h), after incubation at low temperature (26ºC for 24h) and untreated cells as controls. High-resolution maps were built, and differentially expressed protein spots identified by computer-based analysis (Melanie software). More than 1000 protein spots are detected on 2D-PAGE maps using a non-linear wide-range immobilized pH 3-10 gradient for the first dimension gel and a 8-16% (w/v) polyacrylamide gradient for the second. For some of the detected protein spots qualitative or quantitative differences were found by matching the proteomes of those cells. The complete identification and functional characterization of these differently expressed proteins are in progress and may provide new insights into the mechanisms responsible for the effect of caffeine and low temperature on the restoration process of the F508del-CFTR trafficking defect. Work supported by POCTI/MGI/40878 and POCTI/MGI/35737 of FCT & FEDER research grants (Portugal). AS is recipient of BIC-FCT fellowship and MRR is recipient of doctoral fellowship. POSTER SESSION

P-144 Proteomic Approach in Determination of Phenobarbital Effects on Microsomal and Cytosolic Protein Expression Levels

V. Zgoda, I. Kanaeva and A. Archakov Orekhovich Institute of Biomedical Chemistry, Moscow, Russia

Monooxygenase microsomal system is responsible for the oxidative metabolism of a wide variety of chemicals, including therapeutic drugs, carcinogens, pesticides, industrial pollutants, and endogenous compounds, such as steroid hormones. The monooxygenase system is inducible. It is well known that introduction of Phenobarbital to mice leads to induction of few liver microsomal proteins such as cytochrome P450 2B, NADPH-flavoprotein and cytochome b5. Using the proteomic approach we found significantly increased expression level for more than 30 other microsomal proteins. Also we observed increased expression level for some mouse liver cytosolic proteins such as, in particular, for chaperone group. These data may provide more complete understanding of P450 turnover mechanism and pharmaco-molecular response to some drug treatments. POSTER SESSION

P-145 Proteomic Determination of Serum Haptoglobin Phenotype

A. Bruneel1, N. Bosselut1, V. Labas2, J. Vinh2, M. Vaubourdolle1 and B. Baudin1,3 1Service de Biochimie A, Hôpital Saint-Antoine, Paris, France; 2ESPCI, CNRS UMR 7637, Paris, France; 3GRECAN-EA 1772 UFR Sciences Pharmaceutiques, Caen, France

Human haptoglobin (Hp) is a positive acute phase serum protein characterized by a genetic polymorphism concerning the alpha-chain which can appear as three major phenotypes: Hp 1-1 (α1/α1), Hp 2-1 (α2/ α1) and Hp 2-2 (α2/ α2). Although the functional property of Hp in the capture of circulating hemoglobin after hemolysis was fully investigated, haptoglobin polymorphisms, as determined by genotyping or under non-denaturing electrophoresis methods, have been recently associated with the prevalence and the clinical evolution of a broad range of severe diseases such as chronic hepatitis, atherosclerosis, malaria and hemochromatosis. In the present study, the combination of 2D electrophoresis (IPG 4 to 7, and PAGE – SDS 12%) with MALDI-tof mass spectrometry analysis allowed the identification of five distinct a2-chain Hp isoforms (~17 kDa) and strongly suggested the identification of two α1-chain Hp isoforms (~ 8 kDa) enabling accurate determination of Hp phenotypes in human serum. Further, it appeared that Hp phenotyping could be greatly simplified by using standard one-dimensional denaturing PAGE - SDS coupled with Coomassie blue coloration, although Hp isoforms could not be identified. Using 5 to 10 µl of serum, this fast and versatile electrophoresis method w a s applied to the determination of Hp phenotype frequencies associated with primary biliary cirrhosis (PBC), a rare autoimmune liver disease. The preliminary results on 41 sera strongly suggest an over-representation of the phenotype 2-2 in PBC in comparison to the healthy group (n = 16 sera). POSTER SESSION

P-146 Examination of the CD34+ Progenitor Cell Proteome from Umbilical Cord Blood

C. Zenzmaier1, A. Raicht1, B. Gesslbauer2, M. Kollroser3, A. Jandrositz1, K.-H. Preisegger1, and A. J. Kungl2 1Lifecord Inc., Graz, Austria; 2Institute of Pharmaceutical Chemistry and Pharmaceutical Technology and 3Institute of Forensic Medicine, University of Graz, Austria

Umbilical cord blood (UCB) has beed used successfully as an alternative source of haemopoietic progenitor cells (HPC) in stem-cell transplantation. Delayed engraftment has emerged as a limiting factor to more widespread use of UCB as a source of HPC, which might be overcome by increasing the number of HPCs. Ex vivo expansion of progenitor cells seems one way to solve this problem which itself is crucially dependent upon knowing stem cell propagating factors. In order to identify stem cell-specific proteins which might act as such factors, the proteome of human stem cells from umbilical cord blood was explored. For this purpose, 2-D gels of five CD34+ preparations with a purity of > 85% were compared for matching protein spots and approximately only 15% of the proteins were found at identical positions in all samples. The identity of these proteins was determined after excision and in-gel trypsinisation of each protein spot by applying nano-LC coupled to ion trap MS/MS detection. In a second approach, the entire proteome of one CD34+ preparation was investigated by in- solution digest followed again by nano-LC and ion trap MS/MS detection. By this means, 215 proteins were reliably identified with a mowse score >80. This study represents the first comprehensive attempt to identify novel progenitor cell protein markers.

This work was supported by the Austrian Indust. Res. Promotion Fund, grant no. 806244/7966 KA/SA POSTER SESSION

P-147 Inhibition of the Protease Calpain by MDL 28170 Prevents Inflammation-induced Neurofilament Light Chain (NFL) Breakdown in the Spinal Cord and Reduces Thermal Hyperalgesia

S. Kunz1, C. Ehnert1, A. Pfenniger2, J. Kruip2, T. Wendrich2, A. Schmidtko1, I. Tegeder1, G. Geisslinger1 and E. Niederberger1

1pharmazentrum frankfurt, Klinikum der Johann Wolfgang Goethe-Universität Frankfurt, Frankfurt am Main, Germany; and 2Aventis Pharma, Industriepark Höchst, Frankfurt am Main, Germany

Since long term hyperexcitability of nociceptive neurons in the spinal cord has been suggested to be caused and maintained by changes of protein expression we assessed protein patterns in lumbar spinal cord of rats during a zymosan induced paw inflammation employing two- dimensional (2D) gel electrophoresis. 2D PAGE revealed a time-dependent breakdown of scaffolding proteins one of which was neurofilament light chain (NF-L) protein which has been previously found to be important for axonal architecture and transport. NF-L breakdown was prevented by pre-treatment of the animals with a specific inhibitor of the protease calpain (MDL-28170) which has been shown to be the primary protease involved in neurofilament degradation in neurodegenerative diseases. Treatment with the calpain inhibitor also provided anti-inflammatory and anti-hyperalgesic effects in the zymosan-induced paw edema and the Hargreaves model, respectively, suggesting that the activation of this protease is involved in the development of sensitization of nociceptive neurons. This may be partly due to neurofilament breakdown but most likely additionally involves the cleavage of other calpain substrates such as I_B, glutamate receptors or Ca2+-channels. Our results suggest that inhibition of pathological calpain activity may allow for a reduction of adaptive changes involved in the development of chronic pain and therefore indicate a therapeutic potential of calpain inhibitors. POSTER SESSION

P-148 Proteome Analysis of Synovial Fluid and Plasma of Patients Suffering from Juvenile Idiopathic Arthritis

I. Prüfer1, K. Falk1, C. Koy1, S. Mikkat1, J. Oppermann2, R. Nowack2, H.-J. Thiesen3 and M.O. Glocker1 1Proteome Center Rostock, Medical Faculty, University of Rostock, Germany; 2Carl-Thiem- Klinikum Cottbus, Cottbus, Germany; 3Institut for Immunology, Medical Faculty, University of Rostock, Rostock, Germany

Chronic arthropathies are the most common rheumatic diseases in childhood [1]. In Europe and Northern America prevalences range from 0.08-1.1% in children [2,3]. According to the ILAR classification the disease pattern is subsumed to the term "Juvenile Idiopathic Arthritis (JIA)” [4]. In comparison with Rheumatoid Arthritis of adulthood (RA), JIA is considered much more heterogeneous e.g. concerning genetic markers and clinical pathology [2,5]. For our investigations we chose a group of eight patients suffering from JIA that were treated with medicinal standard therapy. The group is composed of five female and three male individuals with the age of 9-19 years. For differential proteome analysis we compared blood plasma (BP) and synovial fluid (SF) for each patient individually. Subsequently, the resulting differential protein patterns were analysed for all patients of the group.We applied 2D-PAGE using immobilized pH-gradients to separate the proteins. Each gel contained about 600 visible spots. By computer-assisted image analysis common differences between BP and SF were identified for all patients of the group. The corresponding protein spots were picked, digested by trypsin and identified using MALDI-TOF mass spectrometry. Proteins that were found higher in abundance in BP encompassed vitronectin precursor and complement factor C4. Proteins with higher abundance in SF were fibrinogen-β-chain degradation products, stromelysin-1 (MMP-3), and ASPIC resp. CRTAC1. Fibrinogen-γ-chain proteins were found shifted to the acidic side in SF. The goal of our differential proteome analysis approach is to search for marker proteins that are suitable for the development of appropriate diagnostic methods.

References: 1 Hulya Bukulmez, Murray H Passo: Classification of Juvenile Chronic Idiopathic Arthropathies: Is It Time to Change Yet? Bull Rheum Dis 2002, 51:1-5 2 Sampath Prahalad, David N Glass: Is juvenile rheumatoid arthritis/juvenile idiopathic arthritis different from rheumatoid arthritis? Arthritis Res 2002, 4(suppl3):303-310 3 Surjit Singh: Chronic Arthritis: Current Perspectives. Indian Padiatrics 2003, 40:3939-397 4 Petty RE et al: Revision of the proposed classification criteria for juvenile idiopathic arthritis: Durban, 1997. J Rheumatol, 1998, 25:1991-1994 5 Sinz et al., Mass spectrometic proteome analyses of synovial fluids and plasmas from patients suffering from rheumatoid arthritis and comparison to reactive arthritis or osteoarthritis. Electrophoresis 2002, 23: 3445-3456 POSTER SESSION

P-149 Lysines and Cysteines are both Targets for Acetylation in the HIV-1 Tat Protein

W. Dormeyer1, A. Dorr2, M. Ott2 and M. Schnölzer1 1Protein Analysis Facility and 2Applied Tumor Virology, Deutsches Krebsforschungszentrum, Heidelberg, Germany

The human immunodeficiency virus-1 transactivator of transcription (HIV-1 Tat) is a small nuclear protein that stimulates transcriptional elongation by transiently binding to an RNA stem loop structure called the transactivation-responsive element (TAR) which is located at the 5' end of all viral transcripts. The first 72 amino acids of Tat are sufficient to transactivate transcription from the viral long-terminal repeat (LTR) and comprise five regions: an N-terminal acidic region, a cysteine-rich region (CRR), a core region, an arginine-rich motif (ARM) and a glutamine-rich region. The CRR and the core region form the cofactor binding region which interacts with cofactors such as cyclin T1 whereas the ARM is essential for TAR binding. Here, we report on the acetylation of Tat by the acetyltransferase activity of p300. In vitro acetylation assays using synthetic peptides corresponding to different Tat regions were performed in the presence of p300 acetyltransferase enzyme and acetyl-coenzyme A (AcCoA) or with AcCoA alone as negative control. Analysis of the acetylation reactions by MALDI TOF MS revealed that the ARM is the only acetylation target of p300 in Tat. Proteolytic digestion in combination with MALDI-TOF MS and sequential Edman degradation mapped the acetylation site of p300 to a single lysine residue, K50, in the Tat ARM. Surprisingly, we also observed a strong acetylation reaction in the absence of p300 mapping to the Tat CRR and to a lesser extent of the Tat core region. MALDI TOF MS of the acetylated Tat CRR indicated that at least three residues were acetylated, exceeding the number of lysines (28K29K) in this region. Furthermore, multiple CRR acetylation occurred independently from the presence of lysine residues since a mutant peptide in which 28K29K were mutated to alanines showed the identical acetylation pattern as the wild type CRR peptide. However, chemical protection of all cysteine residues in the CRR peptide completely inhibited the acetylation reaction implying that an enzyme-independent transfer of the -SH coupled acetyl group from Acetyl-CoA to the side chain of the Tat cysteine residues was responsible for the Tat CRR acetylation. In terms of biological relevance, a coenzyme function of Tat in acetyltransferase reactions might be considered and will be tested in future transacetylation experiments. In general, advertence of the cysteine content of peptides and proteins is important for the interpretation of in vitro acetylation assays and the mapping of acetylation sites. POSTER SESSION

P-150 Proteomic Analysis of the Serum Ligands of the High Mobility Group B1 (HMGB1) Proteins

S. Frontini1, I. Dumitriu1, F. Catalanotti2, L. De Monte3, C. Traversari2, M.E. Bianchi4, A.A. Manfredi1, M. Alessio2, and P. Rovere-Querini1 1Cancer Immunotherapy & Gene Therapy Programme and Clinical Immunology Unit; 2MolMed SpA; H San Raffaele-DIBIT; 3Proteomics and 4Chromatin dynamics Units,c/o San Raffaele University Hospital, Milano, Italy.

The HMGB1 is an abundantly expressed non-histone protein involved in the regulation of transcription in the nucleus. However, it has a double life[1], since it can be leaked or actively secreted in the extracellular environment, where it behaves as a potent inflammatory mediator. This role is possibly particularly important in the pathophysiology of the septic shock, where HMGB1 has been identified as a mediator of systemic inflammation after resolution of the early innate response. Finally, its quantitative release as a consequence of massive cell death justifies the inflammatory effects of uncleared necrotic cells in vivo [2]. These findings (potent inflammatory action of HMGB1, associated to massive release from dying cells) raise the possibility that factors present in the extracellular environment, and in particular in biological fluids, like plasma or liquor, specifically cleave or inactivate HMGB1. To address this possibility, we used the pT7-7-rHMGB1cm plasmid for the expression of full-length HMGB1 in BL21(-) E. coli strain. The protein was then purified as described [2] and endotoxin removed. For expression in eukaryotic cells, full length HMGB1, tagged with 6 histidine at the N-terminal (His- HMGB1), was cloned into mammalian expression vectors and expressed in human CEM cells. His-HMGB1 was purified with a Ni2+-charged chelating sepharose column and ultra-filtrated on polyethersulfone membranes. Immunoblots were performed using anti-His or anti-HMGB1 antibodies and HRP-conjugated anti-mouse or anti-rabbit second step reagents. The recombinant proteins were then incubated for different times in the presence of BSA or human serum, before analysis by western blot or by a sandwich enzyme linked immuno sorbent assay, which relies on antibodies recognizing different epitopes of the molecule located at the carboxyl- or at the amino-terminus. The domains are involved in different functions of HMGB1, including RAGE activation and proinflammatory cytokine induction. The molecule was also evaluated functionally after incubation and retrieved by immunoprecipitation with specific monoclonal or policlonal antibodies. The modification of the protein, and the associated moieties are being actively investigated by proteomic analysis after 2D gel analysis.

References: [1] Muller S, Scaffidi P, Degryse B, Bonaldi T, Ronfani L, Agresti A, Beltrame M, Bianchi ME (2001) New EMBO members' review: the double life of HMGB1 chromatin protein: architectural factor and extracellular signal. EMBO J. 20:4337-4340. [2] Scaffidi P, Misteli T, Bianchi ME (2002) Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 418:191-195. POSTER SESSION

P-151 Proteomics of Oxidative Stress in Endothelial Cells

M.R. Eman1,2, J. Haverkamp1,3, A.J.R. Heck1, A.J. Verkleij2 and J.A. Post2 1Department of Biomolecular Mass Spectrometry and 2Department of Molecular Cell Biology, Utrecht University; 3Unilever Research & Development, Vlaardingen, The Netherlands

Atherosclerosis is one of the main causes of morbidity and mortality in the elderly population. The consequences of atherosclerosis are primarily expressed at older age, however it is generally well accepted that initial stages of atherosclerosis are triggered already at younger age. It is thought that ageing of the vascular cells leads to a lesser functioning of the endothelial cells and makes the vascular system more vulnerable for age related diseases such as atherosclerosis. One of the important factors proposed to be involved in ageing is oxidative stress, which can clearly have a great impact on cellular functioning. Because of the high complexity of the total vascular wall proteome we decided to study one cell type present in the vascular wall. In the vessel wall the endothelial cells are very important in regulating vascular function and for that reason we studied this cell type. Therefore, the aim of this study is to understand the influence of oxidative stress on the proteome of endothelial cells We use a model system with cultured Human Umbilical Vein Endothelial Cells (HUVECs), which are exposed to Buthionine Sulfoxymine (BSO). BSO depletes cellular glutathione (GSH) and will thereby diminish the antioxidant capacity of the cell. In this way the HUVEC will be gradually exposed to an oxidative stress state. By use of two-dimensional electrophoresis and differential analysis with PDQuestTM , in combination with mass spectrometry, we show proteome alterations due to depletion of GSH and which might be relevant in ageing of the human vasculature. Despite the observed differences it is clear that this kind of analyses are extremely difficult when applied to total cell lysates.

This research is funded via the Technological Co-operation program from Senter, an agency of the Dutch Ministry of Economic Affairs. POSTER SESSION

P-152 Proteomics Approach to Study Effects of Mobile Phone Radiation using Two Variants of Human Endothelial Cell Line

R. Kuokka and D. Leszczynski STUK – Radiation and Nuclear Safety Authority, Helsinki, Finland

We have used proteomics to examine mobile phone radiation induced effects in the human endothelial cell line EA.hy926 and its subcloned slow-growing variant EA.hy926v1. Cells were exposed for one hour to GSM 900 MHz signal at 37 ± 0.3 °C. An average SAR of the exposure was 2.4 W/kg. For the two-dimensional gel electrophoresis (2DE) cells were collected immediately after the exposure and lysed (7M Urea, 2M Thiourea, 4% Chaps, 2% IPG buffer 3- 10 NL, 1% DTT) and furthermore protein concentrations were determined using Bradford protocol (Sigma). The 1st dimension of 2DE was performed using IPGphor Isoelectric Focusing System (Amersham) and Immobiline DryStrips having pH gradient range of 3-10 NL and length of 18 cm (Amersham). The isoelectric focusing was carried out using step-n-hold and gradient methods until 65000 volt-hours were achieved. The 2nd dimension of 2DE was performed using 8% SDS-PAGE with ProteanIIxi Multicell apparatus (Bio-Rad). The gels were stained by silver, scanned using GS-710 Calibrated Imaging Densitometer (Bio-Rad) and analyzed using PDQuest 6.2 software (Bio-Rad). Approximately 1300 protein spots were detected in each cell line. Comparison of the sham and exposed samples revealed several tens of protein spots which were statistically significantly (student T-test, p < 0.05, n=10) affected by the exposure (increased or declined expression) in both cell lines. A few of these spots were selected for the mass spectrometry identification using the following criteria: spots needed to be (i) enough separate from the adjacent spots, (ii) sufficiently large and (iii) well focused in both dimensions. According to these criteria 14 spots were selected. By far 6 protein spots have been identified using mass spectrometry (Bruker Biflex MALDI-ToF). These are vimentin (two iso-forms), isocitrate dehydrogenase 3 (NAD+) alpha, heterogenous nuclear ribonucleoprotein H1, tubulin _6, and _-actinin 2 (uncertain due to the low protein content). Presently expression of these proteins is being confirmed using western blot analysis as well as cell stainings to locate the protein expression in the cells. Comparison of all protein spots between the cell lines revealed significant differences. Only ca. 50% of 1300 protein spots could be confidentially matched between the cell lines, even though both cell lines have the same origin. This suggests large protein diversity between the cell lines. Altogether, our study has shown that proteomics is an effective tool in examining the effects of mobile phone radiation. Using this approach it was possible to reveal new potential target proteins responding to the mobile phone radiation. In addition to the protein expression level, the method also showed changes in the protein activity level corresponding to a shift in the pI value of the protein due to post- translational modification (e.g. phosphorylation). However, since the sensitivity of the method is limited and often the difference between the exposed and sham-exposed is not high enough, the method is only able to show potential target candidates responding to the mobile phone radiation. Therefore further experiments using other cell biology methods are needed for target validation. POSTER SESSION

P-153 Extracting and Integrating Proteomic Data from 2DGE Images and Literature Databases Reveals New Pathological Pathways in Renal Cell Carcinoma

S. Fater1, R. Kapitan1, J. Klenk1, G. Schmidt1, A. Harder2, T. Halder2, M. Kersten2, R. Lichtenfels3 and B. Seliger3 1Definiens AG, München, Germany; 2TopLab GmbH, Martinsried, Germany; 3Johannes Gutenberg University, Third Department of Internal Medicine, Mainz, Germany

Comparing the proteome of healthy human renal epithelium with tissue specimen of renal cell carcinoma (RCC) revealed significant changes in the protein expression profiles of the distinct RCC subtypes. The series of human RCC lesions and corresponding normal renal tissue analysed was derived from biopsy material obtained from RCC patients undergoing nephrectomies. The proteins extracted from such samples were separated at TopLab’s proteomic facility using high-quality 2D gel electrophoresis in the mass range of 5kD to 150kD and the pH range of 4 to 7. To ensure statistical significance, five replicate gels were analysed per tissue state/sample. The Definiens image analysis software Proteomweaver automatically detected the protein spots and matched corresponding spots in all gels. To identify differentially expressed proteins, peptide mass fingerprinting with MALDI-TOF/MS after in-gel protein cleavage was performed. The quantitative spot analysis showed a significant up-regulation of the tumor rejection antigen-1 (grp94, gp96; 4.7 fold) and vimentin (4-fold) in the RCC lesions and a 3.9- and 3.7-fold down-regulation of the ribosomal P0 protein and aminoacylase-1, respectively. Using a protein interaction database generated by the Definiens information extraction system Polymind, we obtained new insight into the cellular mechanisms which differ between the malignant and control tissue state. Polymind extracted 53023 protein interactions from the complete Medline corpus which comprises 11403565 abstracts. The system uses an advanced natural language understanding system with deep parsing and a rich ontology compiled from MeSH (NCBI), WordNet (Princeton University) and SwissProt (Swiss Institute of Bioinformatics) to extract proteomic information with unprecedented precision POSTER SESSION

P-154 Toward the Identification of Liver Toxicity Markers: A Proteome Study in Human Cell Culture and Rats

B. Thome-Kromer1,2, I. Bonk1,2, M. Klatt1,2, G. Nebrich1, M. Taufmann1,2, S. Bryant3, U. Wacker1,2* and A. Köpke1,4

1Formerly at WITA Proteomics AG, Teltow/Berlin, Germany; 2EUROGENTEC PROTEOMICS GmbH, Teltow/Berlin, Germany; 3Johnson and Johnson Pharmaceutical Research & Development, Raritan NJ, USA; 4Devgen N.V., Technologiepark, Ghent-Zwijnaarde, Belgium

The effects of toxic and non-toxic compound treatments were investigated by high resolution custom developed 2-11 pH-gradient NEPHGE 2D-electrophoresis. Two models were compared: (i) in vivo rat and (ii) the human cell line HepG2, to test their suitability in proteomics based toxicity marker finding. 163 and 321 proteins were identified from the rat liver and HepG2 proteome. These represent various isoforms of 113 and 194 different NCBI annotated gene sequences, respectively. Nine compounds were selected to induce proteome variations associated with liver toxicity and metabolism. Variant proteins were assessed regarding their usefulness as toxicity marker by evaluating their treatment specificity against multiple control treatments. 13 potential toxicity marker proteins were found in rat liver and eight in HepG2. Catalase and carbamoylphosphatesynthetase-1 isoforms were found significantly changed after treatment by 4/4 and 3/4 toxic compounds in rat liver, respectively. Aldo-keto-reductase family 1, member C1 was implicated for 3/4 liver cell toxic compounds in HepG2. Our approach was able to differentiate the quality of potential toxicity markers and provided useful information for an ongoing characterization of more compounds in a wider number of toxicity classes (Proteomics 3 (10), 2003, in press). POSTER SESSION

P-155 Isolation of Rodent Airway Epithelial Cell Proteins Facilitates in vivo Proteomics Studies of Lung Toxicity

Å. Wheelock1, L. Zhang2, M.-U. Tran3, D. Morin1, S. Penn2, A. Buckpitt1 and C. Plopper3 1Departments of Molecular Biosciences and 3Anatomy, Physiology and Cell Biology, UC Davis, CA, USA; 2Advanced Research Team, Amersham Biosciences Corp., Sunnyvale, CA

Recent developments in genomics, proteomics and metabolomics hold substantial promise for understanding cellular responses to toxicants. Gene expression profiling is now considered standard procedure, but numerous publications reporting a lack of correlation between mRNA and protein expression emphasize the importance of conducting parallel proteomics studies. The cellular complexity of the lung presents great challenges for in vivo proteomics, and improved isolation methods for proteins from specific lung cell-phenotypes are required. To address this issue, we have developed a novel method for isolation of rodent airway epithelial cell proteins, which facilitates in vivo proteomics studies of two target-cell phenotypes of the lung, Clara cells and ciliated cells. The airway epithelial cell proteins are reproducibly solubilized and extracted, leaving the underlying basement membrane and smooth muscle intact as shown by histopathological analyses. The method yields epithelial cell-specific proteins in 5-fold higher concentrations and reduces the yield of non-epithelial airway proteins 13-fold in comparison to samples from microdissected airways. In addition, the number of detectable protein spots when using 2-dimensional Differential Gel Electrophoresis‘ (DIGE) increased with 36%. This new isolation method provides the means for sensitive in vivo proteomics studies of the toxic response to ambient air pollutants such as ozone and naphthalenes, which are known to specifically target the airway epithelium. POSTER SESSION

P-156 The Use of SELDI-TOF in Ecotoxicological Biomarker Discovery

T. Knigge, T. Monsinjon, A. Bjørnstad and O.K. Andersen RF-Akvamiljø, Mekjarvik, Randaberg, Norway

Surface-Enhanced Laser Desorption Ionisation – Time of Flight (SELDI-TOF) is a new proteomic technique to assess potential effects based on the mass-spectra of the entire proteom. It is now extensively used in medical research to find diagnostic markers for diseases. Our objective is to use the same approach within environmental research to identify potential biomarkers of pollution. We have adapted the methodology of the ProteinChip Array system (Ciphergen Biosystems, Fremont, USA) for the use with various tissue samples deriving from marine invertebrates. Protein expression in haemolymph, gill and digestive gland (hepatopancreas) of the blue mussel (Mytilus edulis) and the shore crab (Carcinus maenas) were screened for biomarker profiling. Laboratory experiments with exposure to oil and 4- nonylphenol, a substance known to act as an endocrine disruptor, have been conducted. Also the application of SELDI-TOF for field samples with different pollution history (Metals, PAH) has been tested. The aims of these studies were (i) to find certain proteins which are specifically induced or repressed following the exposure and (ii) the use of patterns of protein expression being typical for a certain pollution scenario. The comparison of protein profiles revealed a number of protein- or peptide-peaks that indicated general toxicological response or that showed differential expression in males and females due to the lab exposure. Employing pattern recognition software, it was possible to distinguish between the contaminated and the reference field sites, based on a set of proteins with highly significant differences in peak intensities. In summary, our results support the idea that this specific proteomic approach could greatly facilitate the discovery of new and more sensitive biomarkers. POSTER SESSION

P-157 Potential of SELDI-TOF in Ecotoxicology

T. Monsinjon, T. Knigge, A. Bjørnstad and O.K. Andersen RF-Akvamiljø, Mekjarvik, Randaberg, Norway

Among the thousands of proteins compromising the proteome, some contain evidence of the pathological changes that a cell or an organ is undergoing. Recently the analysis of the proteome has been successfully employed in the discovery of new biomarkers of human diseases. Addressing the same concept of proteomics to ecotoxicological problems may therefore help to establish new sensitive biomarkers of exposure and effect. For that purpose the SELDI technology combined with the ProteinChip Array system allowed first of all to establish protein expression profiles and then give the ability in providing biomarkers, not only by identifying single proteins, but sets of proteins that react robustly and specifically to chemicals. Such sets of biomarkers should be capable of discriminating between field sites of different contamination, located in Norway. Two polluted field sites were selected; one dominated by metals, originating from an abandoned copper mine (Visnes) and the other by polyaromatic hydrocarbons (PAH), found in the effluent from an aluminium plant (Høgevarde). Two lab exposures, with Oil and Oil+ PAH have also been conducted. Protein expression in gill and hepatopancreas of the blue mussel (Mytilus edulis) was investigated for biomarker profiling. To elucidate statistically significant differences in protein expression a biomarker pattern analysis with a minimum of 30 specimens each was conducted, using a tree building algorithm to classify the most important protein peaks. By this approach, patterns of certain key proteins or peptides, independent of their identity, could be determined, that react specifically to the particular stress situation, present at the respective sites.

This research was supported by the Norwegian Research Council (Grant no 133724/420) POSTER SESSION

P-158 Proteomic Approach to Study the Effect of Crude Oil, PAHs and Alkylphenols in the Blue Mussel Mytilus edulis H. Manduzio1, F. Durand1, L. Gricourt1, M. Hubert2 C. Lange2, P. Cosette3, T. Jouenne3, F. Leboulenger1 and B. Roucher1 1Laboratory of Ecotoxicology, UPRES EA 3222, University of Le Havre, France, 2Laboratory of Spectrométrie de Masse Bio-Organique, University of Rouen, Mont-Saint Aignan, France, 3UMR 6522 CNRS – University of Rouen, Mont-Saint Aignan, France

The aquatic environment suffers from impact caused by the growing number of xenobiotics which are discharged into continental and oceanic waters. In this study, we have developed a proteomic approach in the blue mussel, Mytilus edulis, dedicated to elucidate the effect of pollutants on protein expression. We focused on oil, a mixture of petroleum hydrocarbons supplemented with alkylphenols. Being lipophilic, hydrocarbons are easily absorbed by marine organisms in which they can accumulate. They are also known to cause toxic responses by interfering with cellular processes. Alkylphenols can mimic natural hormones and exert estrogenic-like effects. Mussels were collected in a clean area off the West Norwegian coast and maintained in flowthrough marine water in the AkvamiljØ, RF-Rogaland Research (Stavanger, Norway) facilities. After exposure for 21 days to 0.5 ppm North Sea oil from Statfjord (Norway), or to a mixture of 0.5 ppm North Sea oil, 0.1 ppm alkylphenols (mix of 8 alkylphenols equivalent to that released in water offshore) and 0.1 ppm extra PAHs (mix of 11 PAHs equivalent of main forms present in the North Sea crude oil), the proteomic signatures of each condition were compared. Isoelectrofocusing was performed in a pH 3-10 NL range and SDS-PAGE was carried out using a system which allows the running of 12 gels in parallel. The protein spots were visualized by colloidal blue staining and analysis was performed using the ImageMaster 2D Elite software (Amersham Pharmacia Biotech). The identification of spots were performed by mass spectrometry and microsequencing. Differential expression of 56 proteins (14%) was detected among the 410 spots compared between control and exposed mussels to the first condition. The enrichment of oil with PAHs and alkylphenols induced more variations with a differential expression of 76 spots (22%) among 341 spots compared. We now intend to identify these proteins specifically differentially expressed in order to a better understanding of the molecular mechanisms involved in the effects of oil pollutants. This study provides another evidence that expression of specific protein pattern could be related to chemical discharged and used in environmental bio-monitoring. POSTER SESSION

P-159 Identification of Protein Adducts Generated from Naphthalene and 1-Nitronaphthalene in Dissected Airways of Rhesus Macaques

C. Lin1, B. Boland1, Y. J. Lee2, M. Salemi2, C. Plopper3 and A. Buckpitt1 1Department of Molecular Biosciences and 3Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, 2Molecular Structure Facility, University of California, Davis, CA, USA

Ambient air in urban areas is a complex mixture of allergens, oxidants, and polycyclic aromatic hydrocarbons (PAH). However, the effects and interaction between allergens, O3, and PAH are unknown. Naphthalene (NA), a common PAH, and 1-nitronaphthalene (NN) are ambient air pollutants which undergo bioactivation by pulmonary cytochrome P450 monooxygenases. Reactive metabolites of NA and NN covalently bind to cellular proteins and have been implicated in cellular injury in rodent models. The relevance of rodent models in assessing the importance of chemicals which require bioactivation is not clear because of the 10 to 100 fold lower activities of cytochrome P450 monooxygenases in primate lung as compared to rodent lungs. Recent work by others has established and validated a primate model for human asthma exposing animals to house dust mite antigen (HDMA) and ozone (O3). Accordingly, these studies were designed to measure and identify the formation of reactive metabolite protein adducts and to determine whether exposures alter the rates and or nature of protein adducts generated in rhesus macaques. Airways were isolated from rhesus monkeys exposed to filtered air, O3, HDMA, or a combination of O3 and HDMA (which sensitizes the animals and produces an asthmatic response) and incubated with 14C-labeled NA or NN. Proteins adducted by reactive metabolites were separated by two dimensional gel electrophoresis, blotted to membranes, and the covalent adducts were measured by storage phosphor analysis. Those proteins adducted by reactive metabolites were excised from the gel and digested with trypsin; the tryptic peptides were analyzed by MALDI TOF and QTOF mass spectrometry. Adducts identified from incubations with either NA or NN included a and bactin, tubulin α-1, α-8, β-1, myosin light chain alkali, heat shock 60 and 70 kDa, aldehyde dehydrogenase, 15-hydroxyprostaglandin dehydrogenase, annexin V and VI, peroiredoxin 2, and microfibril-associated glycoprotein 4. Tropomyosin was adducted only by reactive NA metabolites while collagen α-3, actin cytoplasmic 2, β globin, selenium binding protein, protein disulfide isomerase, ubiquinol-cytochrome C reductase complex core protein 1, glutathione S- transferase P, monoamine or phenol-sulfating phenol sulfotransferase, guanine nucleotide binding protein b subunit, leucine-zipper protein, heat shock 27 kDa, 14-3-3 protein (zeta/delta, beta/alpha, gamma, epsilon), and chloride intracellular channel protein 1 were only adducted in incubations containing NN. We also observed that several proteins were targeted by NA or NN metabolites only in those animals exposed to O3 / HDMA or the combination. These studies show that many proteins targeted by reactive NA and NN metabolites in the lungs of rhesus macaques were not adducted in the lungs of rodents, and while many of the proteins were adducted in common by the two toxicants or four exposures, some are unique to the individual agents or different exposures. POSTER SESSION

P-160 Proteomics as a Tool to Investigate Effects Due to Genetic Engineering in the Context of Natural Variability: A Model Study using Arabidopsis thaliana

M.C. Ruebelt1,2, M. Lipp3, K.-D. Jany2, K.-H. Engel1, T.L. Reynolds3, C. George3, and J.D. Astwood3 1Technical University Munich, Germany; 2Federal Research Center for Nutrition, Germany; 3Monsanto Company, St. Louis, MO, USA.

Current tools used to assess the safety of food and feed derived from modern biotechnology emphasize the investigation of possible unintended effects caused directly by the expression of transgenes or indirectly by pleiotropy. These tools include extensive multi-site and multi- year agronomic evaluations, food and feed composition (biochemical) analyses, animal nutrition and classical toxicology evaluations. In order to complement the existing tools, w e are investigating proteome analysis based on two-dimensional gel electrophoresis (2DE). This technique was optimized to obtain well-resolved and reproducible patterns of Arabidopsis thaliana seed proteins. The method resolves proteins with isoelectric points 4 - 9 and Mr in the range of 6 to 120 kDa. The method was validated for gel-to-gel repeatability, linearity of colloidal Coomassie Blue staining and sensitivity. The separation of the proteins was demonstrated to be very reliable with standard deviation of 1.4 mm and 0.4 mm for x-position and y-position, respectively. The average relative standard deviation of the protein volume was found to be 25%. A linear relationship (R2>0.95) between protein amount and spot volume was demonstrated over a 100-fold range for the majority of selected proteins. The lowest quantifiable amount of protein was 5 ng for bovine serum albumin. Twelve Arabidopsis ecotypes with different geographic origin were grown side-by-side under highly standardized environmental conditions. The protein profiles of the twelve ecotypes were compared and catalogued on the basis of the presence, absence and volume of each spot. The computer-assisted image analysis of the proteome patterns detected approximately 600 protein spots for each ecotype; the total number of resolved protein spots found among the 12 ecotypes was 931. Significant natural variability in protein expression was observed; 597 of the 931 detected protein spots were found to vary with regard to their presence/absence among the ecotypes. Spot quantity variation of more than 3-fold was found for 25% of the 334 spots detected in all ecotypes. In the evaluation of unintended effects of genetic modification, we conclude that experimental designs must account for existing natural variability, which in the case of the expressed proteome, can be substantial. An evaluation of the impact of genotype and the subsequent heritability of seed protein profiles should be viewed as prerequisite baseline database information establishing a context for comparative analyses. POSTER SESSION

P-161 Venom Proteomics: the Brown Spider Loxosceles gaucho

L.F. Machado1, S. Melchior2, E.D. Botelho1, W. Fontes1, C.A.O. Ricart1, K. Barbaro3, P. Roepstorff2 and M. V. Sousa1 1Brazilian Center for Protein Research, Dep. of Cell Biology, University of Brasilia, Brazil; 2Protein Research Group, Dep. of Biochemistry & Molecular Biology, Odense University, Denmark; 3Laboratory of Immunopathology, Institute Butantan, São Paulo, SP, Brazil

Brown spiders of the Loxosceles genus are widely distributed in American, European, African and Australasian continents. In Brazil, there are seven species well adapted to temperate climate in Southern states, where the envenomization by Loxosceles venom (loxoscelism) is considered a health problem. The mechanism of action of the Loxosceles venom is poorly understood. Sphingomyelinase D apparently seems to be the principal enzyme responsible for the characteristic dermonecrosis caused by the venom on human skin. In a previous report (Cunha et al., J. Prot. Chem 22, 135-146, 2003), we purified and characterized two isoforms of dermonecrotic toxins (loxnecrogins) from the venom of L. gaucho. Herein we then turned to a higher resolution proteomic approach aimed at obtaining a global view of the venom proteome, with a particular interest in dissecting the loxnecrogin isoforms pattern. Proteomic 2DE maps for L. gaucho, L. intemedia and L. laeta venoms showed a principal region (30 to 35 kDa, pI 3 to 10), where at least eight loxnecrogin isoforms could be separated and identified. Their identification (as sphingomyelinase D) used a combined approach composed of Edman chemical sequencing, MALDI-TOF MS and ES-Q/TOF MS/MS. The venom was also pre-fractionated by gel filtration on a Superose 12 FPLC column, followed by cLC-MS. Again eight possible loxnecrogin isoforms with masses around 30-32 kDa were detected. The identification of the several dermonecrotic toxin isoforms in L. gaucho venom is an important step towards understanding the physiopathology of the envenomization. That could lead to improvements in the immuno- and chemotherapy of loxoscelism.

Financial support: PADCT/CNPq (nº. 62.545/98-4) POSTER SESSION

P-162 Application of Proteomic Tools in Environmental Analysis Quantification of Vitellogenin from zebrafish liver using ICAT-Technology

I. Mistele1, B. Kühl1, G. Brenner-Weiss1, U. Obst1, M. Seifert2 and B. Hock2 1Forschungszentrum Karlsruhe, Germany; 2TU München-Weihenstephan, Germany

Many adverse effects on reproduction observed in the aquatic environment are attributed to endocrine disruptors [1,2,3]. The majority of these effects detected up to now are mediated by estrogen receptors (ER). In the model for estrogen action, the estrogenic compound binds to ER in the cytoplasm of relevant cells followed by the translocation of the hormone-receptor complex into the nucleus to regulate cellular activities by acting as transcription factor. In addition to natural steroid hormones like estradiol (E2) several chemicals and natural compounds have been suspected to exhibit estrogenic effects. For this reason, the characterisation of biomarkers of the exposures to these compounds could result in useful tools for monitoring the aquatic environment. An example of a well-known biomarker is vitellogenin (Vtg), a precursor of yolk proteins normally produced in the liver of oviparous females, whose synthesis is regulated by estrogenic steroid hormones and influenced by endocrine disruptors. Male fish also possess the Vtg gene, but it is normally silent because of the low levels of circulating E2. Vtg synthesis in males can, however, be induced by treatment with exogenous estrogenic compounds. In this work we present our efforts towards the implementation of an alternative procedure for the quantification of Vtg by the isotope-coded approach using the acid cleavable ICAT reagent.

References: [1] Jobling S, Sumpter JP (1993) Aquatic Toxicol 27: 361 [2]Thorpe KL, Cummings RI, Hutchinson TH, Scholze M, Brighty G, Sumpter JP, Tyler CR (2003) Environ Sci Technol 37: 1142 [3]Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R (1999), Nat Biotechnol 17: 994–999. POSTER SESSION

P-163 A Proteomic Approach to Investigate the Response of Candida albicans Towards Environmental Stress

H. Kusch1,2, S. Engelmann2, M. Hecker2, J. Morschhäuser1,3 1Institut für Molekulare Infektionsbiologie, Universität Würzburg; 2Institut für Mikrobiologie, Universität Greifswald; 3Max-von-Pettenkofer-Institut, Ludwig-Maximilians-Universität München; Germany

The yeast Candida albicans is a harmless commensal in most healthy people, but it may cause superficial as well as life-threatening systemic infections in immunocompromised patients. The mechanisms of switching from a non pathogenic to a pathogenic phenotype are far from being completely understood. For a comprehensive understanding of pathogenicity an almost complete description of the physiology of C. albicans is urgently needed. We used two-dimensional gel electrophoresis of protein extracts in order to investigate the cell physiology under different growth conditions (e.g. different growth phases, different growth media or imposition of stress stimuli). Changes of growth conditions may result in a varied synthesis of a characteristic set of proteins. These proteomic signatures describe the cell physiology under defined conditions and are excellent tools to predict the physiological state of a cell under unknown conditions (e.g. yeast to hyphae transition, treatment with antibiotics etc.). In this study we focused on proteomic signatures of cells in the stationary phase of growth and of those exposed to oxidative (5 mM or 10 mM hydrogen peroxide) or osmotic (10 % sodium chloride) stress. Thereby the protein patterns of cells grown in complex medium (YPD) to an OD600 of 1 were compared with those of the varying growth conditions by the dual channel imaging technique. Proteins whose level changed were analysed by mass spectrometry. Whereas under oxidative stress conditions and at the stationary growth phase drastical alterations in the protein pattern were found, the amount of only a few proteins changed when cells were treated with 10 % sodium chloride. Interestingly, some proteins (e.g. GAP1) appeared as additional spots in the more acidic region of the gel under oxidative stress conditions. The observed pI shift might be due to an oxidation or S-thiolation of cystein residues in the respective proteins already described for proteins in other eukaryotes [e.g. Grant et al. (2000), Mol Cell Biol. 19, 2650-2656]. POSTER SESSION

P-164 The Bacterial Struggle Against Antibiotics – A Proteomic Study

J.E. Bandow1*, H. Broetz-Oesterhelt2, H. Labischinski2, and M. Hecker1 1Institut für Mikrobiologie, Ernst-Moritz-Arndt-Universität, Greifswald, Germany; 2Bayer AG, Pharmaforschungszentrum, Wuppertal, Germany *present address: Pfizer Inc., Ann Arbor, MI, USA

Bacterial resistance to antibiotics poses a challenge to the pharmaceutical industry: ever new antibiotics need to be developed to keep pace with the amazing adaptability of bacterial pathogens. The development of structurally novel inhibitors involves the understanding of the mechanism of action and the related cellular response. We chose a proteomic approach to study the complex cellular response of the Gram-positive model organism Bacillus subtilis to 30 antibacterial compounds covering all well-known and emerging target areas. Changes in protein expression profiles captured on 2D gels between untreated and antibiotic treated cells were determined and marker proteins for each treatment were identified by MALDI-ToF-MS. We were able to group the antibacterial compounds based on the number of shared marker proteins. Since this grouping strongly correlated with a grouping based on mechanism of action we were able to classify a structurally novel compound, Bay 50-2369, as peptidyltransferase inhibitor based on marker proteins shared with other peptidyltransferase inhibitors. Many marker proteins could be linked to existing knowledge on bacterial physiology. The Ile- tRNA synthetase inhibitor Mupirocin for instance induced the stringent response and enzymes involved in Ile/Val synthesis. However, each of the protein expression profiles also held some surprises as for instance the pI shift occurring in proteins synthesized after actinonin treatment. POSTER SESSION

P-165 Global Analysis of Protein Synthesis and Turnover in Escherichia coli as Revealed by 2-Dimensional Gel Electrophoresis of Pulse-Labelled Proteins

D. Weichart*, A. Cicek**, K. Stephani and R. Hengge Freie Universität Berlin, Institut für Biologie – Mikrobiologie, Berlin, Germany * Present address: Max Planck Institut für Molekulare Genetik, Mass Spectrometry Group, Berlin, Germany ** Present address: Freie Universität Berlin, Institut für Kristallographie, Berlin, Germany

We have initiated a survey of the global dynamics of protein populations in laboratory strains of Escherichia coli by 2-dimensional electrophoresis (2DE) of in vivo radioactively labelled proteins. In parallel, we employed protein staining with SYPRO Red to quantify protein abundance in the same samples. Because identical sampling and separation protocols were used, a direct overlay of the radioactive and non-radioactive gels was possible. This combination of techniques allows monitoring in parallel of abundance, synthesis rates and turnover for all proteins that can be resolved reproducibly on the gels. Our analysis focussed on protein dynamics in E. coli in different phases of growth. The transition between growth and stationary phase (as caused by carbon starvation) leads to major changes not only in protein synthesis, but also in protein turnover for many proteins. In some of these cases, the degradation rates were reduced in strains lacking a functional copy of the clpA or clpP protease subunit genes, indicating involvement of ClpAP or ClpXP proteases in degradation of the polypeptide. In other cases, it remains to be determined which of these proteins are degraded through proteolysis and in which cases the apparent turnover is based on other mechanisms such as posttranslational protein modifications (merely altering the position of spots on the gels). We have been able to detect one series of unusual posttranslational modifications on our gels: the conversion of the original translation product of the rpsF gene (the ribosomal protein S6) to singly and multiply glutamylated species. Monitoring of the abundance and dynamics of these proteins is possible because the modified products are situated in a well-resolved area of the 2D gels, which allowed the identification of the proteins by peptide mass fingerprinting using MALDI-TOF-MS, and was helped by the fact that this modification reaction had been described in the literature many years ago. This case hence underlines on the one hand the need to identify proteins migrating on 2DE gels in the vicinity of the protein(s) of interest, and on the other hand it emphasizes how proteome research relies on profound biochemical knowledge of the significant biological systems. POSTER SESSION

P-166 Mapping of Minimal Differences in the Alkaline Proteome of Two Lactococcal Strains by Difference In-Gel Electrophoresis

O. Drews1, G. Reil2, H. Parlar2 and A. Görg1 Technische Universität München, 1FG Proteomik and 2Lehrstuhl für Chemisch-Technische Analyse und Analytische Lebensmitteltechnologie, Freising-Weihenstephan, Germany

Lactococcus lactis is widely used in the food industry and last but not least after sequencing its genome has become a model organism. Increasingly, the bacteria was subject to proteomic studies in recent years, but in general, these investigations were limited to pH ranges from 3 to 10 or even from 4 to 7. Despite the fact that about one quarter of the theoretical proteins of Lactococcus lactis have isoelectric points beyond 9, these proteins were not included in the mentioned studies. Therefore, we started to establish a reference map of lactococcal proteins by using narrow immobilized pH gradients spanning pH 9 to 12. After 2D electrophoresis of extracts obtained from exponentially growing cells we were able to detect more than 80 protein spots in that pH gradient. By MALDI-TOF MS more than 50 of those detected could be identified. Their theoretical isoelectric points range as high as 11.3 and as low as 9.2. The majority represented ribosomal proteins, which are of interest with respect to stress response, because they are thought to act as sensors of heat and cold shock [1]. Although in more recent times mapping of diverse proteomes in the alkaline pH range succeeds, ongoing analyses based on these maps are still rather seldom in the literature. This may lead to the presumption that a differential analysis at the alkaline end is an impracticable task. For this reason we accomplished a comparative study in the mapped pH range. The application of lactococcal starter cultures is dependent on the fermentation, which is highly strain specific. We compared the sequenced strain Lactococcus lactis subsp. lactis IL1403 with the strain Lactococcus lactis subsp. cremoris MG1363, which were both grown in chemically defined media under the same conditions. By staining of the resolved proteins after 2D electrophoresis with silver or Coomassie Blue as few as seven qualitative differences in the protein patterns could be detected. Therefore, we tested a new detection technology, which allows to discriminate between different protein extracts loaded onto the same gel. It is called difference in-gel electrophoresis (DIGE) and is based on covalent labeling of proteins with fluorescence dyes prior to the 2D electrophoresis. By this, we were able to detect even minimal differences in the 2D pattern of the two closely related organisms, which could not be detected with silver or Coomassie Blue staining. In total, 13 qualitative differences were determined. For example proteins like the translation initiation factor IF-3 or the 50S ribosomal protein L31 demonstrate only slight differences in the isolelectric point between the two strains. These may indicate single amino acid substitutions or minor posttranslational modifications. Labeling of a mixture of both lactococcal extracts with a third fluorescence dye confirmed each spot position within the same gel by loading all three differently labeled extracts on one gel. Therefore, differences due to labeling artefacts can be excluded. In conclusion we not only set up a proteomic map of two major lactococcal strains in the alkaline pH range, but effectively demonstrate the discrimination between those two even to the smallest difference in the isoelectric point.

Reference: [1] VanBogelen, R.A. et al.: PNAS 1990, 87: 5589-5593 POSTER SESSION

P-167 Changes in Protein Expression of Enterobacter ssp Induced by High Concentrations of Cobalt II Salts: A Proteomic Study

J. Marrero1, L.J. González2, A. Sánchez2, M. Ayala3, D. Paz-Lago2, A. Fallarero1, O. Coto1, L. Castellanos Serra2 1Faculty of Biology, University of Havana, Ciudad Habana, Cuba; 2Division of Physical- Chemistry and 3Division of Immunotechnology, Center for Genetic Engineering and Biotechnology, Havana, Cuba

Traces of heavy metal salts are required as nutrients for essential functions in microorganisms. However, higher concentrations of these cations are generally toxic and can provoke contrasting effects on living organisms. Enterobacter spp. strain C-1, a Gram negative bacterium isolated from Moa mine in Cuba, is able to survive in presence of high concentrations of heavy metals. The proteome maps of Enterobacter spp. strain C-1, grown under aerobic conditions in the presence of cobalt (II) sulphate and in its absence were compared using two-dimensional polyacrylamide gel electrophoresis analysis in the pI range from 4 to 7U and mass range from 15 to 120 kDa. Significant changes in the expression level (> 2 fold) were detected for 15 proteins: nine are up-regulated after treatment with Co (II) and six were down-regulated. Enterobacter ssp has an unknown genome, for this reason, only three distinct proteins were identified by peptide mass fingerprint (PMF). Therefore, all proteins had to be identified by de novo sequencing of selected tryptic peptides followed by databases alignment using MS BLAST program. Of the identified proteins, 92% are involved in cellular oxidative stress probably induced by the presence of Co (II). This is the first step towards an understanding of the role of proteins participating in the toxic response and the resistance mechanism to heavy metals in Enterobacter spp. POSTER SESSION

P-168 A Proteomic Approach to Identify Proteins Involved in the in vitro Interaction Between Commensal Bifidobacteria and the Human Intestinal Tract

E.S. Klaassens, W.M. de Vos, F.H.J. Schuren and E.E. Vaughan Wageningen University and Research Centre, Laboratory of Microbiology, Wageningen, The Netherlands

The human gastrointestinal tract is more densely populated with microorganisms than any other organ and is a site where the microflora may have a pronounced impact on our physiology. In the intestine species of the bifidobacteria are dominant during the entire lifespan of almost every human. Many beneficial effects have been claimed for bifidobacteria, including protection against pathogens, normal development of the immune system and positive nutritional effects for the intestinal cells and the host. However, detailed insight in the activity and function of bifidobacteria in these processes is currently not available. This makes bifidobacteria not only a representative but also a relevant genus to study in relation to gut health. The initial aim of the current study is to analyse the effect of interaction of the human host on the bifidobacterial cells on the protein level. Colonic epithelial cell-lines are used as in vitro models for the human epithelial cells of the large intestine and different species of commensal bifidobacterial species are included in the study. Optimisation of protein extract preparation and proteomics techniques for the bifidobacteria species was first performed. Different sample buffers and isoelectric focussing (IEF) running conditions were applied to the samples to optimise protein separation and obtain gels with a reproducible protein pattern. Bifidobacteria were exposed to the human epithelial cell lines CaCo2 and HT29-MTX for various time periods. These protein extracts as well as the non-colonised controls were subjected to a proteome study. The maps generated by two dimensional gelelectrophoresis, which are used to visualise the total expressed proteome, will be presented. POSTER SESSION

P-169 Comparative Proteomics of the Human Pathogen Campylobacter jejuni

M.G. Brubacher1, R.T. Cole2, M.Y. White2, D.E. Garfin1, S.P. Djordjevic3 and S.J. Cordwell2 1Bio-Rad Laboratories, Hercules, CA, USA ; 2Australian Proteome Analysis Facility, Sydney, Australia; 3Microbiology and Immunology Section, Elizabeth Macarthur Agricultural Institute, Camden, Australia

Campylobacter jejuni is an important human pathogen leading to diseases such as gastrointestinal disorders, including food poisoning, and outcomes as severe as Guillain-Barré Syndrome. Proteomics has been successful in comparing strains of bacteria with different phenotypes, including infectivity, antibiotic resistance and medical outcome. Here we present a comprehensive catalog of C. jejuni proteins using a variety of proteomics methodologies as a basis for a protein map of this organism. C. jejuni proteins were pre-fractionated using commercially-available kits into whole cell, cytosolic, soluble / insoluble and membrane fractions and subjected to wide- and narrow-range IPG two-dimensional gel electrophoresis (2-DGE) prior to protein identification by MALDI-TOF mass spectrometry (MS). Fractions were also digested with trypsin and the resulting peptides analyzed by two-dimensional liquid chromatography (2-DLC) and electrospray-ionisation (ESI) MS/MS. The two methods provide a highly complementary approach to protein ‘cataloguing’ of bacterial proteomes. We also conducted comparative proteomics of membrane fractions from two clinical isolates – a gastrointestinal isolate and a Guillain-Barré Syndrome isolate to determine potential markers for rapid strain identification. Western blotting was also performed on these fractions to characterize highly immunogenic proteins. Identification of proteins from strains of C. jejuni will provide an important first step in characterizing strain differences potentially responsible for different disease outcomes associated with this organism. POSTER SESSION

P-170 Proteomic Analysis of Wild Type and phoB Mutants of Vibrio cholerae O1: Effect of the Phosphate Limitation

F.S.N. Manta, G.S.C. Vasconcelos, C.M. Batista, M.R. Soares, D.P. Carvalho, W.M.A. von Krüger and P.M. Bisch Rede Proteômica do Estado do Rio de Janeiro, Unidade Multidisciplinar de Genômica, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil

Vibrio cholerae, a Gram-negative bacterium, can survive in aquatic reservoirs and in intestine of human and animal models. It adapts to different environments by coordinating gene expression. Limitation of inorganic phosphate (Pi) induces expression of genes involved in Pi transport, metabolism and colonization in a process that requires the two-component system PhoB/PhoR. PhoB is a transcriptional activador, which, phosphorylated in its N-terminal domain, binds DNA through its C-terminal domain. Two types of phoB mutant of V. cholerae 569B were obtained: (i) a knockout mutant (WK3), with a kanamycin resistance cassette into the phoB gene; (ii) a substitution mutant (WK4), in which 6 aminoacids were substituted by 10 other aminoacids. Comparison of wild type and phoB mutant strains in a mouse colonization assay showed that phoB mutations greatly reduced colonization ability. This work describes proteomic analysis of total cell extracts and subcellular fractions from 569B (wild type) and phoB mutants (WK3 and WK4) of V. cholerae O1 in response to Pi limitation mediated by PhoB. This analysis was carried out by 2D electrophoresis and MALDI-TOF mass spectrometry. 2D gel electrophoresis was performed on an immobilized pH gradient 3 to 10 or 4 to 7 for the first dimension and SDS-PAGE on a 12-14% gradient gel for second dimension. The protein differentially expressed were digested with trypsin and analyzed by MALDI-TOF MS. The peptide masses were compared with a non-redundant proteobacteria protein database and proteins were identified by sequence similarity to putative gene products from V. cholerae ElTor O1, N16961. Some of the proteins expressed by wild type and not the mutants were identified. The periplasmic protein of Mr 60 kDa and pI 5.4 is the product of the gene VCA0033, a hypothetical protein of V. cholerae, and presents homology to alkaline phosphatases from some Vibrio species and other microorganisms. It may the responsible for the alkaline phosphatase activity expressed by the wild type strain in low phosphate medium. Another identified periplasmic protein of Mr 25 kDa and pI 5.5 is the "phage shock” product of VC1678 with no assigned function. The products of loci VC2563 (Mr 70 kDa, pI 5.4), VC01549 (Mr 51 kDa, pI 6.4) and VCA0603 (Mr 35 kDa, pI 6.3) were also identified and certainly are involved in Pi metabolism. The outer membrane protein of Mr 38 kDa and pI 4.7, product of locus VCA1008, presents no homology to porins induced by low Pi in other bacteria, therefore, is a newly identified porin of V. cholerae. Many other proteins from the wild type and mutant strains have been identified. Their corresponding genes are probably under PhoB control in low phosphate conditions. The presence of some proteins only in the mutant strains suggests that PhoB can also repress the expression of some genes, directly or indirectly, in low phosphate conditions. POSTER SESSION

P-171 Functional Characterisation of the Secretome of the Human Pathogen Listeria monocytogenes

M. Trost1, D. Wehmhöner1, U. Kärst1, G. Dieterich2, J. Wehland1 and L. Jänsch1 German Research Centre for Biotechnolog (GBF), 1Dept. of Cell Biology, and 2Dept. of Structural Biology, Braunschweig, Germany

The secretory proteins of microbial pathogens are the frontline in host-pathogen interactions. Especially the secretion into cytosolic compartments of the host seems to be important for the pathogenicity of many virulent microbes. We analysed the secretome of Listeria mono- cytogenes, a Gram-positive, food-borne, facultative intracellular pathogen, causing listeriosis leading to meningo-encephalitis and to abortions in pregnant women. Both 2D-Gel-Electrophoresis and LC-MS experiments were used for the characterisation of secretory proteins of wt, a prfA deletion mutant and a constitutive prfA overexpressing strain. Since PrfA regulates most known virulence factors of L. monocytogenes and is absent in avirulent Listeria strains, we expected to find new putatively PrfA-regulated proteins. About 60 different proteins were identified by 2D-PAGE and mass spectrometry and about 115 by LC-MS experiments. All known secreted virulence factors as well as other putatively PrfA-regulated proteins were identified by software-based expression analyses of the 2D gels and the usage of different concentrations in the LC-MS experiments. Furthermore, several protein species were unexpectedly found in the culture supernatant. Their presence and function in the secretome will be discussed. POSTER SESSION

P-172 Proteomic Analysis of Surface Associated Proteins in Clostridium difficile

A. Wright, E. Calabi, K. Brown and N. Fairweather Department of Biological Sciences, Centre for Molecular Microbiology and Infection, Imperial College, London, UK.

Clostridium difficile is now established as the aetiological agent of pseudomembraneous colitis and antibiotic associated diarrhoea. It is thought that antibiotic treatment disturbs the natural gut flora, encouraging the colonisation and overgrowth of C. difficile. Little is known about the conditions necessary for colonisation of the gut or the identity of the bacterial factors necessary for the initial stages of pathogenesis.

We have taken a proteomics approach to identify and characterise the surface associated proteins from C. difficile. S-layer proteins can be removed from the cell wall by a variety of treatments, e.g. 0.2M glycine, 10 - 50mM EDTA, 1 - 5M LiCl and 8M urea. This reveals the presence of two predominant proteins, as seen by 1D SDS-PAGE, that have been identified as the two S-layer proteins. However, 2D SDS-PAGE analysis of these protein preparations reveals over 15 spots, which we hypothesise, are surface associated proteins. We have identified two of these proteins as FliC (a flagellin) and Orf2, a protein located downstream of the gene for the S-layer protein, slpA, in the genome. We will present further data on our analysis of the S-layer associated proteome of C. difficle. POSTER SESSION

P-173 Subproteomes of Mycobacterium tuberculosis: Comprehensive Analysis of Culture Supernatant Proteins of Virulent M. tuberculosis H37Rv and Attenuated Mycobacterium bovis BCG Copenhagen

J. Mattow1, U.E. Schaible1, F. Schmidt1, F. Siejak1, E.-C. Müller2, G. Haeselbarth3, P.R. Jungblut1, and S.H.E. Kaufmann1 1MPI for Infection Biology, Berlin, Germany; 2Max-Delbrück-Centre for Molecular Medicine, Berlin, Germany; 3Max-Volmer-Institute, Technical University, Berlin, Germany

The secreted proteins of Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, represent immunodominant T-cell antigens and are crucial for pathogenesis. They are considered superior protective antigens as compared to the cell-associated ones, and it is assumed that in-depth knowledge of these proteins and their secretion pathways will facilitate the design of novel tuberculosis vaccines, diagnostic tests and tuberculostatic drugs. Here, a comprehensive analysis of culture supernatant (CSN) proteins of M. tuberculosis H37Rv was accomplished by combination of two-dimensional electrophoresis (2-DE), mass spectrometry and N-terminal sequencing by Edman degradation. Analytical 2-DE gels resolved approximately 1,250 protein spots from CSN of M. tuberculosis H37Rv, 381 of which were identified by mass spectrometry and/or Edman degradation. This study revealed 137 different proteins, 42 of which had previously been described as secreted. Comparative proteome analysis of CSN from virulent M. tuberculosis H37Rv and attenuated Mycobacterium bovis BCG Copenhagen identified 39 M. tuberculosis-specific spots containing 27 different proteins, representing candidate antigens for novel vaccines and diagnostics in tuberculosis. These included 5 proteins encoded by open reading frames absent from M. bovis BCG, e.g. early secretory antigen target (Esat6), as well as 22 novel differential proteins, such as acetyl-CoA C-acetyltransferase (Rv0243) and 2 putative Esat6 like proteins (Rv1198, Rv1793). The latter ones are currently tested in our laboratory for their potential to induce protective immunity in mice. POSTER SESSION

P-174 Characterization of the Protein Synthesis of C. pneumoniae in a Persistent Infection Induced by Iron Deficiency

W. Wehrl, P. Jungblut, T.F. Meyer, and A.J. Szczepek Max-Planck Institute for Infection Biology, Dept. Molecular Biology, Berlin, Germany

Chlamydophila pneumoniae is an obligate intracellular pathogen implicated in a variety of acute and chronic diseases. Long-term infections are associated with a persistent life stage, in which bacteria can stay for years. They are less accessible to antibiotic treatment but still prone to sustain an inflammatory response. In vitro, different cell-models have been established to mimic and characterize the non- productive, quiescent life stage of persistency. For C. pneumoniae and C. trachomatis altered metabolic activities and changed antigenic profiles compared to acute infections have been reported. Most of the studies inlcuding transcriptome and proteome analysis described persistency induced by IFN_-treatment. Here, we use iron depletion of the infected cell culture that also leads into persistence. We describe differently regulated proteins found by substractive proteome analysis by comparing two early stages of infection with and without addition of the iron chelator deferoxamine- mesylate (DAM). While only 1 bacterial protein was upregulated under iron deficiency up to 24h post infection (p.i.), 11 were found to be up- and 8 to be downregulated from 24-48h p.i. Two downregulated proteins could be identified by peptide-mass-fingerprinting as Thioredoxin Reductase (TrxB) and Chromosome Partitioning protein (ParB), the latter involved in chromosome segregation. Thus, using comparative approach we identified on a proteome level downregulation of ParB in persistent chlamydial forms, which is in agreement with previous results describing changes in cell division and atypical altered morphology of persistent Chlamydiae. POSTER SESSION

P-175 Proteomics of the Outer Membrane of Moraxella catarrhalis and Direct Straintyping using MALDI-TOF MS

A. Schaller1,3, R.S. Coimbra2, C.Aebi1,2, S. Gallati1,3, and P. Stutzmann Meier2 1University Children’s Hospital, 3Divison of Human Genetics, Bern, Switzerland; 2Institute for Infectious Diseases, University of Bern, Bern, Switzerland

Background: M. catarrhalis is a major mucosal pathogen of the human respiratory tract, both in children and in adults. Outer membrane proteins (OMP) have been evaluated as possible vaccine candidates and at least 8 individual OMP have been characterized in detail so far. The aim of this study was to establish a proteome map of the M. catarrhalis outer membrane from different strains to be able to identify further vaccine candidates. In addition, we performed a direct straintyping of various M. catarrhalis strains by analyzing whole cells using MALDI-TOF MS, allowing us a rapid identification of different strains. Methods: OMP of different M. catarrhalis strains were prepared using the standard EDTA/buffer method and displayed by 2D-PAGE. Individual spots were further characterized by MALDI-TOF MS and Western blot. Additionally, whole cells of different M.catarrhalis strains either grown on plates or in broth were directly analyzed by MALDI-TOF MS. Results: 2D-PAGE analysis of M. catarrhalis OMP revealed an average of approx. 124 spots per strain. In contrast to 1D SDS-PAGE, 2D-PAGE revealed some differences in the protein pattern between individual strains. 5 out of approx. 124 spots could be assigned to the known M. catarrhalis OMP UspA2, CopB, TbpA, OMP CD and OMP E. We are currently in the process of annotating the genome of M. catarrhalis and constructing a database for the identification of the remaining spots. At least 100 ORFs with significant similaritiy to membrane proteins (inner and outer membrane) of other species could be identified in the genome of M. catarrhalis. For a rapid straintyping of the different M. catarrhalis strains, we analyzed whole cells in a MALDI-TOF MS and obtained characteristic spectra for each strain. The spectra obtained from bacteria grown on plates or grown in broth were identical. Furthermore, the spectra of one particular strain did not change significantly during different growth phases. Conclusions: 2D-PAGE is an efficient method for displaying OMP of different M. catarrhalis strains. This method allows the screening of potential vaccine targets present on all M. catarrhalis. Furthermore, analyzing whole cells of M. catarrhalis by MALDI-TOF MS gives us a rapid tool for straintyping of different in vivo isolates. POSTER SESSION

P-176 System Analysis of Helicobacter pylori Clinical Isolates

V. Govorun1, K. Momynaliev2, O.V. Smirnova2, V.G. Zgoda1, M.V. Serebryakova1, O.V. Tikhonova1, S.A. Moshkovskii1 and A.I. Archakov1 1V.N. Orekhovich Institute of Biomedical Chemistry, Moscow, Russia; 2Institute of Physico-Chemical Medicine, Ministry of Health, Moscow, Russia

Nucleotide sequence diversity of H.pylori exceeds that for all other bacteria studied to day. Therefore, the projects which deal with studies of the regulation of both mRNA and protein expression require data about nucleotide diversity both on the gene and nucleotide level. In this study, four clinical isolates of Helicobacter pylori and its reference strain J99 were analyzed by different methods. Genome scanning and expression level estimation were performed for this isolates using DNA array technology. Simultaneously, proteomes of these isolates were studied. A genome comparison of different H.pylori strains has shown critical variety in certain gene groups including cag pathogenicity island. Several DNA array data were confirmed on proteomic level. In addition to absence of protein spots for certain genes, the proteome mapping has revealed significant pI shifts of many protein spots, as well as expression level variety between clinical strains. The correlation between protein content in the individual spot and nucleotide substitutions (mutation frequency of synonymous or non- synonymous sites) has been observed. Moreover, the new rules of expression regulation may be proposed when we observe the absence or presence some H.pylori restriction and modification (methylation) system and expression level of targeted genes. The main conclusion of this study is that the diversity of H.pylori may be expressed not only by lists of data, but by some relations between different parameters noted in this project. POSTER SESSION

P-177 Identification of Disease Related Proteins Using 2-DE, Immunoblotting and MALDI-TOF MS

M. Utt Institute of General and Molecular Pathology, University of Tartu, Estonia

H. pylori proteins were separated by two dimensional electrophoresis (2-DE) on the pH range 6-11 and 10 % PAGE. Gels were silver stained or blotted to PVDF membrane. The membranes were probed with sera from H. pylori positive persons with atrophic corpus gastritis and gastric cancer patients with intestinal type of adenocarcinoma of different localisation. Silver stained gels and 2D immunoblots were analysed with Melanie 3 software and pI as well as Mr were calculated for immunoreactive H. pylori proteins. Totally 328 immunoreactive proteins were found in the Mr range 27-33 kDa and 45-84 kDa. Differences in immune response between two patient groups were found. Specific proteins were identified using MALDI-TOF MS fingerprinting in combination with CAF-MALDI-TOF MS. 2D immunoblotting could be used to identify disease related antigens for diagnostic use and seroepidemiological studies. POSTER SESSION

P-178 Identification of Yersinia pestis Surface Proteins by Biotinylation and High-throughput Proteomics

S. Smither1, B. Crossett2, K. Brown2, R. Titball1 and J. Hill1 1Microbiology Department, Dstl, Porton Down, UK; 2Centre for Molecular Microbiology and Infection, Imperial College London, London, UK

The outer membrane of Gram-negative bacteria acts as the interface between the microorganism and its environment. The outer membrane is a complex barrier of proteins, lipids and other molecules that are important for bacterial survival and, for some pathogenic bacteria, may have a role in virulence. The surface of the bacterium is exposed to the immune system. Components of the outer membrane could therefore act as targets for immunoassay based detection systems. A means of identifying novel surface proteins would therefore be beneficial. In silico methods exist such as the computer prediction program PSORT which categorises proteins according to their likely subcellular location. Here we describe a high through-put method to identify novel surface proteins of Yersinia pestis, the causative agent of plague. The cell surface of Y. pestis was labelled with biotin. Bacterial whole-cell lysate was then separated by two-dimensional electrophoresis. By staining and blotting gels in parallel it was possible to identify labelled proteins through probing with streptavidin-horseradish peroxidase conjugate and detecting via chemiluminescence. Automated spot-picking and in-gel trypsin digestion was performed and MALDI-TOF Mass Spectrometry was used to identify proteins. The completed genomes of two Y. pestis strains were subjected to PSORT analysis to generate a theoretical list of outer membrane proteins for comparison of in silico and practical approaches to protein identification. Bacterial biotinylation was simple and quick to perform and approximately forty labelled proteins were observed. PSORT predicted over four hundred proteins to be outer membrane located. This shows that a combination of theoretical and practical approaches are complementary and may be used as a means to select cell surface proteins as targets for bacterial vaccines or detection systems. POSTER SESSION

P-179 Proteome Analysis of Virulent and Avirulent Strains of Rickettsia Prowazekii

D. Chelius1, C.-C. Chao2, T. Zhang1, L. Daggle2 and W.-M. Ching2 1Thermo Finnigan, San Jose, CA; USA; 2Rickettsial Diseases Department, Infectious Diseases Directorate, Naval Medical Research Center, Silver Spring, MD, USA

Introduction: Rickettsia prowazekii, the etiologic agent of epidemic typhus, has the potential to be selected as a biological weapon by terrorists and rouge countries. The threat of typhus as a biological weapon lies in its stability in the dried louse feces and in its infection by inhalation of an aerosol. Although the genomic DNA sequence of avirulent strain of R. prowazekii (Madrid E) has been recently completed, the factors that are responsible for the avirulence of Madrid E have not been investigated. Using DNA microarray to compare the genomic DNA between virulent Breinl strain and avirulent Madrid E strains has indicated 25 genes with significant difference. Characterization of the variations in the identity of the expressed proteins and the expression levels of the same protein between virulent and avirulent strains of R. prowazekii may provide a global view for better understanding of the virulence of R. prowazekii. Methods: The two-dimensional LC/MS method was used to identify proteins expressed in two different strains of Rickettsia. R. prowazkii Madrid E (avirulent) and Breinl (virulent) strains were purified by Dr. Dasch in Naval Medical Research Center. The purified cells were lysed followed by reduction, alkylation and trypsin digestion using standard procedures and analyzed with a modified version of the ProteomeX workstation. A strong cation exchange (SCX) column was used as the first dimension from which peptides were eluted onto a hydrophobic peptide-trap at high flow rate and desalted. The bound peptides were resolved on a reversed-phase column (75 µM ID) at a 200 nl/min flow rate. The packed tip column is placed directly in front of the mass spectrometer, limiting peak dispersion after the column. While peptides were eluted from one peptide trap and packed tip column, a second peptide trap was loaded with peptides from the SCX column and equilibrated, reducing analysis time by half. Results: Over 120 proteins could be identified in both samples. The identified proteins represent over 15% of the 834 open reading frames. Several proteins were unique to the virulent strain or the avirulent strain. GroEL chaperonin (60 kD), outer membrane protein B, peptidoglycan-associated lipoprotein precursor, elongation factor tu, and DNAK protein are among the proteins identified. These identified unique proteins maybe important for the difference in the virulence of Madrid E and Breinl strain. POSTER SESSION

P-180 Comparative Proteome Analysis of Two Pseudomonas aeruginosa TB Clone Isolates Found in Cystic Fibrosis Patients

C. Arevalo-Ferro1, J. Buschmann1, G. Reil2, A. Görg3, B. Tümmler4, L. Eberl1, and K. Riedel1 Technische Universität München, 1Department of Microbiology, 2Department of Chemical- Technical Analysis and Chemical Food Technology, and 3Proteomics Group, Freising- Weihenstephan, Germany; 4Klinische Forschergruppe, Medizinische Hochschule Hannover, Hannover, Germany

Cystic fibrosis (CF) is the most common severe inherited disease among the Caucasian population. The genetic lesion in CF causes an impaired epithelial chloride ion transport. This, in turn, leads to the production of sticky dehydrated mucus in the ducts of exocrine glands. As a consequence, mucociliary and alveolar clearing are impaired and colonization of the lung epithelium by opportunistic bacterial pathogens such as Pseudomonas aeruginosa is facilitated. Most individuals become chronically infected with a single clonal lineage (defining a set of genetically related strains) of P. aeruginosa. The genome of these clonal lineages often comprises pathogenicity islands, which contain large hypervariable clusters of virulence genes not present in closely related non-pathogenic strains. These gene islands often encode relevant functions for bacteria-host interactions, metabolic functions, or transporters. Comparative two-dimensional polyacrylamide gel electrophoresis (2-DE) was employed to study the intraclonal proteome diversity of the two P. aeruginosa TB clones TBCF10839 and TBCF121838, both isolated from CF-patients. These clonal variants differ dramatically in their pathogenic potential; in contrast to TBCF121838, clone TBCF10839 is highly virulent and capable to proliferate in polymorphonuclear granulocytes (PMNs). In this study we compared the protein patterns of the intracellular, extracellular, and surface protein fractions of TBCF10839 with those of TBCF121838. Proteins exclusively expressed in the TBCF10839 are expected to represent important virulence factors or gene products involved in special environmental adaptation. Work currently under way aims at the identification and further characterization of these proteins by means of mass spectrometry. POSTER SESSION

P-181 Characterisation of the Burkholderia pseudomallei Proteome

B. Crossett1, K.E. Keith1, P.C.F. Oyston2, M.I. Richards2, R.W. Titball2 and K. Brown1 1Centre for Molecular Microbiology and Infection, Department of Biological Sciences, Imperial College London, South Kensington Campus, London, UK. 2dstl, C.B.D., Porton Down, Salisbury, Wiltshire, UK

Burkholderia pseudomallei is Gram negative facultative anaerobe which is endemic to equatorial areas of the world between 20° North and South. It is capable of infecting a wide range of mammals, including humans, and is the causative agent of the disease melioidosis. The disease is particularly prevalent in South East Asia and Northern Australia. In North East Thailand, for example, it is estimated to cause 20% of community acquired septicaemia. Treatment of the disease is prolonged, the fatality rate can be as high as 40%, relapses are common and there is currently no licensed vaccine. In conjunction with the sequencing of the B. pseudomallei genome by the Sanger Institute a project to investigate the proteome was undertaken. B. pseudomallei was cultured under a variety of conditions. Following cell lysis, the proteome was fractionated by serial extraction with reagents of increasing solubilising power. The first fraction was extracted in 40 mM Tris, while the second fraction was extracted with 8 M urea, 4% (w/v) CHAPS, 40 mM Tris and 0.2% (w/v) Bio-Lyte 3/10 ampholyte. The third fraction was extracted with 5 M urea, 2 M thio urea, 2% (w/v) CHAPS, 2% (w/v) SB-3-10, 40 mM Tris and 0.2% (w/v) BioLyte 3/10 ampholyte. The samples were then analysed by two-dimensional SDS-PAGE. After staining, protein spots were excised using the GelPix robotic spot cutter (Genetix Ltd, UK). In gel trypsin digests were performed using a Multiprobe HT II liquid handling station (Packard Biosciences, UK) and Montage Zip plates (Millipore, UK). Protein identifications were assigned by MALDI-TOF peptide fingerprint analysis. POSTER SESSION

P-182 Mapping the Membrane Proteome of Burkholderia cepacia Strain J2315

K.E. Keith1, B. Crossett1, B. Bryne2, P.C.F Oyston3, N.F. Fairweather1, R.W. Titball3 and K.A. Brown1 1Centre for Molecular Microbiology and Infection, Department of Biological Sciences, Imperial College, London, UK. 2Membrane Protein Crystallography Group, Department of Biological Sciences, Imperial College, London, UK. 3dstl, C.B.D., Porton Down, Salisbury, Wiltshire, UK

B. cepacia is a Gram-negative phytopathogen first associated with a soft rot of onion bulbs. More recently B. cepacia has been intensively studied as a bioremediation agent and also emerged as an important opportunistic pathogen in immuno-compromised patients and patients with cystic fibrosis (CF). B. cepacia infection in humans is associated with a wide range of clinical outcomes from chronic symptomatic carriage to fulminant and sometimes fatal pneumonia. With prevalence rates in some CF centres as high as 40%. B. cepacia’s pathogenicity is multi-factorial and a number of secreted and membrane- associated proteins have been implicated. The membrane contains a number of antigenic proteins involved in host recognition and host-cell immune system evasion, the type III secretion apparatus and a wide range of potential drug and vaccine candidates. In this study, the methodologies required to isolate the B. cepacia membrane protein fraction have been optimised. The methods are also suitable for use with B. mallei and B. pseudomallei. Work has also been initiated using two-dimensional gel electrophoresis, spot excision, trypsin digestion and mass spectrometry analysis by MALDI-TOF to map the membrane proteome. The availability of annotated genome sequence will enable high throughput proteomic analysis of B. cepacia membrane proteins. Further work will aim to access the membrane protein proteome for immuno-reactive proteins using patient sera. POSTER SESSION

P-183 Investigation of the Metabolic Pathway of Phenol Degradation of an Indigenous Soil Pseudomonad by MALDI TOF-MS

I. Tsirogianni1, M. Aivaliotis1, M. Karas2 and G. Tsiotis1 1Division of Biochemistry, Department of Chemistry, University of CreteHeraklion, Greece; 2Institut für Pharmazeutische Chemie, Instrumentelle Analytische Chemie, Johann Wolfgang Goethe Universität, Frankfurt am Main, Germany

Large quantities of hydrocarbons are present in the environment, many of which must be removed from contaminated sites because of their toxicity. Bacteria, which have the capability of degrading aromatic compounds, are important in various procedures of bioremediation of waters, soils and municipal wastes [1]. These organisms induce the expression of a series of proteins that participate in the metabolic pathway of the degradation of aromatic pollutants, in order to use those compounds as the only carbon source for their survivor. Studies of the aerobic catabolism of phenol in bacteria have revealed that there are two major pathways, the meta and the ortho cleavage pathway [2]. The key enzymes catechol 2,3-dioxygenase (C2, 3O) and catechol 1,2 dioxygenase (C1, 2O) catalyze the extradiol and intradiol cleavage of catechols to 2-hydroxymuconate semialdehyde and muconic acid, respectively [3]. A new soil bacterium referred as Pseudomonas sp. Strain phDV1 is able to grow in phenol as sole carbon and energy source. The aerobic catabolic pathway was investigated by identification of enzymes involved in the metabolism of phenol. The key enzyme in the phenol degradation meta cleavage pathway C2, 3O was isolated using sucrose density centrifugation and anion exchange chromatography. The C2, 3O was detected and identified by absorption spectroscopy and MALDI TOF MS. Sucrose density centrifugation was used to reduce the complexity of the total protein mixture. 1D Tricine page electrophoresis separation in combination with MALDI TOF MS was used for the identification of the enzymes involved in the metabolic pathway [4].

References [1] Bouwer EJ., Zehnder AJB., (1993) Trends Biotechnol. 11: 360-7 [2] Harayama S, Kok M, Neidle EL., (1992). Annu Rev Microbiol. 46: 565-601 [3] Eltis LD, Bolin JT. (1996) J Bacteriol. 178: 5930-7. [4] Harry JL, Wilkins MR, Herbert BR, Packer NH, Gooley AA, Williams KL. (2000) Electrophoresis 6: 1071-81 POSTER SESSION

P-184 Initial Phase of a Proteome Project to "fuel” the Understanding of Gluconacetobacter diazotrophicus Physiology

L.M.S. Lery, F.C. Viana, M.R. Soares, S.C.S. Rossle, K.R.S. Teixeira, W.M.A. von Kruger and P.M. Bisch Rede de Proteômica do Estado do Rio de Janeiro, Unidade Multidisciplinar de Genômica, Universidade Federal do Rio de Janeiro, Rio de Janeiro - Brasil

Gluconacetobacter diazotrophicus is an N2-fixing, aerobic bacteria found as an endophyte in roots, stems and leaves. It was first isolated from sugar cane, but has been found in association with coffee plant, potato-candy and pineapple. A growth-limiting factor for any organism is its ability to fix nitrogen into innumerable nitrogen compounds, such amino acids and proteins. G. diazotrophicus is of great interest not only due its capacity to assimilate N2, but because it is a good model to study symbiosis of bacterium with plant. Also, is suggested that G. diazotrophicus produces plant growth hormones and bacteriocins. Moreover, it has an economical importance, since a better understanding of its physiology will allow the development of more efficient methods for its inoculation into vegetal species, increasing crop productivity. A given genome has information for the synthesis of various proteomes, in response to environmental stimuli. In an attempt to characterize the molecular events associated with different metabolic states of G. diazotrophicus, 2D gel electrophoresis was used to analyze global protein expression during phases of growth under both non-fixing and fixing conditions. In the non fixing condition the total amount of proteins expressed by bacteria is smaller than in its absence. Computer-aided analysis of 2D gels, revealed complex proteomes, with a differential expression of about 40 proteins between the initial and the logarithmic phases of growth, both in fixing and non-fixing conditions. Moreover, at least 25 proteins were specifically induced in the absence of nitrogen compounds in logarithmic phase of growth. Membrane associated processes appears to be of major importance for the bacterial metabolism, because many proteins were detected when outer membrane preparations were analyzed by SDS-PAGE. Initial analysis of periplasmic and culture supernatant proteins showed different expression pattern in different growth conditions. Mass spectrometry has allowed us to identify proteins from 2D gels, including a gluthatione synthetase, an important enzyme of aminoacids metabolism, and ModC, an ATPase of the molybdate transport system, witch may helps couple ATP hydrolysis to active molybdate transport. With this approach we hope to find, in the future, proteins from G. diazotrophicus that might be involved in biological processes of economic, agronomic and even social interest.

Financial support: FAPERJ, CNPq POSTER SESSION

P-185 Proteome Analysis of Methylobacterium extorquens AM1 - A Comparison of Cells Grown under Methylotrophic and Non-methylotrophic Conditions

M. Laukel1,2, G. Borderies3, M. Rossignol3, U. Völker4, and J.A. Vorholt1 1Laboratoire des Interactions Plantes-Microorganismes, LIPM, INRA-CNRS, Castanet,- Tolosan, France; 2Max-Planck-Institut für Terrestrische Mikrobiologie, Marburg, Germany, 3UMR 5546 CNRS/Université P. Sabatier, Castanet-Tolosan, France; 4Universität Greifswald, Greifswald, Germany

Aerobic methyltrophic bacteria are able to use reduced one-carbon compounds such as methanol and methane as growth substrates. These bacteria are widespread in nature and found in aquatic and terrestrial habitats as well as in the phyllosphere of plants. They are of ecological relevance with respect to the global carbon cycle, of interest for biotechnological applications due to the relatively inexpensive feedstocks methane and methanol, and for bioremediation processes of halogenated hydrocarbons. A central feature of the metabolism of methylotrophic bacteria is that the carbon flow proceeds via the central intermediate formaldehyde, which is highly toxic due to its nonspecific reactivity with proteins and nucleic acids. The knowledge concerning the biochemistry and physiology of methylotrophic bacteria has increased significantly with the discovery of a formaldehyde-oxidation pathway in the model methylotroph Methylobacterium extorquens AM1 [1,2]. This C1 transfer pathway is linked to tetrahydromethanopterin and methanofuran, that were previously thought to be restricted to methanogenic archaea. The availablity of the unfinished genome sequence of M. extorquens AM1 (http://www.integratedgenomics.com/genomereleases.html#list6) prompted us to investigate the proteome of M. extorquens AM1 under both methylotrophic and non-methylotrophic growth conditions (i.e. methanol-grown versus succinate-grown cells). Protein spots were analysed on 18 cm silver stained gels with overlapping pH ranges. Identification was realised by mass finger print analysis using ProteinProsector (http://prospector.ucsf.edu/) that was implemented for the Methylobacterium proteome. We identified 84 proteins that were induced upon methylotrophic growth, while 90 proteins were identified that were higher expressed in succinate-grown cells. 184 proteins were identified that are expressed to about similar levels under both growth conditions. Among the proteins that are overexpressed in the presence of methanol, we found methanol dehydrogenase, all the proteins known to be directly involved in formaldehyde oxidation to formate, and ten proteins known to be involved in formaldehyde assimilation. Our study identified new potential candidate proteins of methylotrophy that have not been studied so far.

[1] Chistoserdova L, Vorholt JA, Thauer RK, Lidstrom ME (1998) C1 transfer enzymes and coenzymes linking methylotrophic bacteria and methanogenic Archaea. Science 281:99-102. [2]Vorholt JA. (2002) Cofactor-dependent pathways of formaldehyde oxidation in methylotrophic bacteria. Arch. Microbiol. 178:239-249. POSTER SESSION

P-186 Identification of Copper-response Proteins in Methylococcus capsulatus (Bath) by 2-Dimensional Gel Electrophoresis

O.A. Karlsen1, F. Berven1, Ø Larsen1, A. Fjellbirkeland1, H. Dalton2, J.C. Murrell2, J.R. Lillehaug1 and H.B. Jensen1 1Department of Molecular Biology, University of Bergen, Norway; 2Department of Biological Sciences, University of Warwick,UK.

In the methanotrophic bacterium Methylococcus capsulatus (Bath), copper ions regulate the switch between the metabolic enzymes, the soluble methane monooxygenase (sMMO) and the particulate methane monooxygenase (pMMO). Copper ions are also required for the catalytic activity of the pMMO, and are necessary for the production of intracytoplasmic membranes. Clearly, copper is an essential nutrient in the biology of M. capsulatus. Recently, The University of Bergen and The Instititute for Genomic Research (TIGR) have in a collaborative effort sequenced the genome of M. capsulatus (www.tigr.org). A complete sequenced genome has initiated the work for characterisation of the proteome of the bacterium. In this study we have used 2-dimensional gel electrophoresis (2-DE) as an approach to map proteins differentially expressed due to variations in the copper concentration in growth medium. The identification of the genes encoding the differentially expressed proteins, may result in fundamental knowledge of the regulatory mechanisms of expression in M. capsulatus. POSTER SESSION

P-187 Proteomic Characterization of Frankia Cells During Hydrogen Evolving Stage

A. Mohapatra and A. Sellstedt* UPSC, Department of Plant Physiology, Umeå University, Umeå, Sweden

Frankia is a soil organism with the ability to fix nitrogen in free-living aerobic conditions as well as in symbiosis with higher plants. Hydrogenase is a common enzyme in Frankia. The hydrogen metabolism in Frankia is interesting in two aspects: in the efficiency of nitrogen fixation and in the production of hydrogen. Hydrogen is produced concomitantly as nitrogen is fixed via nitrogenase and an oxidising hydrogenase is advantageous for nitrogen fixation. On the other hand, in the absence of an active hydrogenase net hydrogen is produced via nitrogenase. A reversible hydrogenase can in vivo both oxidise and produce hydrogen. Both hydrogen uptake and production was recorded in Frankia. We present result that indicates the presence of a reversible hydrogenase in Frankia. Biological hydrogen metabolism has attracted great interest, as the clean source of energy, hydrogen, may be valuable as an alternative energy source to the fossil fuels used today. Hydrogenases, oxygen-sensitive enzymes that can make hydrogen gas, are key to the function of hydrogen-producing organisms, which become functional in anaerobic conditions. Here we have used a 2D Gel based proteomics method to the study of hydrogen evolution in Frankia. POSTER SESSION

P-188 A Comparative Analysis of the Mitochondrial Proteome Expression in Chlamydomonas reinhardtii Grown under Iron Deficient/Sufficient Conditions

R. Jain and M. Eriksson Umeå Plant Science Center (UPSC), Umeå University, Umeå, Sweden

Iron deficiency is an actual problem for plants and other biological system as it can lead to impairment of many important enzymes. The aim of this study is to characterize the response to iron deficiency in plants, a mechanism that is poorly understood. As a model system w e have used the eukaryotic unicellular green alga Chlamydomonas reinhardtii, which is an excellent system to study cellular functions in a photosynthetic organism. We have profiled the mitochondrial proteome of C. reinhardtii grown under iron deficient/sufficient medium by 2DE, DIGE, and MALDI for a comparative analysis of protein expression under these conditions. POSTER SESSION

P-189 Investigation of Plasma Membrane Proteins in Chlamydomonas reinhardtii under Different Iron Nutrition

I. Reinhardt1, S. Haebel2, A. Herbik1 and T.J. Buckhout1 1Humboldt-Universität zu Berlin, Institut für Biologie, AG Angewandte Botanik, Berlin, Germany; 2Universität Potsdam, Interdisziplinäres Forschungszentrum für Biopolymere, Golm; Germany

The unicellular green algae Chlamydomonas reinhardtii is used as a model to study the mechanism of adaptation to Fe deficiency stress in plants. A proteomics approach has been chosen to gain insight into the molecular events at the plasma membrane that are involved in Fe metabolism. Chlamydomonas cells were cultivated under different Fe nutritional conditions and were fractionated into soluble, microsomal and plasma membrane fractions. The plasma membrane (PM) was of preferential interest for studying changes in Fe uptake. The PM vesicles were purified by differential centrifugation and subsequently by two-phase partitioning of microsomal membranes. Proteins from the PM and soluble fraction could be separated reproducible on 1D and 2D-gels. In both fractions a number of polypeptides were induced or repressed under Fe deficiency. For identification of these proteins, either the masses of tryptic peptides or the amino acid sequences determined by MALDI-TOF MS or ESI-MS/MS were used for searching databases. In this study we present the current state of our analysis of plasma membrane proteins. Only few proteins were strongly regulated by the Fe nutrition status of the cell, and only a subset of these were unique to cells grown without Fe. Combining mass spectrometric analyses with in silico analyses of EST databases, the corresponding genes could be assigned to all proteins. For most proteins, changes in translation at the protein level corresponded to changes in gene transcription at the mRNA. However at present, the function of most of the proteins, whose synthesis changed in response to the Fe nutritional status, is still unknown. POSTER SESSION

P-190 H+-ATP Synthase Dimers in the Chloroplast of Chlamydomonas reinhardtii

H. Seelert1, S. Rexroth1, J. Meyer zu Tittingdorf1, H. Schwaßmann1, F. Krause1, R. Schlichting2 and N.A. Dencher1 1Physical Biochemistry and 2Institute of Botany, Darmstadt University of Technology, Darmstadt, Germany

While most proteomic approaches focus merely on expression levels, protein localization and protein interactions play important roles for the metabolic function. The H+-ATP synthase located in unstacked regions of the thylakoid membrane has a central role in chloroplastidic ATP production. While dimeric or oligomeric association of ATP synthase complexes in mitochondrial inner membrane was demonstrated by electron microscopy and biochemical investigations, chloroplast ATP synthase was proposed to exist merely as a monomer. The ATP synthase dimer was proposed to be a unique feature of mitochondria. Functions like protein complex stability, control of enzymatic activity and the formation of tubular cristae inside the mitochondria have been connected to dimerization of ATP synthase [1]. However, it is surprising that the closely related protein complexes show severe differences in structural organization. Using optimized isolation conditions we observed the dimer of chloroplast ATP synthase. By 2D-BN/SDS-PAGE and protein analysis by peptide mass fingerprinting the dimer was unambiguously identified. While in mitochondria dimer specific subunits were found, the subunit composition of chloroplast ATP synthase is not altered by dimerization. In comparison to the stable mitochondrial dimer the chloroplast ATP synthase dimer is very fragile.

Reference: [1] Schägger, H., Biochim. Biophys. Acta 2002, 1555, 154-159. POSTER SESSION

P-191 Ecs, an ABC Transporter, Affects Protein Secretion in Bacillus subtilis

T. Lundén, E. Lindberg, M. Sarvas and V.P. Kontinen National Public Health Institute (KTL), Vaccine Development Laboratory, Helsinki, Finland

We have identified an ABC transporter, Ecs, in the gram-positive bacterium Bacillus subtilis [1, 2]. Ecs regulates both protein secretion and expession of at least some exoprotein genes. ecs is a three cistronic operon. Mutants of the ecsA and ecsB cistrons are defective in secretion of some overproduced model proteins and also in sporulation and they do not become competent for DNA uptake. Mutations in ecsC do not show any phenotype. These results suggest that Ecs is an important component of the global regulatory network of B.subtilis. So far it is unknown whether Ecs is an importer or exporter and the compounds it transport remains to be found. Our aim is to characterize molecular mechanisms of action of Ecs. In the present work the effect of different ecs mutations on the secretion of endogenous exoproteins was studied with two-dimensional electrophoresis and the results were compared to the transcriptome of same mutants. 2-DE results indicated decreased levels of several exoproteins in cultures of ecs mutants compared to the wild type. DNA macroarray analysis of transcriptomes of ecs mutants indicated several changes in gene expression patterns. However, there was no effect on the epression of genes encoding exoproteins. This indicates that the Ecs-dependent regulation of protein secretion takes place mainly at a post-transcriptional level.

References: [1] Leskelä, S., Kontinen, V. and Sarvas, M. (1997). Microbiology 142: 71-77 [2] Leskelä, S., Wahlström, E., Hyyryläinen, H-L, Jacobs, M., Sarvas, M., Kontinen, V.P. (1999). Mol. Microbiol 31: 533-544 POSTER SESSION

P-192 Application of Protein-protein Interaction Approaches to Study the Global Network in Nitrogen Control of Corynebacterium glutamicum

M. Silberbach, J. Strösser, R. Krämer and A. Burkovski Institute for Biochemistry, University of Cologne, Cologne, Germany

Corynebacterium glutamicum is a Gram-positive soil bacterium with outstanding industrial importance based on its capability to produce high amounts of L-glutamate, L-lysine and several other amino acids. Since the cellular metabolism and especially the biosynthesis of amino acids is strictly dependent on the availability of nitrogen sources, the understanding of the corresponding regulatory mechanisms is essential. The main components of nitrogen assimilation and pathways regulating nitrogen metabolism have been identified during the last few years [1, 2, 3]. The aim of this project is to complete the current knowledge about nitrogen metabolism in C. glutamicum by functional analysis of protein-protein interactions. For this purpose, we started an approach to establish a Tandem Affinity Purification (TAP) protocol for C. glutamicum analogous to the TAP method developed for Saccharomyces cerevisiae [4, 5] The C. glutamicum method will be validated by the investigation of known interactions first and extended to a general approach later. Besides this molecular biology based assay, we started a biochemical approach in parallel, the co-immunoprecipitation of proteins by magnetic beads. As a model, we used the GlnK protein. GlnK as the central transmitter protein of the nitrogen control is subject to several modification/demodification processes which change its function and binding properties. First results show the applicability of this method.

References: 1. Schmid R. et al. (2000): Response to nitrogen starvation in Corynebacterium glutamicum. FEMS Microbiol Lett 187: 83-88 2. Nolden L. et al. (2001). Sensing nitrogen limitation in Corynebacterium glutamicum: the role of glnK and glnD. Mol Microbiol 42: 1281-1295 3. Meier-Wagner J. et al. (2001). Multiplicity of ammonium uptake systems in Corynebacterium glutamicum: role of Amt and AmtB. Microbiology 147: 135-143 4. Puig O. et al. (2001). The Tandem Affinity Purification (TAP) Method: A general procedure of protein complex purification. Methods 24: 218-229 5. Gavin A.-C. et al. (2002). Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415:141-147 POSTER SESSION

P-193 Towards a Database of Very Alkaline Corynebacterium glutamicum Proteins

C. Lück1, G. Reil2; H. Parlar2 and A. Görg1 Technische Universität München, 1FG Proteomik and 2Lehrstuhl für Chemisch-Technische Analyse und Analytische Lebensmitteltechnologie, Freising-Weihenstephan, Germany

While genomics has been a tremendous challenge, the analysis of the corresponding proteomes is technically even more challenging due of the fact that the number of proteins expressed at a given time in a cellular system is at least several thousands for ‚simple‘ procaryotic organisms, and up to 50,000 in eukaryotes. The major challenges for successful proteome analysis are: (i) the ability to analyze very alkaline and/or hydrophobic proteins with high resolution under steady-state conditions, (ii) the ability to detect minor components in the presence of large quantities of housekeeping proteins, (iii) methods for protein quantitation that are sensitive, rapid, simple, reliable and inexpensive, iv) simplification and automation of protein separation procedures and the ability to perform high-throughput analysis. During the past ten years, 2-D electrophoresis with immobilized pH gradients (IPG-Dalt) has constantly been refined to accomplish at least several of these goals, e.g. by the development of basic IPGs up to pH 12 for the analysis of very alkaline proteins, or the introduction of overlapping narrow IPGs to stretch the first dimension for higher resolution and the analysis of minor components, as well as the development of ready-made IPG strips and automated procedures [Görg et al. Electrophoresis 2000, 21: 1035-1053]. Corynebacterium glutamicum is a Gram-positive soil bacterium with outstanding industrial importance based on its capability to produce high amounts of amino acids such as glutamate and lysine, and which also serves as a model organism for mycolic acid-containing actinomycetes such as Mycobacterium tuberculosis and Corynebacterium diphtheriae. The aim of the present study was to establish a 2D gel database of the ‚alkaline‘ proteome of Corynebacterium glutamicum. Theoretical 2-D profiles calculated from its sequenced genome indicate that a considerable number (up to 30%) of proteins with pI values between pH 8 and pH 12 are present. In classical carrier ampholyte generated 2-D PAGE alkaline proteins can only be separated by non-equilibrium pH gradient gel electrophoresis (NEPHGE) [O’Farrell et. al., Cell, 1977], however at the expense of resolution and reproducibility. In contrast, well resolved, highly reproducible 2-D patterns of these proteins are obtained using steady-state IEF with immobilized pH gradients (IPGs). This is accomplished using either narrow range pH 10-12 or pH 9-12 IPGs, or non-linear, wide-range IPGs flattened at the basic end (IPG 4-12and IPG 6-12). Different optimization steps with respect to protein extraction and prefractionation, IEF running conditions, pH gradient engineering and gel composition had to be performed to obtain highly reproducible 2-D patterns. 2-D gel separated proteins were identified by MALDI- ToF-MS (Ettan MALDI TOF, Amersham Biosciences). A 2-D database of very alkaline Corynebacterium glutamicum proteins has been initialized and is continuously updated.

This study was supported by the BMBF project ‘New and Efficient Technologies for Functional Proteome Analysis’ POSTER SESSION

P-194 A Proteomic Approach to the Understanding of Bacterial Biofilms

K. De Vriendt, B. Devreese and J. Van Beeumen Laboratory for Protein Biochemistry and Protein Engineering, Ghent University, Ghent, Belgium

It is now a common belief that in natural, industrial and medical habitats, bacteria do not grow as free-living individuals (planktonic cells). They rather exist as an organized, sessile community, called a biofilm. Biofilms are formed by attachment of cells to solid material and their spreading along the surface. They can occur on a wide variety of surfaces, including catheters in hospitals where they provide a risk to nosocomial infections. Biofilms are marked by specific developmental stages and by some phenotypical differences in comparison to planktonic cells, but how these differences are linked to a different protein profile is still poorly understood [1]. In this work, we used the gram-negative bacteria Shewanella oneidensis MR-1 as a model to study bacterial biofilm. S. oneidensis can grow aerobically and anaerobically, using different organic and inorganic compounds as electron acceptor, including the metals Fe(III) and Mn(IV). S. oneidensis is, as a biofilm, able to colonize metal pipes, leading to corrosion and enormous costs for the industry [2]. To unravel the phenotypical differences and to identify the differentialy expressed proteins between the S. oneidensis biofilm cells and the planktonic cells, a proteomic approach using 2-D polyacrylamide gel electrophoresis (2-D PAGE) in combination with mass spectrometry, was set up, aided by the fact that the genome of S. oneidensis has been completely sequenced, thus facilitating protein identifications [3]. The biofilm was grown in Luria-Bertani medium on silicone tubings. The bacteria were circulated through the tubings for 7 days to complete saturation of the inner surface. Planktonic cells were grown overnight up to the stationary phase in Luria-Bertani medium in shaking flasks. For both types of cells, three replicates were prepared to be analyzed simultaneously. The soluble proteins were extracted with a 9M Urea/4% Chaps lysis buffer, separated using 2-D PAGE and visualized using Coommasie Brilliant Blue staining. After 'in-gel' digestion with trypsin of selected spots, the peptides were analysed by mass spectrometry. To do this, we used a novel MALDI-TOF/TOF mass spectrometer (4700 Proteomics Analyzer, Applied Biosystems), which allows acquiring high quality MS/MS spectra of selected peptides. Protein identifications are in progress.

References: [1] Stickler D., 1999, Curr. Opin. Microbiol. 2: 270 - 275 [2] Potekhina JS, et al, 1999, Appl. Microbiol. Biotechnol 52: 639 - 646 [3] Heidelberg JF., et al, 2002, Nat. Biotechnol. 20: 1118 - 1123 POSTER SESSION

P-195 Novel Glutathione S-Tranferase Proteome Analysis in Biofilms and Microorganisms by HPSEC and Fluorescence Microscopy after Reactions with Specific Fluorescence-labelled Antibodies

E. Hoque, J. Fritscher and G. Teichmann GSF – Forschungszentrum für Umwelt und Gesundheit, Institut für Hydrologie, Neuherberg, Germany

While exploring stress ecological significance of microbial communities in cold sulfidic and anthropogenically-influenced spring waters, we investigated Glutathione S-Transferase (GST) as main biomarker detoxifying proteomes. For this purpose, we developed a simple fluorescence-based HPSEC and microscopic immunogenic proteome analysis method for GST proteomes (crude, cytosolic and microsomal proteins; native proteins) in biofilm matrix and microorganisms, especially in the major biofilm-constituting fungus Mucor hiemalis strain EH5 adapted to low-temperature and sulfide-sensitive ecosystem of cold sulfidic spring waters. The biocompatible HPSEC system consisted of a polymeric hydrophilic gel as stationary phase, UV-VIS and fluorescent detectors. The column was run in isocratic mode with 20 mM NaH2PO4, 200 mM NaCl and 0.1% methanol (total ionic strength: 0.22 M) as a mobile phase and calibrated with molecular weight standards. Results showed appearance of fluorescently- Alexa-Flour®‚ labelled GST-antiGST-complex peaks with concomitant reduction of GST peaks, when GST standards and protein fractions of biofilms and M. hiemalis EH5 are pre-treated with fluorescently-labelled polyclonal antiGSTs. Online detection of absorption peaks at 214, 260 and 495 (λex for Alexa-Fluor®) nm as well as specific fluorescence at 519 nm allowed detection of GSTs and GST-AntiGST-complexes from crude, cytosolic and microsomal protein fractions as well as of antiGSTs. Detected signals of GST-AntiGST-complexes correlated with GST activities of these fractions. Additionally, high-resolution fluorescence microscopy showed the occurrence of GST proteomes in native states, especially in the activated fungal spores of Mucor hiemalis EH5. Results of GST proteome analysis are discussed in connection with special ecotoxicology of cold sulfidic spring waters. POSTER SESSION

P-196 Proteomics Analysis of Insecticidal Toxin Proteins from Novel Egyptian Bacillus thuringiensis Isolates

W.S.A. Maaty1, K. Ray2, S.A. Mostafa1, H.A. El-Itriby1, and M.A. Madkour1

1Agricultural Genetic Engineering Research Institute, Giza, Egypt; 2Department of Molecular and Cell Biology, Michigan State University, East Lansing, USA

Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS) was used to identify and characterize the crystal protein toxins contents from different novel Egyptian Bacillus thuringiensis (Bt) strains. 12 pairs of gene specific primers were used to detect the presence of any known cry genes. Furthermore, the 130 and 65 kDa bands from the 5 Bt isolates were cut and trypsin digested. The peptide masses, obtained after in-gel trypsin digestion for all these proteins, matched correctly to the corresponding proteins. Also, LC/MS/MS was able to resolve and identify multiple Cry toxins of very similar molecular weights and highly similar isoelectric points, from a single protein band. Furthermore, in novel Bt strains for which PCR techniques were unable to detect the cognate genes, this method was able to detect wide range of Cry toxins in the novel isolates e.g., Cry1Ca, Cry1Aa, Cry1Ab, Cry9Aa, Cry2Ac, Cry1Ac, Cry1Ad, Cry2Aa, Cry1Ea, Cry1Ag and Cry1Ba. Hence, present data clearly suggest that LC/MS/MS could be used as a tool for identifying Cry toxins from novel Bt strains. POSTER SESSION

P-197 Supramolecular Organization of COX- and AOX-Dependent Respiratory Chains in the Filamentous Fungus Podospora anserina

F. Krause1, C.Q. Scheckhuber2, A. Werner2, S. Rexroth1, N.A. Dencher1 and H.D. Osiewacz2 1Department of Chemistry, Darmstadt University of Technology, Darmstadt, Germany; and 2Botanical Institute, Johann-Wolfgang Goethe-University, Frankfurt am Main, Germany

To elucidate the molecular basis of the link between respiration and longevity we have studied the organization of the respiratory chain of a wild-type strain and of two long-lived mutants of the filamentous fungus Podospora anserina. This established aging model is able to respire by either the standard or the alternative pathway. In the latter, electrons are directly transferred from ubiquinol to the alternative oxidase (AOX) and thus by-pass complex III. We show that the cytochrome oxidase pathway is organized according to the mammalian "respirasome”- model [1]. In contrast, the alternative pathway is composed of distinct supercomplexes of complexes I and III, i.e., I2 and I2III2, which have not been described so far. Enzymatic analysis reveals distinct functional properties of complexes I and III belonging to either COX- or AOX- dependent pathways. By a gentle colourless-native PAGE almost all of the ATP synthases were isolated in the dimeric state. These data are of significance for the understanding of both respiratory pathways as well as of lifespan control and aging.

Reference: [1] Schägger, H. and Pfeiffer, K. (2000) EMBO J., 19: 1777-1783. POSTER SESSION

P-198 Reconstuction of Carbohydrate Catabolism in Rhodopirellula baltica by Proteomics

D. Gade1, D. Theiss2, H. Lehrach2, R. Amann1, J. Gobom2 and R. Rabus1 1Max-Planck-Institute for Marine Microbiology, Bremen, Germany; 2Max-Planck-Institute for Molecular Genetics, Berlin, Germany

Rhodopirellula baltica is a member of the deep-branching phylum Planctomycetes. These bacteria are abundant in the marine water column and possible keyplayers in the breakdown of polysaccharides, the dominant constituents of biomass. R. baltica was selected as a promising model organism since it is nutritionally specialized in carbohydrates. R. baltica was adapted to single carbohydrates by repeated passaging, and metabolic adaptation was studied by two-dimensional gel electrophoresis (2DE). Differences in protein abundance were determined with the 2D DIGE system. Protein identification was achieved by MALDI-TOF-MS. Eight different carbohydrates were used as single growth substrates. In comparison to the protein pattern of glucose grown cells, several distinct proteins were highly upregulated in response to each of the other growth substrates. Identification of these regulated proteins revealed mostly dehydrogenases. In addition the central metabolic routes of R. baltica were reconstructed from more than 200 apparently constitutively synthezised proteins. With only two exceptions, all enzymes of glycolysis were identified. Except for citrate synthase and fumarase, all enzyme of the TCA cycle were detected. Almost all enzymes of the pentose phosphate cycle were identified, whereas no indication for the presence of the Entner Doudoroff pathway was found. These results are in full agreement with the in silico predictions from the genome sequence of R. baltica. POSTER SESSION

P-199 Expression Proteomics of Soluble Proteins in two Leishmania infantum Isolates with Different Virulence

M.A. Dea-Ayuela1, S. Rama-Iñiguez1, A. Pitarch2 and F. Bolás1 1Department of Parasitology, and 2Department of Microbiology II, Faculty of Pharmacy, Complutense University, Madrid, Spain

Leishmania spp. are obligated protozoan parasites of mammalian macrophages. L. infantum is the causative agent of a zoonotic disease affecting dogs and humans in the Mediteranean basin, varying from simple self-healing cutaneous lesions to a severe visceral dissemination. In order to obtain an overall view of the impact of an avirulence isolate on proteome of Leishmania infantum and examine the underlying changes in gene expression, a proteomic approach was implemented. Soluble proteins of stationary promastigotes of two L. infantum isolates with different virulence were extracted by sonication, and subsequently analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using an immobilized linear pH 4-7 gradient for isoelectric focusing. A total of 500 major protein spots were resolved in this pH range. As expected, comparative analysis of 2-D gels of both isolates revealed different profiles of protein expression depending on the strain used. Differential expressed proteins were excised, in-gel digested with trypsin and then identified by mass spectrometric techniques. Since not many L. infantum proteins are annotated in the public domain databases, no conclusive identifications were yielded by means of peptide mass fingerprinting. Thus, matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI- TOF/TOF MS) analyses were carried out in an attempt to attain further sequence information for the protein spots of interest. We unambiguously characterized several specific proteins, such as (i) a putative L. infantum mitochondrial peroxiredoxin that was highly expressed and only stained in gels from the virulent isolate, or (ii) a protein with high homology to a L. major protein encoded by a gene with unknown function that was only detected in the avirulent isolate. This approach represents a first step towards the characterization of L. infantum genes associated with virulence or pathogenicity. POSTER SESSION

P-200 Proteomic Analysis of Leishmania mexicana Differentiation

S.A. Karsani1, J. Tempero1, R. Wait2, D.F. Smith1, and P.G. Nugent1 1Wellcome Trust Laboratories for Molecular Parasitology, Centre for Molecular Microbiology and Infection, Imperial College, London; UK.; 2Faculty of Medicine, Kennedy Institute of Rheumatology, Imperial College London, UK

The parasite Leishmania causes a spectrum of human diseases, the Leishmaniases, with a range of clinical symptoms. Leishmania are digenetic protozoa that inhabit two different hosts – as flagellated promastigotes in the gut of the sandfly vector and as intracellular amastigotes in phagolysosomes of mammalian macrophages. The genus Leishmania is endemic in at least 88 countries across tropical and subtropical regions of Africa, the Americas, the Indian subcontinent, the Mediterranean and South-west Asia. In all, 350 million people are at risk of infection, with 15 million people infected and over 2 million new cases annually. While Leishmaniasis is a treatable disease, current drugs have toxic side effects and are difficult to administer. There is no effective vaccine to-date. We are using proteomic approaches to identify differentially expressed proteins in the amastigote stage of Leishmania mexicana, as new targets for drug and vaccine development. This species can be induced to differentiate in culture and the amastigotes can be purified free of contaminating host proteins. The Leishmania Genome Sequencing Project (www. sanger.ac.uk/Projects/L_major/) has predicted more than 7000 ORFs over the 36 chromosomes of the 36 Mb genome of L. major. Two-dimensional electrophoresis has been carried out on cell lysates using three overlapping pI ranges (3-6, 4-7 and 6-11). More than 2000 individual protein spots have been resolved, and the expression of 67 protein spots found to be developmentally regulated in the amastigote stage. Twenty-two spots are upregulated, 27 down regulated and 18 uniquely expressed when compared with the metacyclic stage proteome. The identity of these and marker proteins is being determined by mass spectrometry. POSTER SESSION

P-201 Analysis of Ixodes ricinus (Acari: Ixodidae) Nymphs by Two Dimensional Gel Electrophoresis

J. Vennestroem Department of Genetics and Microbiology, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark

I. ricinus is vector of different pathogens e.g. Borrelia and Ehrlichia causing diseases in animals and humans. The three year life cycle of the tick consists of three instars. Developments from one instar to the next demands a blood meal from a host. In the time span of the individual instar the life is in the vegetation where the tick lives of the resent acquired blood meal. The term physiological age is used in describing the amount of reserve nutrition left from the blood meal. Assessment of physiological age during the nymph and adult instars of I. ricinus are important when describing behavior and activity of ticks. These parameters play a key role in transmission analysis of pathogens. Traditionally, physiological age is based on visual estimates of morphological characters. To estimate the quality of the standard method for determination we wished to have biochemical verification. The verification was conducted by 2D-electrophoresis. 29 ticks were analyzed and compared. 20 proteins were found to be important for age determination of ticks. The results demonstrate a high concordance between the two methods. Some of the 20 proteins were analyzed by MALDI-TOF-TOF. We further wish to analyze other interesting proteins by MALDI-TOF-TOF to reveal their specificity and to become better acquainted with the physiological parameters which are turned on and of during the ageing of an individual instar. POSTER SESSION

P-202 A Web-Based Proteome Database System for Microbial Research

K.-P. Pleißner, T. Eifert, S. Buettner, F. Schmidt, M. Boehme, S.H.E. Kaufmann, T.F. Meyer, and P.R. Jungblut Max Planck Institute for Infection Biology, Berlin, Germany

Classical proteome investigations such as 2-DE / MS and LC-MS / MS result in complex data sets containing information on thousands of proteins. Laboratory information management systems (LIMS) are often used to acquire and to control experimental data. Bioinformatics approaches have to be applied to analyze and to disseminate the huge amount of information. Existing proteome databases organized in flat-file format limits the application of intelligent data mining methods. The usage of relational database concept overcomes these restrictions. We have developed a web-accessible, relational proteome database system combined with a LIMS for microbial research. The LIMS is connected to the database system via a graphic user interface written in Java. The database system comprises a 2-DE gel protein database "2D- PAGE”, an isotope coded affinity tag LC-MS database and a functional classification database for proteins of Mycobacterium tuberculosis, Helicobacter pylori and other microorganisms. The databases are hyperlinked with public genomic, metabolic and other knowledge sources in molecular biology such as NCBI, KEGG, BioCyc and PEDANT. Different access methods such as clicking on a spot in a 2-DE gel or the formulation of complex biological questions enable an intelligent data mining. Additionally, annotations of proteins and information on protein identification by peptide mass fingerprinting may be retrieved. The database system was constructed using MySQL (http://www.mysql.com/) and open source software tools. Applying the language for statistical computing and graphics R (http://www.r-project.org/) a comprehensive analysis and high-quality visualization of data stored in the databases has been accomplished. Thus, virtual 2-DE plots based on theoretical pI / Mr-values and overlaid with the results of database interrogation may be dynamically generated. Furthermore, linear/non-parametric regression models that fit the GC-content vs. pI-values or histograms of the GC-content distributions may be also calculated on-the-fly. The proteome database system is accessible at http://www.mpiib-berlin.mpg.de/2D-PAGE and includes proteome information on seven microorganisms. POSTER SESSION

P-203 Towards to the Paracoccus denitrificans 2-DE Protein Database

P. Bouchal1, P. Precechtelova1, Z. Zdrahal2 and Igor Kucera1 1Department of Biochemistry, 2Laboratory of Mass Spectrometry of Biomolecules, Faculty of Science, Masaryk University, Brno, Czech Republic

Paracoccus denitrificans is a non-fermentative, facultatively autotrophic soil bacterium often studied in the field of bioenergetics, particularly due to resemblance of its aerobic respiratory chain to that of mitochondria. Also an aspect of a great nutritional adaptability was discovered, related to the ability of exploiting various electron donors and electron acceptors for maintenance and growth. To discuss mechanisms underlying regulation of gene expression by growth conditions, a high-throughput proteome mapping is one of the most effective tools to-date. For 2-DE gel analysis of whole-cell extract and its membrane fraction we optimized new methods using immobilized pH gradient. Before the whole-cell proteome analysis, cells were disrupted using french-press. Released proteins were precipitated by ice-cold acetone and the pellet was solubilized using 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 2 mM tributylphosphine, 15 mM TRIS base, 0.8 % (v/v) Bio-Lyte 3/10 and 0.8 % (v/v) Bio-Lyte 8/10. For analysis of membrane fraction, cells were disintegrated by osmotic and enzymatic lysis, membrane proteins were than extracted using sample solution containing 7 M urea, 2 M thiourea, 1 % ASB 14, 1 % TRITON X-100, 2 mM tributylphosphine, 15 mM TRIS base, 1 % (v/v) Pharmalyte 3/10 and 0.5 % (v/v) Pharmalyte 8/10. Mixture of broad range (3-10) and basic (8-10) ampholytes improved resolution of some basic proteins, especially in the membrane fraction. After in-gel rehydration, proteins were separated by isoelectric focusation using nonlinear immobilized pH gradient (pH 3-10). In the second dimension, 12 % homogenous SDS-PAGE gels were used. Whereas the analysis of whole-cell extract using Laemmli buffer system provided good results, it was necessary to use taurine buffer system for the successful analysis of membrane fraction. Image analysis was performed and spot database created by PDQUEST software. We were able to detect 306 protein spots in the standard whole- cell gel and 515 protein spots in the standard membrane gel. Among them, 36 protein spots have been already analyzed by peptide mass fingerprinting, eventually also by MALDI-post source decay or liquid chromatography-tandem mass spectrometry. However, the genome of P. denitrificans have not been completely sequenced yet and, currently, information about only 97+127 proteins is available from Swiss-Prot/TrEMBL database. Due to this fact, we were able to identify only 6 proteins up to-date. Among them, nitrite reductase is a well-known enzyme involved in denitrification. In order to follow the dynamics of protein expression under different growth conditions, we compared complex protein composition of whole cells of P. denitrificans cultivated (i) aerobically, (ii) anearobically with nitrate (as electron acceptor), (iii) anaerobically with nitrite, (iv) anaerobically with nitrous oxide. We also observed effect of azide on protein expression, which is known to induce some denitrification enzymes under aerobic conditions. For this reason, we compared protein composition of whole cells as well as its membrane fraction grown (i) aerobically, (ii) aerobically with addition of 0.4 mM sodium azide, (iii) anaerobically with nitrate, (iv) anaerobically with nitrite (whole cells only), (v) anaerobically with nitrite and addition of 0.4 mM sodium azide (whole cells only). Student t-test that is implemented in PDQUEST 6.2 was used for determination of significant differences at the levels of p < 0.05; the results are shown. For example, we found that azide induces 5 proteins of the whole-cell extract and 10 membrane proteins. We also compared expression data of nitrite reductase obtained by proteome analysis with measurement of nitrite reductase enzyme activity. These data were in a good agreement.

This work was supported by grants No. 203/01/1589 from the Grant Agency of Czech Republic and No. 740/2002 from Czech Ministry of Education. POSTER SESSION

P-204 A Two-dimensional Electrophoresis Protein Database for Nicotiana tabacum cv. Bright Yellow-2 (BY-2)

K. Laukens, P. Deckers, H. Van Onckelen and E. Witters University of Antwerp, Dept. Biology, Antwerpen, Belgium

The cell line Nicotiana tabacum cv. Bright Yellow-2 (tobacco BY-2) is widely used as a model system to study the growth and development of plant cells. By means of two-dimensional gel electrophoresis and electrospray tandem mass spectrometry the proteome of this cell suspension culture is investigated. With an optimized protocol for sample preparation and 2-DE a reference gel was produced (pI range 3-10). An automated LC ESI QTOF MS-MS set-up allowed the identification of a range of spots from this map. The obtained data were integrated in a federated 2-DE database, which can be accessed at http://tby2-www.uia.ac.be/tby2/. On the online reference map the identified protein spots are hyper-linked to individual protein entries. Each protein entry contains all identity information, as well as links to relevant entries in other public databases. The database can easily be queried with keywords by using the built-in search functions. This BY-2 proteome database is continuously being expanded with more data. New identified protein spots are added regularly. Gels from other pI ranges are being included. A specific topic of interest in our group consists of the BY-2 phospho-proteome, which is investigated using phospho-specific protein stains. The data arising from these experiments, as well as data from planned glyco-proteome studies will also be integrated in this database. A more narrow integration with the new tobacco EST-databases is another scope for the near future. Such an online proteome reference database can be a convenient source of protein information, especially for unsequenced but widespread model organisms like tobacco BY-2. The expansion and the inclusion of post-translational information in this resource will make this a powerful tool for anyone who is interested in the proteome of this system. POSTER SESSION

P-205 A Proteome Approach Defines Protective Functions of Tobacco Trichomes

S. Amme, B. Schlesier and H.P. Mock Institute of Plant Genetics and Crop Plant Research Gatersleben, Gatersleben, Germany

Trichomes are epidermal appendages typically found on the surface of the most terrestrial plants and which comprise a diverse set of structures in a multitude of forms and size. They are uni- or multicellular structures found on vegetative as well as on reproductive tissues. The process of trichome development is complex and involves genes that regulate their spacing, density and morphology. In addition intraspecific variations of trichome types and density are known in many species. Functionally, trichomes play a role in the protection against biotic and abiotic stresses and may also complement the chemical defense of a plant by possessing glands to exude toxic secondary metabolites, which include terpenoids, sucrose esters and alkaloids. The morphological characterisation of trichomes from Nicotiana tabacum was accomplished by scanning electron microscopy and showed several types as previously described [1, 2]: short trichomes with an unicellular stalk and a multicellular head and tall trichomes with a multicellular stalk possessing uni- or multicellular heads. Fluorescence microscopy demonstrated that chlorophyll is absent in short trichomes; however small quantities of this pigment are accumulated in the heads of the tall trichomes indicating residual photosynthetic activity. For biochemical analysis trichomes were mechanically abraded from leaves using glass beads of appropriate size and purified from contaminating leaf tissue by filtration. Trichomes from young and older leaves were separately harvested to investigate the influence of leaf development on the metabolite and proteome profile of trichomes. The purity of isolated trichomes was assessed using the amount of chlorophyll as a relative measure for contamination by leaf tissue. Analysis of the alkaloid nicotine demonstrated several-fold increased contents of this toxic compound in trichomes, relative to leaves. HPLC phenylpropanoid profiling showed a pronounced increase of the main compounds chlorogenic acid, the flavonoid rutin as well as of three other yet unknown compounds. As a global approach for the characterization of plant trichomes we performed a proteomic study on trichomes using 2-D gel electrophoresis to separate proteins of leaves and trichomes. Comparative image analysis of the protein patterns indicated a number of spots which were highly enriched in trichomes relative to leaves. These proteins represented potential candidates contributing to trichome-specific functions and were excised for identification by mass spectrometry. Trichome-specific expression of identified proteins was confirmed by Western blotting as well as by measuring enzymatic activities. Among the proteins identified so far, several are related to stress defence mechanisms.

References: [1] Akers CP, Weybrew JA, Long RC (1978) Amer J Bot 65: 282-292 [2] Meyberg M, Krohn S, Brühmer B, Kristen U (1991) Flora 185: 357-363 POSTER SESSION

P-206 Proteome Analysis of the Tobacco BY-2 Cell Cycle

P. Deckers, K. Laukens, H. Van Onckelen and E. Witters University of Antwerp, Dept. Biology, Antwerpen, Belgium

The highly synchronisable and cytokinin autonomous plant cell suspension culture tobacco Bright Yellow-2 (TBY-2) is widely used for studies of plant cell cycle, growth and other cellular events. Two-dimensional electrophoresis currently remains the method of choice for analysing ‘complete’ proteomes. Using two-dimensional "Fluorescence Difference Gel Electrophoresis” (2D-DIGE) different stages of the cell cycle are being studied. This differential display technology is based on the use of two different fluorescently labelled protein populations run on the same gel. The relative intensities of the differently coloured spots are indicative for the differential abundance of the proteins. Using a third fluorescent dye to label the standard, representing a known portion of all protein samples, multiple gel experiments can be done. Using this powerful technique, different kinds of experiments are running: - Comparative research of the different stages in the cell cycle - Study the effect of endogenous cytokinins on the G2-M transition after treatment with cytokinin-synthesis inhibitors - Multiplexed proteomics: by using different in-gel glycosylation stains and phosphoprotein stains, possible post-translation modification effects of cytokinins during G2-M transition in plant cell cycle are being investigated. Data generated from these experiments will be partially integrated in our BY-2 proteome database, which is already accessible at http://tby2-www.uia.ac.be. By analysing the BY-2 proteome using different strategies, we try to contribute to the existing knowledge of the regulation mechanisms in plant cell cycle. POSTER SESSION

P-207 A Proteomic Approach to Study Leaf Senescence in Arabidopsis thaliana

R. Hebeler1, K. Stühler1, P. P. Dijkwel2, H. E. Meyer1 and B. Warscheid1 1Medical Proteom-Center Ruhr-University Bochum, Germany; 2Department Molecular Biology of Plants, Research school GBB, University of Groningen, The Netherlands

Arabidopsis thaliana is a small flowering plant that is used as a model organism to study the onset of leaf senescence. In general, this process is controlled by leaf age but can be promoted significantly by ethylene within a specific age window [1]. Onset of leaf death (old) mutants of Arabidopsis thaliana have been studied by SAG expression (senescence associated genes), ion leakage and chlorophyll degradation. Advanced leaf senescence was revealed not only in ethylene-treated old2 plants but also in ethylene-treated as well as in air- grown old1 and old3 plants [1]. To obtain a deeper insight into the underlying processes, a genetic model has been developed linking the three OLD genes and ethylene into a regulatory pathway. Nonetheless, information on the protein expression levels is needed, and a strategy was developed to obtain protein profiles of Arabidopsis thaliana with normal and altered leaf senescence.

Plant tissues were processed under liquid N2 and in the presence of protease inhibitors to minimise protein degradation. After centrifugation of the cell extracts obtained, the soluble sample fraction was collected comprising mainly cytosolic proteins. Membrane and DNA associated proteins were extracted from the pellet by additional homogenation and centrifugation [2]. Protein fractions were further separated via two-dimensional gel electrophoresis (2-D gel) following the protocol by Klose et al. [3]. To obtain further information on the identity of the proteins separated, distinctive spots were cut off the gel, in-gel digested and tryptic peptides generated were extracted with 50% ACN/0.1%TFA for analysis by matrix assisted laser/desorption ionisation mass spectrometry (MALDI-MS). The analytical method developed in this study will be combined with in vivo 15N-labelling techniques to determine protein expression level alterations of mutants and the wild type of Arabidopsis thaliana.

References: [1] Jing, H.C., et al., Arabidopsis onset of leaf death mutants identify a regulatory pathway controlling leaf senescence. Plant J, 2002. 32: 51-63 [2] Giavalisco, P., et al., Extraction of proteins from plant tissues for two-dimensional electrophoresis analysis. Electrophoresis, 2003. 24: 207-16 [3] Klose, J. and U. Kobalz, Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome. Electrophoresis, 1995. 16: 1034-59 POSTER SESSION

P-208 Characterization of F-box Protein Mutants and Identification of their Degradation Substrates

K. Schwager, L.I.A. Calderon V., S. Knierer, C. Kuhnle und C. Schwechheimer Zentrum für Molekularbiologie der Pflanzen, Universität Tübingen, Germany

Ubiquitin–mediated protein degradation is an important regulatory mechanism in eukaryotes. Proteins are destined for degradation through the conjugation of poly-ubiquitin chains. E3 ubiquitin ligases (E3s) assure the specificity of the process in functioning as receptor for the degradation substrates. One type of E3 enzymes are SCF complexes, consisting of a SKP1 homologue, a Cullin, RBX1 and a F-box protein (FBP). The FBP is the subunit responsible for the substrate recognition. Remarkably more than 2% of the Arabidopsis genome, about 700 genes, encode for FBPs. However, only a few processes are known to be controlled by FBP- mediated proteolysis. Our lab is interested in identifying processes that are regulated by protein degradation. In this project, we analyse a family of four FBPs with unknown biological function. To identify their role in plant growth and development, we want to investigate this biological function using FBP mutants. As we expect that FBP degradation substrates accumulate in theses mutants, w e use different proteomic approaches to compare their protein patterns. These include 2D gelelectrophoresis, Differential gelelectrophoresis (DIGE) and Far Western on 2 D Gels. By Far Western we want to provide further evidence that differentially accumulated proteins represent FBP substrates. In order to identify FBP interactors like SCF complex subunits and FBP degradation substrates, we want to purify the FBP using Tandem–affinity-purification (TAP-tag). So far a FBP w a s cloned into a plant transformation vector containing the TAP-Tag (pEXSGTAP) by Gateway technology. Further analysis is in progress.

1. Gagne et al., The F-box subunit of the SCF E3 complex is encoded by a diverse superfamily of genes in Arabidopsis. PNAS (2002) 17: 11519-11524 2. Görg et al., The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis (2000) 21: 1037-1053 3. Yoshida et al., E3 ubiquitin ligase that recognizes sugar chains. Nature (2002) 418: 438-442 4. Rigaut et al., A generic protein purification method for protein complex characterization and proteome exploration. Nature Biotech. (1999) 17: 1030-1032 POSTER SESSION

P-209 Comparative Proteomics to Reveal Naturally Occurring Variability in Arabidopsis Accessions

F. Chevalier, A.-D. Devauchelle, S. Barteau, N. Sommerer, O. Martin and M. Rossignol Laboratoire de Protéomique, UR 1199, INRA, Montpellier, France

Plant evolution to a specific ecosystem results in a genetic adaptation to environmental conditions. These non-genetically modified plants could be of a great interest for physiologists and agronomists thanks to the natural diversity of their characteristic traits, limiting the use of highly restricting mutants or transformed plants. In this study, a comparative proteomics approach was used to establish genetic distance between eight different wild type accessions of the model plant Arabidopsis thaliana. The comparison of the total root proteome of the Col-0, Col-4, Cvi-0, Ler-1, Ws-1, Be-0, Rld-1 and Ll-0 accessions was carried out using two-dimensional electrophoresis (2-DE). Computer analysis of 2-DE gels permitted a precise quantification of protein patterns in each extract. A statistical treatment of spots volumes allowed a discrimination between genotypes in accordance with their phenotypical and physiological features. Common, variable and specific protein spots among the different accessions are used to identify those metabolic pathways affected by environmental status and to give insights into the underlying adaptative processes. Results confirm the utility of comparative proteomics in the genetic relationships establishment of species. POSTER SESSION

P-210 Proteomic Approach to Investigate Mitochondrial Protein Complexes in Arabidopsis thaliana

H. Eubel, U.K. Schmitz, L. Jänsch and H.-P. Braun Abteilung Angewandte Genetik, Universität Hannover, Hannover, Germany

Plant mitochondria have many unique functions which partially are related to photosynthesis, e.g. they have special possibilities to oxidize malic acid and glycine, they can synthesize several prosthetic groups, they have a very much branched respiratory chain, they have a comparatively large genome and they have unusual ways of importing proteins. Most of these functions are based on the presence of protein complexes. Using protein solubilizations with various detergents, protein separations by 2D blue-native / SDS PAGE and protein identifications by mass spectrometry, the protein complexes of plant mitochondria were systematically characterized. The obtained data allow new insights into the function of plant mitochondria: (i) the respiratory protein complexes I and III form a very stable supercomplex, that possibly regulates the alternative respiration pathway via the alternative oxidase (AOX), (ii) the complex III of the respiratory chain contains the two subunits of the mitochondrial processing peptidase and therefore is bifunctional, (iii) cytochrome c oxidase occurs in two different forms, that seem to differ with respect to activity, (iv) complex II contains several plant specific subunits of so far unknown function (v) the preprotein translocase of the outer mitochondrial membrane (the so-called TOM complex) only contains a single type of preprotein receptor, which occurs in different isoforms (vi) some plants contain a homodimeric 500 kDa complex representing acetyl-CoA carboxylase within their mitochondria and therefore are capable of complete de novo fatty acid biosynthesis. The unique features of these protein complexes will be discussed with respect to special functions of mitochondria in plant cells.

References: Eubel, H., Jänsch, L. and Braun, H.P. (2003) New insights into the respiratory chain of plant mitochondria: supercomplexes and a unique composition of complex II. Plant Physiol., in press. Werhahn, W., Jänsch, L. and Braun, H.P. (2003) Identification of novel subunits of the TOM complex from Arabidopsis thaliana. Plant Physiol. Biochem. 41: 407-416. Werhahn, W. and Braun, H.P. (2002) Biochemical dissection of the mitochondrial proteome from Arabidopsis thaliana by three-dimensional gel electrophoresis. Electrophoresis 23: 640- 646. Kruft, V., Eubel, H., Werhahn, W., Jänsch, L. and Braun, H.P. (2001) Proteomic approach to identify novel mitochondrial functions in Arabidopsis thaliana. Plant Physiol. 127: 1694-1710. POSTER SESSION

P-211 "Respirasome"-Like Supercomplexes in Mitochondria of Higher Plants

F. Krause, N. Reifschneider, D. Vocke, H. Seelert and N.A. Dencher Physical Biochemistry, Department of Chemistry, Darmstadt University of Technology, Darmstadt, Germany

Higher plant mitochondria are unique in many aspects compared with their animal and fungal counterparts. This is to a large extent related to the close functional interdependence of mitochondria and chloroplasts, in which the two ATP-generating processes of oxidative phosphorylation and photosynthesis, respectively, take place. Previously, results obtained by blue-native electrophoresis of digitonin-solubilized mammalian mitochondria led to proposal of the "respirasome”-model, which postulates that in the inner mitochondrial membrane the standard respiratory chain forms a network of the two supercomplexes I1III2IV4 and III2IV4 in a 2:1 ratio [1]. To date, nothing is known about the occurrence of supercomplexes in the branched plant respiratory chain, which has additional alternative respiratory enzymes besides the respiratory complexes I-IV. By analysis of digitonin-extracts from partially purified spinach mitochondria using blue- and colourless-native electrophoresis, we demonstrate that most of complexes I, III and IV are assembled into "respirasome"-like supercomplexes. Furthermore, a small proportion of the FOF1-ATP synthase is found in the dimeric state.

Reference: [1] Schägger, H. and Pfeiffer, K. (2000) EMBO J., 19:1777-1783 POSTER SESSION

P-212 Identification of Oxidised Mitochondrial Proteins by Immunoprecipitation and 2D-LC/MS/MS

B.K. Kristensen1, P. Askerlund2, N.V. Bykova3, H. Egsgaard1 and I.M. Møller1 1Plant Research Department, Risø National Laboratory, Roskilde, Denmark; 2Jönköping University, Jönköping, Sweden; 3Department of Physics and Astronomy, University of Manitoba, Manitoba, Canada

All stress phenomena are accompanied by an increased production of Reactive Oxygen Species (ROS) and this can lead to damage to proteins, lipids and DNA. We are developing methods for the identification of oxidised proteins in mitochondria, one of the major ROS- producing sites in the eukaryotic cell. We isolated highly purified mitochondria from green 7- day-old rice leaves. The mitochondria were sonicated and the matrix fraction isolated as the supernatant after centrifugation (100 000 g x 60 min). Part of the fraction was left untreated while the other part was subjected to a mild oxidative treatment (0.5 mM H2O2 + 0.2 mM CuSO4 for 10 min at room temperature). The oxidised proteins in both samples were tagged with dinitrophenylhydrazine (DNP), which forms a covalent bond with carbonyl groups. The DNP- tagged proteins were immunoprecipitated (IP) using anti-DNP antibodies linked to magnetic beads. The precipitated proteins were digested with trypsin and the mixture of peptides injected into a nano-HPLC system coupled online to an ESI-Quad-TOF mass spectrometer. The peptides were separated by stepwise ion exchange chromatography followed by reverse phase chromatography (2D-LC), and analysed by MS/MS. Proteins were identified by un- interpreted fragment ion database searches. Using this approach we identified around 20 and 60 oxidised proteins in the control and oxidised sample, respectively. Western blots of 2D-gels of the same samples prior to IP verified that the oxidation treatment increases protein oxidation also for specific proteins. We conclude that a number of proteins are oxidised in vivo and further identify a large group of proteins that are particularly susceptible to mild oxidation. POSTER SESSION

P-213 Proteomics of Metal Hyperaccumulator Thlaspi caerulescens

M. Tuomainen, A. Tervahauta, V. Hassinen, S. Lehesranta and S. Kärenlampi Institute of Applied Biotechnology, University of Kuopio, Kuopio, Finland

Introduction: Proteomics was used in this study to characterise proteins and protein networks related to plant metal exposure and accumulation. The hyperaccumulator plant Thlaspi caerulescens used for the molecular profiling belongs to the same taxonomic family (Cruciferae) as does Arabidopsis thaliana and Brassica, which improves the chances to identify the proteins of interest. The main objectives of the present study were to screen and compare protein patterns of various Thlaspi ecotypes to find and identify metal responsive proteins putatively related to metal hyperaccumulation. Materials and Methods: T. caerulescens ecotypes Monte Prinzera, Lellingen and La Calamine with different characteristics of metal transport and accumulation were exposed to Zn deprivation, 500 µM and 1000 Zn µM and 70 _M Cd in hydroponics. Total soluble proteins were extracted from the leaves and roots. A 2-dimensional electrophoresis was run and gels were stained with Sypro orange fluorescent stain. The protein pattern analysis was done with PDQuest software and using more thorough statistical analysis: variance, Kruskal-Wallis and principal component analysis. Proteins of interest were identified with HPLC-ESI-MS/MS mass spectrometry. Results: On the basis of PDQuest data, 33 interesting protein spots from leaves and 53 spots from roots were subjected to MS analysis. Of the analysed proteins 23 from the leaves and 14 from the roots were identified. The identification was based mainly on homologous genes in Arabidopsis and Brassica. Statistical approach showed that the greatest differences in protein expression can be seen mainly among ecotypes whereas the effects of different exposure levels were less pronounced. Conclusions: Proteomics provides a powerful additional tool for the detection and identification of proteins induced or repressed under metal stress. This study also showed that proteomic approach combined with advanced statistical analyses offers great possibilities for a large scale comparison of protein patterns of various ecotypes. The hyperaccumulator plant Thlaspi provides a good model since many of the proteins can be identified based on the homology to Arabidopsis or Brassica genes. On the other hand, it was apparent that Thlaspi contains many genes and proteins for which homology was not found from databases. These proteins may be of particular interest for further studies of metal tolerance, uptake and accumulation.

References: Assunção AP et al. (2001) Plant Cell Environ. 24: 217-226 Koistinen K et al. (2002) New Phytologist 155: 381-391

Acknowledgements: EU project PHYTAC contract nº: QLRT-2001-00429; Academy of Finland project nº: 53885; The Finnish Graduate School in Environmental Science and Technology POSTER SESSION

P-214 Effect of H+ Stress on the Activity of Plasma Membrane H+ ATPase in Corn Roots

C. Krämer, J. Wiese, F. Yan and S. Schubert Insitute of Plant Nutrition, Interdisciplinary Research Center (IFZ), Justus Liebig University, Giessen, Germany

A high proton activity in the rhizosphere limits plant growth by reducing the capability of maintaining a stable intracellular pH. However, corn (Zea mays L.) is able to adapt to such conditions by enhancing the activity of root plasma membrane H+ ATPase, as could be shown with intact plants and with isolated plasma membrane vesicles (Yan et al., 1992; Yan et al., 1998). The background of this adaptation to high H+ activities is still unknown. Several explanations for the change in H+ ATPase activity are conceivable: - a variation in gene expression of one or several specific isoforms of the plasma membrane H+ ATPase, - a posttranslational change, - a variation in gene expression of regulatory proteins. To address this question, corn (Zea mays L., cv. Blizzard) is cultivated under controlled conditions at pH 6 and pH 3.5 for three weeks in hydroponic culture. To achieve an adaptation of the corn roots to the low pH the H+ concentration is enhanced gradually by 50 µM per day. Possible isoforms of the plasma membrane H+ ATPase will be separated by means of 2D- gelelectrophoresis and detected and quantified using antibodies directed against H+ ATPase. Preliminary results show up to four different isoforms of the plasma membrane H+ ATPase. To identify the given spots they have to be examined by MALDI-TOF-TOF. Furthermore, by using specific and degenerated primers against known and unknown ATPase isoforms H+-stress specific isoforms of the plasma membrane H+ ATPase will be isolated. The expression of those isoforms will be examined during adaptation to proton stress. Combination of the molecularbiological and proteomic results will lead to a better understanding of the adaptation processes to H+ stress. POSTER SESSION

P-215 Analysis of the Heat Stress Response of Populus euphratica’s leafs through 2D-PAGE Approach

S. Ferreira and M.S. Pais Plant Biotechnology Unit, ICAT – FCUL, Lisbon, Portugal

Temperature is one of the major abiotic factors that influence plant growth and development. Heat is most often associated with loss of crop productivity and loss of sweetness of fruits. Euphrates poplar has an unique capacity to adapt very quickly to sudden thermal changes, surviving to 45ºC as the absolute maximum temperature that it can handle. One of the effects of long-term exposure to high temperature is the recruitment of mRNA of constitutive genes into heat stress granules in the cytosol of eucaryotic cells, and the suppression of their translation, concomitantly with the induced expression of heat stress genes (Kirschner et al, 2000). The analysis of heat-induced protein patterns by 2D-PAGE can give relevant clues about the proteins being differentially synthesized during heat stress, so, the study of total proteome of heat-stressed leafs of Euphrates poplar seemed adequate and of great interest. Mature leaves collected from plants produced by grafting, with 3 to 4 month-old shoots, and maintained in a greenhouse, were exposed to 25ºC (control) and 40ºC in the dark, in a phosphate buffer solution (Panchuk et al, 2002) at 60 rpm. Two independent replicates were produced and total protein contents from both situations were extracted with TCA-acetone. Solubilization prior to IEF was performed in a urea-thiourea buffer and protein quantification was performed with the Bradford Standard Assay (BioRad). The preliminary results through observation and reproduction of the analytical pattern in 7 centimeter IPG Dry Strips (Pharmacia Biotech) stained with Coomassie Brilliant Blue were promising and showed relevant differences between control and stress situations. To obtain a higher resolution, IPG Dry Strips of 18 centimeter and pH 3-10 NL were, then, loaded with 800 micrograms of protein through in gel rehydration and runned on Multiphor II for IEF coupled to an EPS 3500 unit as power source (Pharmacia Biotech). The second dimension of these strips is now being performed with a 24 centimeter - Protean xi Cell II unit (BioRad), with 3 replicates for each experimental situation. The resulting gels will be analysed with PDQuest 2-D Gel Analysis Software, Version 6 (BioRad).

References: Kirschner M, Winkelhaus S, Thierfelder JM, and Nover L (2000). Plant J 24: 397-411 Li-kuo F, Jian-ming J (Ed). 1992 Beijing, Science Press Panchuk II, Volkov RA, Schoffl F (2002). Plant Physiol 129: 838-853 Görg A, Boguth G, Obermaier C, Weiss W (1998). Electrophoresis 19: 1516-1519 Rabilloud T., Adessi C., Giraudel A., and Lunardi J. (1997). Electrophoresis 18: 307-316 POSTER SESSION

P-216 Proteome Analysis of Sugar Beet Leaves under Drought Stress

M. Hajheidari1, Gh. Hosseini Salekdeh1, M. Heidari1, M. Abdollahian-Noghabi2, and S.Y. Sadeghian2 1Agricultural Biotechnology Research Institute of Iran, 31535-1897, Karaj, Iran; 2Sugar Beet Seed Institute, PO.Box 31585-4114 Karaj, Iran

Drought is a major factor limiting sugar beet production in Iran and it is increasing in importance in irrigated environments as a result of water shortages and poor maintenance of infrastructure. The genetic complexity of drought tolerance, environment variability and strong genotype - environment interactions, and the difficulty of imposing uniform drought stress have hampered breeding efforts. We applied a proteomics approach to analyze the responses of sugar beet leaves to drought stress. Two inbred lines of sugar beet (Beta vulgaris L.) differing in drought responses were cultivated in the field. The line-source irrigation system was used and water stress levels were applied in strips parallel to the sprinkler line. The leaf samples were collected from well watered and stressed lines in three replications. Changes induced in leaf proteins were studied by two dimensional gel electrophoresis and quantitatively analyzed using image analysis software. Seventy-nine proteins out of over 1000 leaf proteins examined were found to change reproducibly in abundance in response to drought stress. Of 15 drought responsive proteins analyzed using liquid chromatography tandem mass spectrometer, 12 proteins were identified. We describe the expression of proteins involved in several pathways and discuss their possible roles in adaptation of plants to drought stress. POSTER SESSION

P-217 Proteome Analysis of Rice Leaf Response to Drought Stress and Recovery

Gh. Hosseini Salekdeh1, J. Siopongco2, L. J. Wade3, B. Ghareyazie1, and J. Bennett2 1Agricultural Biotechnology Research Institute of Iran, 31535-1897, Karaj, Iran; 2International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines; 3School of Plant Biology (Plant Sciences), Faculty of Natural and Agricultural Sciences, The University of Western Australia, Australia

Proteomic analysis offers a new approach to discovering the genes and pathways that are crucial for stress responsiveness and tolerance. We applied this approach to rice cultivars that differ in their drought responsiveness and tolerance mechanisms. Three-week-old plants of rice (Oryza sativa L. cv CT9993 and cv IR62266) developed gradual water stress over 23 d of transpiration without watering and 10 d of recovery. Out of about 1000 spots reproducibly detected, 42 proteins showed significant and reproducible changes in abundance in at least one stage of the drought/re-watering cycle compared with well-watered controls harvested at the same time points. These proteins could be grouped into clusters based on their response patterns. About 50% more proteins significantly responded to drought in IR62266 than in CT9993. The most common expression pattern was to increase in CT9993 or decrease in IR62266. Thirty-eight proteins were analyzed by MALD-MS, supplemented where necessary by ESI-Q-TOF MS/MS. Mass spectrometry helped to identify 23 of these proteins including proteins involved in basic metabolic pathways such as ATP production, photosynthesis and protein synthesis, as well as proteins of oxidative stress tolerance and cytoskeleton reorganization. The most abundant drought-induced protein did not register a database hit from the MS data but was identified after the isolation of full-length cDNA using PCR primers based on a Q-TOF peptide sequence. The corresponding S-like RNase gene in barley was also isolated. They were found to be S- like RNase homologue but they lacked the two active-site histidines required for RNase activity. POSTER SESSION

P-218 Proteome Analyses of Spring Wheat Reveal Different Responses to Amount and Composition of N- and S-Fertiliser

K. Hollung1, A.K. Uhlen2, E. M. Færgestad1 and E. M. Magnus1 1MATFORSK, Norwegian Food Research Institute, Ås, Norway; 2Department of Horticulture and Crop Sciences, Agricultural University of Norway, Ås, Norway

Unpredictable variation in protein quality is a major concern for the bread wheat producers and the baking industry. To study effects of nitrogen (N)- and sulphur (S)-fertilisation on wheat protein yield and quality, a pot experiment consisting of different amounts and different timing of N- and S-fertiliser was conducted. The spring wheat cultivar Bastian of strong gluten quality was used in the experiment. The protein content analysed by near infrared spectroscopy (NIR), varied according to amount and timing of the N-fertiliser applied, as well as to variations in yield. The SDS sedimentation volume, however, was greatly affected by S-fertilisation and the N/S- ratio in the grains. Selected samples representing extremes in N/S-ratio was analysed for rheological properties and for protein size distribution by size-exclusion fast protein liquid chromatography (SE- FPLC). However, to explore the effects of N/S-fertilisation on the protein composition in more detail, two-dimensional (2D)-electrophoresis was performed. This gave a better resolution than the SE-FPLC and revealed several specific protein spots differing in appearance between the wheat samples. Comparisons of the protein patterns by image analyses indicate that some proteins are regulated by type of fertiliser and some proteins by timing of the addition of fertiliser. The results suggest that proteome analyses by 2D-electrophoresis provide additional information about variations in protein composition due to differences in fertilisation. The results also indicate potentials of S-and N-fertilisation given as a late application in the growth period. POSTER SESSION

P-219 Comparative Transcriptome and Proteome Analysis of the Crown Root Mutant rtcs in Maize (Zea mays L.)

M. Sauer and F. Hochholdinger Eberhard-Karls-Universität, Center for Plant Molecular Biology (ZMBP), Department of General Genetics, Tübingen, Germany

Plant roots serve important functions such as water and nutrient uptake and anchorage in the soil. The genetic analysis of root formation its due to its complexity and variability still in its infancy. The maize root system consists of four major root types: the embryonic primary- and seminal- roots and the postembryonic shoot-borne roots which consist of crown- and brace- roots which make up the major backbone of the root system. Crown-roots are formed at consecutive underground stem nodes starting at the coleoptilar node. The coleoptilar node is the first stem node that is formed early during seedling development. Brace-roots are formed late in development from aboveground stem nodes. The monogenic recessive mutant rtcs (rootless concerning the crown and seminal roots) completely lacks all shoot-borne roots including crown-, brace- and seminal-roots. The mutant rtcs is affected at an early stage of root development since for none of the missing root types primordia are initiated (Hetz et al., 1996). The mutant rtcs was used as a model to compare gene and protein expression profiles of 10-day-old wild-type and mutant coleoptilar nodes. Transcriptome analyses with microarrays containing 12,000 different maize cDNAs and proteome analyses comparison differentially accumulated proteins via 2D gel and subsequent MALDI-ToF and ESI analyses were carried out. The results will be presented and discussed.

Reference: Hetz W., Hochholdinger F., Schwall M., Feix G., Plant Journal 10: 1996, 845-857 POSTER SESSION

P-220 Investigating the Xylem-proteom: Gain and Use of Pure Xylem Proteins from Barley J. Wiese, S. Karl and S. Schubert Universität Giessen, Institut für Pflanzenernährung, Giessen, Germany

Our research deals with a apoplastic sub-proteom, the xylem. In the literature the xylem is usually only regarded as a transport compartiment for anorganic nutrients, carbohydrates and phytohormones. Although described as important eg. in pathogene defence, apoplastic proteins are an underestimated protein fraction in literature. Among apoplastic proteins xylem proteins are the least prominent. There is scarce work about their function in pathogene defence and regulation of stomatal opening. A systematic approach to investigate the function of xylem proteins lacks in the literature. We are using a system of stress treated plants to isolate proteins and polypeptides possibly involved in the transduction of signals from the root to the shoot of barley plants. In previous experiments we demonstrated, that barley (Hordeum vulgare cv. Ingrid) grown with low pH in the root medium show an enhanced resistance against powdery mildew infection of the leaf. Since H+ is not directly transported to the leafs the question arises how the stress signal is transported from the roots to the shoot and converted into pathogen resistance. We approach the question by investigating the xylem proteom which is the transport compartment connecting root and shoot, looking for a potential signal of polypeptide nature. The focus of this poster is to demonstrate a simple method to gain xylem sap of high purity from barley and a reliable method for the purity control by using RUBISCO as a marker. POSTER SESSION

P-221 Two-dimensional Gel Electrophoresis as a Tool to Characterize and Identify Different Oak (Quercus ilex) Spanish Provenances

C. Gómez-Pinto1, D. Ariza2, R.M. Navarro2 and J. Jorrín1 1Dpto. Bioquímica y Biología Molecular, ETSIAM, Universidad de Córdoba, Campus de Rabanales, Edificio Severo Ochoa Córdoba, Spain; 2Dpto de Ingeniería Forestal, ETSIAM, Universidad de Córdoba, Spain

Forest restoration and reforestation is, nowadays, an objective of top priority. It has caused and important demand of forest species and has favoured research on their biology and nursery production (Navarro et al., 1998). Protein composition and content (reserve and stress proteins) is very important after seedling transplanting to the field, as they depend almost exclusively on the endogenous pool and are subjected to different biotic and abiotic stresses (Marschner et al., 1996). The use of biochemical and molecular biology techniques in forest ecophysiological studies is gaining interest. Such approaches are directed, from a basic point of view, to know different aspects of their biology, and for a practical one, to characterize and catalogue genotypes, lines, provenances. Protocols for 2-DE of oak leaves have been optimise and used for analysing different Spanish provenances (Extremadura, Alcarria, Sierra Morena) (Gómez-Pinto et al., 2003). The protein profile depended on the provenance, individual plant, developmental stage and leaf orientation. Dramatic changes in Rubisco large subunit have been observed in adult plants along a 24 h cycle in leaves of different orientation (north, south, west and east).

References: Gómez-Pinto C, et al. (2003) Protein profile in Quercus ilex leaf tissue. Analysis by two dimensional gel electrophoresis. Seminars in Proteomics UCO-2003, Córdoba, Spain, February 2-4. Marschner H, et al. (1996). Effect of mineral nutritional status on shoot-root partitioning of photoassimilates and cycling of mineral nutrients. J Exp Bot 47: 1255-1263 Navarro RM, et al. (1998) Caracterización de calidad final de planta de encina, alcornoque, algarrobo y acebuche, en cinco viveros de Andalucía. Consejería de Agricultura y pesca, Junta de Andalucía, Sevilla. POSTER SESSION

P-222 A Proteomic Approach to Study Plant Responses to Parasitic Angiosperms. Studies with the Pisum sativum-Orobanche crenata Pathosystem

M.A. Castillejo1, N. Amiour2, D. Rubiales3, E. Dumas-Gaudot2 and J. Jorrín1 1Dpto. Bioquímica y Biología Molecular, ETSIAM, Universidad de Córdoba. Campus de Rabanales, Edificio Severo Ochoa (C6), Córdoba, Spain; 2UMR 1088 INRA/U. Bourgogne & FRE CNRS 2625, INRA/CMSE, PME (-Plant Microbe Environnement), Dijon, France; 3Instituto de Agricultura Sostenible, IAS-CSIC, Córdoba, Spain

Broomrapes (members of the genus Orobanche) are parasitic plants that threaten agricultural production in many parts of the world. The most damaging broomrapes are O. crenata, O. cumana, O. aegyptiaca, O. ramosa and O. minor. O. crenata is the most dangerous and the most widespread Orobanche species in the Mediterranean region and West Asia. It is a major constraint for faba beans, field peas, lentils, vetches and various forage legumes (Rubiales, 2001). The search for plant material resistant to this weed race and its introduction in commercial varieties is a main priority in breeding programmes. Resistance to O. cumana in legumes seems to be multigenic (Rubiales, 2001). A deeper knowledge of biology of the plant- parasitic weed interaction and the molecular bases of the specifity and host resistance and pathogen virulence will be necessary in order to develop more resistant varieties or alternative control strategies (Jorrín et al., 2000). Such objective can be covered by using a proteomic approach. Two pea genotypes: Messire, highly susceptible, and Ps642, moderately resistant to crenata broomrape were used. Plants were inoculated by using a Petri dish bioassay (Pérez de Luque, 2002) and roots sampled 3 weeks after inoculation. Proteins were extracted by using the phenol protocol (Bestel-Corre et al., 2002) and subjected to 2-DE (IEF, pI 3-10, as first dimension, and SDS-PAGE, 12% polyacrilamide gel, as the second one), being the gels silver stained. The protein profile differed among genotypes and treatments, with 38 differentially expressed proteins between infected and non-infected Messire, 16 between infected and non-infected, and 24 between both lines. Protein identification by mass spectrometry will open new perspectives in identifying plant response mechanisms to parasitic angiosperms.

References: Bestel-Corre G, et al. (2002) Proteome analysis and identification of symbiosis-related proteins from Medicago truncaula Gaertn. By two-dimensional electrophoresis and mass spectrometry. Electrophoresis 23: 122-137. Jorrín J, et al. (2000) How plants defend themselves against root parasitic angiosperms: molecular studies with Orobanche spp. In Resistance to Orobanche: the state of the art (JI Cubero et al., eds.). Consejería de Agricultura y Pesca, Junta de Andalucía, Sevilla, España. Pérez de Luque A (2002) Caracterización de los mecanismos de resistencia a jopo (Orobanche crenata Forsk.) en guisante (Pisum spp.), habas (Vicia faba L.) y otras leguminosas de interés agronómico. Ph. D. Thesis, University of Córdoba. Rubiales D. (2001). Parasitic plants: an increasing threat. Grain Legumes 33: 10-11. POSTER SESSION

P-223 Peptide Mass Fingerprinting of Differently Expressed Proteins in Uromyces striatus Infected and Non-infected Medicago truncatula Leaf Tissue

M.A. Castillejo1, N. Imin2, D. Rubiales3, J. Jorrín1 1Dpto. Bioquímica y Biología Molecular, ETSIAM, Universidad de Córdoba. Campus de Rabanales, Edificio Severo Ochoa (C6), Córdoba, Spain; 2Genomic Interactions Group, Research School of Biological Sciences, Australian National University, Australia; 3Instituto de Agricultura Sostenible, IAS-CSIC, Córdoba, Spain

The alfalfa relative Medicago truncatula (annual medic) is gaining interests as a model plant for structural and functional genomics directed to help in the identification of agronomically important genes in crop legumes, including those for root symbioses, disease/pest resistance, plant architecture, seed quality, and production of specific secondary metabolites (Frugoli and Harris, 2001). Alfalfa rust incited by U. striatus is an important disease in many areas, being particularly damaging in alfalfa grown for seed. Many other rust species of the genera Uromyces are important constraints for grain and forage legumes, such as U. viciae-fabae (faba bean rust), U. pisi (pea rust), U. appendiculatus (common bean rust) and U. vignae (cowpea rust) among others. Plant response to biotrophic fungal pathogens in legumes are being studied by us at the molecular level by using a proteomic approach (Torres et al., 2002). . The protein profile of healthy and rust infected Medicago truncatula leaves from the resistant cultivar Paraggio has been analyzed by 2-DE (Castillejo et al., 2003), and some differentially expressed proteins have been identified by peptide mass fingerprinting. Those correspond to: malate dehydrogenase, acidic phosphatase, glutathione peroxidase, glutathione transferase, phospholipid hydroperoxide glutathione peroxidase, and an unknown protein from Arabidopsis thaliana.

References: Castillejo MA, et al. (2003) Plant pathogen interaction studies in Medicago truncatula. Two dimensional electrophoresis of proeins in Uromyces striatus infected leaf tissue. Frugoli J, Harris J (2001) Medicago truncatula on the Move. Plant Cell 13: 458-463. Torres A.M., V. Geffroy and D. Rubiales, 2002. Medicago truncatula to help progress for resistance against pathogens and pests in legume crops. Grain Legumes 38: 18-19. POSTER SESSION

P-224 Glutamine Starvation Induces Proteomic Changes in a Myeloid Cell Line

M. Pokar, M.M. Eliasen and R. Oehler* Surgical Research Laboratories, University of Vienna, Austria *Corresponding author

Given the central role of protein synthesis in cellular function, it is likely that intricate mechanisms exist to detect and respond to amino acid deprivation. However, the current understanding of amino acid dependent control of gene control in mammalian cells are limited. The affected expression in monocytes of a few gene products, e.g. HLA-DR and Hsp70 , during critically illness can be associated with reduced availability of glutamine in plasma of these patients. In this initial study we investigated the consequences of a reduction of glutamine (Gln) from 2mM to 0.6mM and 0.2mM, respectively, on the protein expression profile in monocytic U937 cells. Cells were cultured with the reduced Gln concentrations for either 24 h or 96 hours at the three different Gln concentrations. Soluble proteins were prepared by digitonin extraction followed by precipitation in ethanol. Proteins were resolubilised in 7M Urea/2M Thiourea containing 4% CHAPS and 30mM Tris pH 8.8, and the samples were dialysed before isoelectric focussing. The experiment was repeated three times, yielding n = 3 for each group. The three identical samples were pooled before two-dimensional electrophoresis (2D-E), thus each detected protein represent the mean value of three experiments. We used immobilised 13cm IPG-strips, pH 3-10L for IEF and 11% continuous SDS-PAGE, and we made 4 parallel gels with 100 µg protein from each pool. Approximately 600 proteins with PI between 3.0 - 10.0 and Mr from 25 kDa - 250 kDa were reproducibly resolved and visible with ruthenium staining. A specific protein pattern, characteristic for Gln starved cells, was identified. These proteins constitutes approximately 2% of the visible proteome. This study shows that Gln depletion induces a comprehensive starvation response in monocytic cells. Our data indicates that there exist a general control mechanism that initiates the starvation response in monocytes and that this response leads to regulation on the protein expression level.

References: Spittler, A. et al.: Postoperative glycyl-glutamine infusion reduces immunosuppression: partial prevention of the surgery induced decrease in HLA-DR expression on monocytes. Clin. Nutr. 2001, 20: 37-42. Oehler, R. et al.: Glutamine depletion impairs cellular stress response in human leucocytes. Br. J. Nutr. 200, 87 Suppl 1: S17-21 POSTER SESSION

P-225 Inhibited Eukaryotic Elongation Factor-2 Protein Synthesis Coincides with High Hydrostatic Pressure-induced Decrease in Protein Synthesis

M.A. Elo1, H.M. Karjalainen1, R.K. Sironen1, L. Valmu2, H.J. Helminen1, and M.J. Lammi1 1Department of Anatomy, University of Kuopio, Finland; 2Institute of Biotechnology, Biocenter Viikki, University of Helsinki, Finland

High continuous hydrostatic pressure causes many responses in mammalian cell cultures. Accumulation of heat shock protein 70 mRNA and protein in the pressurised cells without transcriptional gene activation is one of the events taking place in many mammalian cells. Pressure also causes a considerable inhibition of total protein sythesis. In this study, our goal was to identify pressure-regulated proteins with the use of two-dimensional gel electrophoresis/mass spectrometry. Before exposure of HeLa cervical carcinoma cells and T/C28a4 cells (SV40 immortalized human chondrocytic cell line) to hydrostatic pressure, pressurisation medium was made by mixing 1 part of DMEM containing methionine and cysteine and 9 parts of DMEM without methionine and cysteine, and medium w a s supplemented with 30 µCi/ml Tran35S-label, 4 mM glutamine, fetal calf serum and penicillin/streptomycin. After replacement of the medium and sealing of the culture dishes, continuous 30 MPa hydrostatic pressure for up to 12 h was used for the experiments. The isoelectric focusing was performed using 13 cm long 3-10 non-linear IPG strips. After electrofocusing, the gels were equilibrated and proteins were further separated in 10% SDS- polyacrylamide gels. The gels were dried, the radioactivity signal was analysed with PhosphorImager™, and finally the gels were stained with PlusOne™ Silver Staining kit. Protein spots selected for further analysis were reduced and alkylated with iodoacetamide before overnight digestion with sequencing-grade trypsin, and the peptide mixture was desalted using Millipore ZipTip™ µ-C18 pipette tips. Mass mapping of the peptides was performed with a Biflex™ MALDI-TOF mass spectrometer in a positive ion reflector mode using α-cyano-4- hydroxycinnamic acid as the matrix. In the LC-MS/MS analysis, the peptides were first separated by microbore reversed-phase HPLC on an 0.075 x 150 mm PepMap column by elution with a linear gradient of acetonitrile in 0.1% formic acid. Chromatography was performed at a flow rate of 0.25 µl/min, and the eluent was directly injected into a Q-TOF mass spectrometer equipped with an electrospray ionization source. MS/MS spectra were acquired by colliding the douply charged precursor ions with argon collision gas accelerated with voltages of 30-45 V. Database searces were carried out by using programs ProFound or Mascot MS/MS ion search. Analysis of metabolically labeled samples showed that biosynthesis of eukaryotic elongation factor-2 (eEF-2) was particularly inhibited in HeLa and T/C28a4 chondrocytic cells pressurised with 30 MPa continuous hydrostatic pressure, also in Western blot analysis its total protein level was decreased within 12 h of the pressure treatment in HeLa cells. Steady-state mRNA level of eEF-2 was not affected by the pressure. In conclusion, this study suggests that inhibition of eEF-2 in pressurised cell cultures may be involved with the inhibition of the general protein synthesis. Since the activity of eEF-2 in translocation of ribosome along the specific mRNA is dependent on its phosphorylation state, analysis of eEF-2 phosphorylation is warranted. POSTER SESSION

P-226 Proteome Analysis of CHO Cells During a Biopharmaceutical Process

D. Krawitz Genentech, Inc, South San Francisco, CA, USA

In the biopharmaceutical industry, recombinant protein drugs are commonly produced in Chinese hamster ovary (CHO) cells. In order to better understand CHO cell physiology and metabolism during a production process, we analyzed protein expression patterns from three independent CHO cell lines. Each cell line was grown under standard culture conditions that mimic a drug production process. Quantitative analysis of over 1300 cellular proteins that were resolved by two-dimensional gel electrophoresis was performed. The protein expression profile of each CHO cell line was compared at different stages of the culture during the bioreactor process. These comparisons define certain protein expression patterns that are unique to individual CHO lines and others that are shared between CHO lines. Additionally, we analyzed the changes in protein expression over the course of a production culture. These changes reflect the cells’ physiological response to the culture conditions. Protein spot identifications were determined by using MALDI-TOF mass spectrometry and peptide mass fingerprinting. Because the Chinese hamster genome has not yet been sequenced in its entirety, spot identifications from MALDI analysis were made by searching a rodent database and confirmed by MS/MS analysis. Data will be presented on the reliability of using MALDI for identification of CHO cell proteins. POSTER SESSION

P-227 The Zebrafish Oocyte: A Surprising Vitellogenin Heteorgeneity

L. Bianchi1, S. Liberatori1, L. Bini1, M. Fabbrini1, F. Argenton2, P. Soldani1, V. Pallini1 and P. Neri1 1Dipartimento di Biologia Molecolare, Università degli Studi di Siena, Siena, Italy; 2Dipartimento di Biologia, Università di Padova, Padova, Italy

The zebrafish, Danio rerio, has recently achieved the pantheon of genetic model organisms [1]. The frenetic sequencing rush of its genome, leading to the identification of thousands of genes and EST sequences, has made D. rerio an easily accessible source of gene sequences. In spite of this, it results quite difficult to bridge zebrafish genome, transcriptome and proteome. Toward a more integrated and complex biology knowledge of D. rerio we proposed to proceed with proteomic methodologies. Unfertilised oocytes and embryos at early developmental stages were analysed by 2D-electrophoresis, 2D-immunoblotting and Mass Spectrometry. According to the zebrafish telolecithal egg nature, the most information were collected about vitellogenin (VTG) and its derived polypeptides. In order to better understand vitellogenin heterogeneity and its involvement in embryo development, a 2D gel of unfertilized oocytes was electroblotted onto a nitrocellulose membrane and immunostained with anti- zebrafish VTG antibody (VTG-Ab). 242 immunoreactive spots were found; they clearly clustered in five main groups of complex isoelectric series (A: pI 6.20–9.00/Mr 200–138.6 kDa; B: pI 6.35–8.20/Mr 53 kDa; C: pI 5.85–7.30/Mr 42.9 kDa; D: pI 5.60–6.10/Mr 39.2–34.3 kDa; E: pI 4.80–6.80/Mr 18–12.5 kDa). By Mass Spectrometry other 23 not immunostained VTG spots were identified. Even if this VTG spots abundance seems to confirm supposed high-level of co- and post-translational modifications and proteolytic egg processing of vitellogenin, it is not yet clear the role of VTG heterogeneity during the zebrafish development. Matching the oocyte immunoreactive spots with the early embryo silver stained ones, remarkable differences of VTG-derived polypeptides were found. This suggests a probable preference of the embryo for using specific VTG-moieties during different developmental stages.

Reference: [1] Fishman, M. C., (1999) Proc. Natl. Acad. Sci. USA, 96: 10554-10556 POSTER SESSION

P-228 Protein Identification From Synaptic Vescicles Isolated From Rat Hippocampal Neurons

V. Corti1, G. Piccoli4, Y. Sánchez Ruiz2, A. Bachi2, A. Bergamaschi3, A. Malgaroli3, and M. Alessio1 1Proteomics Laboratory, 2Mass Spectrometry, and 3Unit of Neurobiology of Learning, San Raffaele Scientific Institute, Milan, Italy; 4Consiglio Nazionale delle Ricerche Institute of Neuroscience, Cellular and Molecular Pharmacology, Department of Pharmacology, University of Milan, Italy

Long-lasting changes in synaptic functions are supposed to underly learning and memory in the mammalian brain. The molecular mechanisms sustaining physiological long-term potentiation (LTP) are not fully understood. Evidence that de-novo protein synthesis is a requirement for the late phases of LTP has come from experiments with protein synthesis inhibitors. In order to reveal the nature of the molecules involved in LTP we have generated reference maps for both synaptosomal (S) and cytosolic (HSS) fractions of the neurons. For these experiments we made large-scale hippocampal cultures from CA3-CA1 region of the rat hippocampus which were kept in vitro for 2 weeks. Proteins belonging to the HSS and S fractions were then isolated and purified by standard biochemical methods, resolved using 2D-PAGE (pH 3-10 NL, 9-16% gradient gel) and finally identified either by mass spectrometry (after tryptic digestion) or by Western Blot. We identified 101 spots corresponding to 82 different proteins; they can be classified as: neuronal (about 20%), mitochondrial (21%), cytoskeletal and cytoskeletal-associated (8%); involved in lipid (2%), aminoacidic (2%) and oxidative (3%) metabolism; involved in cell growth (2%), protein degradation (3%), signaling (5%), protein folding (10%) and glycolysis (10%). About 3% of the total proteins analysed are unknown. By using zwittergent solubilization w e were able to identify integral membrane and tightly membrane associated proteins that represent about 20% of the total. These reference maps are currently used to compare the protein pattern of resting synapses to that of synapses after LTP induction. POSTER SESSION

P-229 Analysis of Activity and Sequence Structure of Cytosolic Enzymes from Mouse Liver by Non-denaturing Two-dimensional Electrophoresis and Mass Spectrometry

Y. Shimazaki, Y. Sugawara and T. Manabe Department of Physics and Chemistry, Faculty of Science and Venture Business Laboratory, Ehime University, Matsuyama, Japan

After cytosol proteins in the mouse liver were separated by non-denaturing two-dimensional electrophoresis (2-DE), activities of multiple enzymes such as fructose bisphosphatase and malate dehydrogenase, sorbitol dehydrogenase and transferase, or several dehydrogenases were analyzed on the same 2-DE gel. Further, peptidase activity was examined by matrix- assisted laser desorption /ionization-time of flight-mass spectrometry (MALDI-TOF MS) after protein separation by 2-DE. Enzymes separated by 2-DE were identified by peptide mass fingerprinting using MALDI or electrospray ionization tandem mass spectrometry or both. Combination analysis of activity and sequence structure verified the accurate position and activity range of the separated enzymes on the non-denaturing 2-DE gel. By using this combination analysis, the activities of both malate dehydrogenase and sorbitol dehydrogenase were inhibited not by AMP, but fructose bisphosphatase activity was inhibited. These results indicate that analysis of enzymes by combination analysis can be used to screen substances that affect enzyme activities. POSTER SESSION

P-230 Two-Dimensional Separation and Detection of VDAC from Bovine Spermatozoa

V.A. Aires1, X. Schneider1, M. Fijak2, E. Hinsch1, V. de Pinto3 and K.D. Hinsch1 1Centre of Dermatology and Andrology, Biochemistry of Reproduction, Justus Liebig University, Giessen, Germany; 2Institute for Anatomy and Cell Biology, Justus Liebig University, Giessen, Germany; 3Dept. of Chemistry, Laboratory of Biochemistry and Molecular Biology, University of Catania, Italy

Voltage-dependent anion channels (VDCAs) or porins are abundant 30-35 kDa pore-forming proteins that form the major pathway for metabolite flux (e.g. ATP) across the outer mitochondrial membrane. Eukaryotes express multiple VDACs, e.g. humans and mice exhibit three isoforms (VDAC1-3) encoded by different genes. Porin subtypes show high sequence homology and similar electrophysiological properties. In an earlier investigation we reported the generation of antibodies directed against synthetic porin peptides and detected porin type 1 and 2 proteins in spermatids as well as in spermatozoa. The goal of the present study was the identification of porin isoforms in bovine spermatozoa using two-dimensional electrophoresis and well characterised subtype-specific anti-porin antibodies. Proteins were extracted from fresh ejaculated bovine spermatozoa and subjected to 2-DE. The first dimension was carried out using immobilised pH gradient (IPG, pH range 3-10). Proteins were then separated by SDS-PAGE on 12% acrylamide gels. Detection of VDAC was achieved by immunoblotting. Our preliminary data suggest that porin subtypes with different pIs are present in bovine spermatozoa. Porin subtypes in spermatozoa might be involved in the regulation of different sperm functions, e.g. motility and acrosome reaction. However, the role of porins in sperm remains to be elucidated. POSTER SESSION

P-231 Are Telomerase Overexpressing Endothelial Cells Young? A Proteomic Approach Using 2D-DIGETM

M. Chang, C. Mayrhofer, R. Voglauer, H. Katinger and J. Grillari Institute of Applied Microbiology, University of Natural Resources and Applied Life Science, Vienna, Austria and Institute for Analytical Chemistry, University of Vienna, Austria

Serial passaging of human primary cells in vitro ends up in an irreversible growth arrest, called replicative senescence, where the cells are still viable for months. The main cause for the limited lifespan of normal cells is supposed to be the shortening of telomeres, since overexpression of the catalytic subunit of human telomerase (hTERT), that prevents this telomere erosion, leads to immortalization of a variety of primary cells. Additionally, ectopic telomerase expression maintains the non-tumorigenic and highly differentiated phenotype of the normal counterparts. However, changes of the whole protein profile associated with telomerase overexpression have not been under investigation yet. Therefore, we have transfected human umbilical vein endothelial cells (HUVECs) with plasmids containing hTERT and a vector control. While the telomerase negative control cells reached replicative senescence 15 population doublings after transfection (PDpT), hTERT overexpressing cells have reached 86 PDpT so far and are still growing. Together with young subconfluent, young quiescent and senescent HUVECs, our newly established hTERT overexpressing cell line was analyzed using 2-dimensional-fluorescenct-difference-gel- electrophoresis (DIGE). Only 10 gels were necessary for obtaining statistically significant differences of the protein profiles of these cells. The results obtained within this study showed that Cytokeratin 7, reticulocalbin 1 and calumenin for example were found at similar protein levels in hTERT and young cells and changed in senescent cells. On the other hand we also found proteins in our immortalized cells whose occurrence are typical for senescent HUVECs. Even if our hTERT overexpressing cell line showed an overall young morphology and phenotype, the reaction to TNF-a (tumor necrosis factor) stimulation was not completely the same as of young HUVECs. This leads us to the conclusion that hTERT overexpression extends the life-span of HUVECs, sustains an endothelial cell specific differentiated phenotype, but does not prevent all changes associated with a senescent phenotype. POSTER SESSION

P-232 Proteomic Analysis of Foetal Rat Epididymis and Vas Deferens: Identification of Growth-related and Andogen-regulated Proteins

A. Umar1, T.M. Luider2 M.P. Ooms1, J.A. Grootegoed1 and A.O. Brinkmann2 1Department of Reproduction and Development and 2Neurology, Erasmus MC, Rotterdam, The Netherlands

Epididymis and vas deferens are structures that are part of the male internal genital tract, which are derived from the Wolffian duct anlagen in the developing embryo. Growth and differentiation of the Wolffian duct is dependent on the action of androgens since absence of androgen action results in a female phenotype. To understand the downstream effects of androgens it was our aim to identify androgen-regulated and growth-related proteins. We have used epididymides and vasa deferentia from rat embryos from day 17 of gestation (E17) to day 21 of gestation (E21). Between 25-40 tissues per time point were collected. This group (group 1) was used to study growth-related changes in protein expression profiles. In addition, we have used E19 epididymides and vasa deferentia for organ cultures (group 2). Tissues were either cultured in the absence of androgen for 9, 24, or 48 hours or in the presence of 10 nM synthetic androgen (R1881) for 9, 24, or 48 hours. For each condition 25 tissues were collected. This second group was used to study androgen-regulated protein expression profiles. An amount of 100µg protein from total cell lysate was used to generate 2- dimensional gels and protein expression profiles were analysed using PDQuest software. All gels were performed in duplicate. Proteins of interest were identified using a matrix-assisted laser desorption/ionisation time-of-flight mass spectrometer. The study of group 1 showed dramatic changes in protein expression profiles between E17 gels and E21 gels. 15 protein isoforms were found to be gradually down-regulated in time, whereas 25 protein isoforms were found to be up-regulated in time. 5 protein isoforms could not be detected in E17 gels and were found to be expressed later on during development. In group 2, 9 proteins were up-regulated and 1 was down-regulated in the presence of androgen. In addition, 4 unique proteins were present in either one situation. Most of the proteins could be identified and categorised as cytoskeletal proteins, enzymes, signalling molecules, transcription factors or household proteins. We are currently focussing on proteins that are regulated in group 1 as well as in group 2. In conclusion, the combination of organ culture and proteomic analysis of foetal reproductive tract tissues is a successful approach to identify abundantly expressed androgen-regulated proteins that may be involved in growth and differentiation. POSTER SESSION

P-233 Analysis of the Mouse Lens Proteome at the Protein Species Level

W. Hoehenwarter1, N. Kumar2, J. Klose3, U. Zimny-Arndt1 and P.R. Jungblut1 1Max-Planck-Institut für Infektionsbiologie, Berlin, Germany; 2Department of Ophthalmology and Visual Sciences, Chicago, IL, USA; 3Institut für Humangenetik, Berlin, Germany

Recent developments in the field of proteomics have led to a strong emphasis on high throughput, fully automated data collection and evaluation. One must not forget, that while providing rapid protein characterisation of a biological state, this tends to remain superficial, not fully appreciating the inherent complexity of protein species abundance and interaction. We have begun a full characterisation of the proteins of the mouse eye lens analyzing primarily but not exclusively the crystallins, focusing on the genetic variability between the strains M. musculus c57bl and 129SvJ and M. spretus as well as looking into the effects of a cataract mutation. Lenses of ten day old mice were homogenized and separated by 2 DE. Spots were excised, trypsin digested and analyzed by MALDI-TOF and ESI (tandem) mass spectrometry. We found the previously unreported mouse homolog of the full human protein phakinin CP49, gi 4502995 in both c57bl and 129SvJ mice. Further, we detected a previously unreported beta B1 crystallin gi 12963789 protein species in cataract mutant strains, possibly due to calpain cleavage. Finally, we analyzed the gamma E/F crystallin genetic situation on the protein level clarifying present ambiguities. In total, the mouse lens was separated into about 1400 spots of which 30 contained crystallins. The construction of a mouse lens 2 DE database as a basis for the further analysis of cataract is underway. POSTER SESSION

P-234 Identification of Proteins in Goat Milk

P. Roncada1,2, F. Carta3 and G.F. Greppi2 1Istituto Sperimentale Italiano L. Spallanzani, Milano; 2Dipartimento di Scienze Cliniche Veterinarie, Università degli Studi di Milano; 3Porto Conte Ricerche, Alghero, Italy

The composition of goats’ milk have been suggested to have several nutritional advantages over milk from cattle. Goats’milk is often utilised when infants show allergic reactions to both cows’ milk and soy-based formulae and suggested that goats’ milk is a viable dairy option to meet nutritional requirements of infants, children and adults. Goat milk casein differs in its amino acid composition and its more digestible than cow milk casein. Goat milk is uniqueness, and his potentiality in human nutrition and medicine has been reported. With the advent of several tools by proteomics, aim of this work is to identified some goat milk protein by t w o dimensional electrophoresis coupled with MALDI-TOF technologies and try to draw a virtual map of this proteins, because this techinique is powerful, highly reproducibile and actually f e w work about milk are reported. POSTER SESSION

P-235 The Modal Distribution of Protein Isoelectric Points Reflects Amino Acid Properties Rather than Sequence Evolution

G.F. Weiller1 and G. Caraux2 1Research School of Biological Sciences, Australian National University, Canberra, Australia; 2Département d'Informatique Fondamentale et Appliquée; LIRMM, Montpellier, France

Two-dimensional gel electrophoresis (2DE), a routine application in proteomics, separates proteins according to their molecular mass (Mr) and isoelectric point (pI). As the genomic sequences for more and more organisms are determined, the Mr and pI of all their proteins can be estimated computationally. The examination of several of these theoretical proteome plots has revealed a multimodal pI distribution, however, no conclusive explanation for this unusual distribution has so far been presented. We have examined the pI distribution of 115 fully sequenced genomes and observed that the modal distribution does not reflect phylogeny or sequence evolution, but rather the chemical properties of amino acids. We provide a statistical explanation of why the observed distributions of pI values are multimodal. POSTER SESSION

P-236 Identification of Binding Media in Paintings by SDS-PAGE and Mass Spectrometry

M. N. Kayali-Sayadi1, B. Ramírez Barat2, I. Blasco Castiñeyra2; B. Cañas Montalvo3 and L. M. Polo-Díez3 1Mass Spectrometry Service, Faculty of Chemistry, 2Department of Painting and Restoration, Faculty of Fine Arts, and 3Department of Analytical Chemistry, Faculty of Chemistry, Universidad Complutense de Madrid, Madrid, Spain

The identification of painting materials, specially binding media, is very important to approach conservation processes and methodology. In tempera and secco techniques, several binding media (egg yolk, egg white, gelatin, casein...), with different behaviour, may be used. In this communication we present the results obtained in the analysis of several binding media prepared using egg yolk, egg white, gelatin and casein. Samples were aged in the laboratory by natural aging during 30 days, applying ultraviolet radiation, or temperature at 70ºC during 624 hours. Redisolution and sample preparation was not straightforward and different methodologies were assayed to analize the proteins in dried, artificially aged, binding media. Best results were obtained when extraction was performed with 5 M urea in Laemmli solution and resolving protein components by 1D SDS-PAGE. The excised gel bands were digested with trypsin and the digest purified using a Zip-tip C18. Samples were analysed by peptide mass fingerprinting using MALDI-TOF MS and nonredundant protein databases were consulted using search engines like Mascot or Profound. Results were confirmed by MS/MS of selected peptides using an Ion-Trap MS fitted with a nanoelectrospray source. POSTER SESSION

P-237 Reproducible Sample Preparation for 2D Gel Electrophoresis: A Key Step in Proteome Analysis

C. Loske2, J. Anders1, M. Wehsling1, D. Matheis1, S. Andrecht1 and R. Hendriks1 1Merck KGaA, Life Science Products R&D MDA, Frankfurter Str. Darmstadt, Germany and 2Merck Biosciences GmbH, Schwalbach, Germany

In proteome analysis, two-dimensional-electrophoresis (2DE) remains the highest resolution technique for protein separation when complex samples need to be arrayed prior to characterization by mass spectrometry. The prerequisites for an efficient sample preparation method for 2DE are reproducible solubilization of all types of proteins, prevention of protein degradation as well as a thorough removal of contaminating nucleic acids e.g. by enzymatic digestion. For this reason, we have developed all-in-one solutions comprising the ProteoExtract™ Complete and Partial Proteome Extraction Kits for efficient denaturing sample preparation for 2DE, an optimized digestion protocol for efficient in-gel tryptic digestion and the 100 µm Chromolith® CapRod® monolithic silica capillary columns for fast and efficient LC/MS analysis of tryptic digests. Tailor-made Partial ProteoExtract™ Kits for the standardized extraction of either complete or partial proteomes from a broad variety of biological samples including bacteria, yeast, mammalian tissue culture cells and mammalian tissues have been established. Complete proteome extraction with the ProteoExtract™ complete proteome extraction kits yield an extract containing virtually all proteins of a cell in one sample and is ideally suited for overview gels. Partial proteome extraction using the ProteoExtract™ partial proteome extraction kits fractionates the complete proteome into four partial proteomes enriched in proteins with decreasing solubility. Since more proteins can be visualized after 2D gel electrophoresis of partial proteomes as compared with complete proteomes, the use of the ProteoExtract™ partial proteome extraction kits for sample preparation raises the chance to visualize low-abundant proteins. Thus the developed kits provide researchers in the discovery proteomics field with valuable tools for sample preparation. POSTER SESSION

P-238 PST® - The ProteoSHOPTM Solution for Membrane Proteins

K. Kuhn, C. Baumann, J. Schäfer, T. Prinz, R. Moraga, J. Müller, A. Thompson, J. Schwarz, U. Bauer, C. Hamon and T. Neumann Proteome Sciences plc, Coveham House, Cobham, Surrey, UK

In the post genomic era, the increasing knowledge of the complete genome sequences has led to considerable effort increasingly being devoted to Proteomics, the study of protein expression and function. A number of potential markers and targets have already been delivered to pharmaceutical and biotechnology companies. However, in the field of drug discovery, the need of reducing cycle times and still delivering valuable data in a shorter period of time still constitutes a big challenge for proteomics. To meet this challenge, Proteome Sciences plc has assembled a toolkit of proteomics technologies, ProteoSHOP™. The ProteoSHOP™ toolkit has been designed to build sensitivity, speed and throughput into proteomics by offering a complete toolkit for the detailed analysis of all classes of soluble proteins, such as cytosolic and secreted, as well as for the more challenging membrane proteins. To address the soluble proteins in cell or tissue extracts and secreted proteins in body fluids, we have developed proprietary methods for high-output 2-dimensional gel electrophoresis (2DE). This process is centered around the Proteomics Software System® (PSS®), an integrated LIMS and data capture/analysis package that controls the entire 2DE process. For the quantitative analysis of hydrophobic membrane proteins and very basic DNA-binding proteins, we have developed our proprietary Protein Sequence Tags (PST®) [1], a gel-free technology based on mass spectrometry and Tandem Mass Tags® (TMT®) [2] for a precise quantification (see also TMT Poster). PST® is a new protein profiling approach which addresses cellular and membrane proteins. Using purified plasma membranes or microsomal fractions, the membrane proteins are pre-processed and extracted during sample preparation. The membrane proteins are labelled selectively with the PST® mass tag and subjected to enzymatic cleavage. The N-terminal fragments of the resulting peptide mixture are selected, analysed by LC-MS and LC-MS/MS and identified by SEQUEST searches of the MS/MS data against the PST® database. By studying only one specific peptide sequence per protein or protein fragment, PST offers the advantage of speed, reproducibility, sensitivity and considerable data reduction. The LC-MS raw data is transferred to the PST® software for storage and further analysis. Using PST® algorithms, the data is then denoised by detecting the background noise level and removing the noise. In a further deconvolution step (see also Deconvolution Poster), the masses of the peptides are calculated. By comparing different PST® profiles, a fast differential expression analysis can be achieved by mass spectrometry (see also Differential Display Poster). By studying only one specific peptide sequence per protein or protein fragment, PST offers the advantage of speed, reproducibility, sensitivity and considerable data reduction. For an accurate quantification TMT® has been developed. The principle of TMT® is to provide different labels for proteins from test and control samples which have identical physico-chemical properties in LC and MS modes, but which release different specific TMT® fragments after collisional activation. The specific TMT® fragment ions from test and control have different masses, and the ratio between them is directly proportional to the ratio of parent protein in the test and control samples. Based on this principle TMT® allows simultaneous determination of both the identity and relative abundance of peptides (and the corresponding proteins). The poster presented here gives an overview of the ProteoSHOP™ toolkit with focus on the PST® technology and its application. [1] Kuhn, K.; Thompson, A.; Prinz, T.; Mueller, J.; Baumann, C.; Schmid, G.; Neumann, T.; Hamon, C. J. Prot Res. 2003, 2: in press. [2] Thompson, A.; Schäfer, J.; Kuhn, K.; Kienle, S.; Schmidt, G.; Neumann, T.; Hamon, C. Anal. Chem. 2003, 75: 1895-1904. POSTER SESSION

P-239 Monitoring of Regulatory Protein Topology using ProteoExtract™ Subcellular Proteome Extraction Kit

A. Abdolzade-Bavil1, S. Hayes2, L. Goretzki3, J. Anders1 and R. Hendriks1 1Merck KGaA, Life Science Products R&D MDA, Darmstadt, Germany; 2EMD Biosciences Inc., Novagen, Madison, WI, USA; 3EMD Biosciences Inc, Calbiochem, San Diego, CA, USA

One of the major challenges in functional proteomics is the separation of complex samples prior to expression profiling, detection of post-translational modifications or determination of subcellular localization. The prerequisite for the success of proteome analysis is the existence of a standardized and reproducible protocol for sample preparation. Having this in mind, w e have systematically studied subcellular proteome extraction methods as tools for the sequential extraction of proteins in their native state according to the subcellular localization using tissue culture cells. The developed method takes advantage of the differential solubility of certain subcellular compartments in either one of four select reagent mixtures. Cell extraction with these buffers yields four subproteomes enriched in (a) cytosolic, (b) membrane and membrane organelle-localized, (c) soluble and DNA-associated nuclear and d) cytoskeletal proteins. The presented method thus increases the chance of visualizing low- abundance proteins in the respective subcellular compartment. During extraction, the integrity of cytoskeletal networks is protected and the method is simple, highly reproducible, labor sparing, and ultracentrifugation-independent. The efficiency and selectivity of subcellular extraction was demonstrated by fluorescence microscopy, immunoblot and enzyme assay analysis. More than 80% of representative marker protein was detected in the dedicated subcellular fraction. Thus, the developed method provides researchers in the proteomics field with a valuable tool for the standardized isolation of proteins according to their subcellular localization. As the extracted fractions are functional, it allows subsequent analysis of proteins by various means, including 1DE/2DE, immunoblotting and enzyme activity assays POSTER SESSION

P-240 High Throughput Proteomic and Genomic Sample Preparation Using AcroPrep™ 96 Multi-well Plate With Ultrafiltration Membrane

N. Tang, D. Wilson and K. Seeley Pall Life Sciences, Ann Arbor, MI, USA

Ultrafiltration (UF) is a technique using a membrane with defined pore sizes to separate molecules according to size. It is a gentle method for the purification and concentration of proteins and nucleic acids. Because the separation is based on mechanical rather than chemical interactions, sample concentration and separation can be achieved without the addition of buffers and salts. Historically, UF devices could only process individual samples; however, now, Pall Life Sciences is introducing a novel UF device called AcroPrep 96, which is capable of processing 96 samples simultaneously. AcroPrep96 is a 96-well filtration plate, available with a full-line of UF membranes corresponding to various molecular weight cut-offs (MWCO). This study demonstrates the use of AcroPrep96 with UF membranes for a variety of proteomic and genomic sample preparatory procedures, including sample purification, desalting, and concentration. POSTER SESSION

P-241 Albumin Removal Kits: Performance Analysis by 2D Gel Electrophoresis

B. Thome-Kromer, M. Taufmann, M. Klatt, P. Bolon, U. Wacker and W. Krömer EUROGENTEC PROTEOMICS GmbH, Teltow/Berlin, Germany

Since the high abundance of albumin is one of the key bottlenecks for proteome studies using serum or plasma we used both fluids to compare 4 commercially available albumin removal kits. Their albumin (immunoglobulin) removal capacity as well as the binding of non-albumin components was compared between kits. Eluate and retentate fractions from the different kits were separated by NEPGHE based 2 D electrophoresis pH 2-11. By using gel sizes of up to 40 x 30 cm, proteins from each of the two fractions were identified by mass spectrometry with a focus on basic proteins. The removal principles of each kit rely on different processes and the efficiencies will be demonstrated, as well as identifications of proteins associated with non-specific binding. POSTER SESSION

P-242 The ProteoExtractTM Albumin/IgG Removal Kit: A Highly Efficient Sample Preparation Method for Body Fluid Proteome Analysis

J. Anders, M. Wehsling, A. Purohit, D. Matheis, S. Andrecht, R. Hendriks and D. Leiss Merck KGaA, Life Science Products R&D MDA, Darmstadt, Germany

One of the major challenges in functional proteomics is the separation of complex samples prior to comparative analysis e. g. for disease marker identification. The best sources for potential disease markers are body fluids because they are easily available in sufficient amounts for analysis. However, the main drawback of a comprehensive analysis of body fluids is the high abundance of serum albumin and IgG. Serum albumin can constitute 50-70 % of the total serum protein and immunoglobulines can constitute 10–25 %. The concentration of these proteins causes loss of resolution in one-dimensional electrophoresis (1-DE), two- dimensional electrophoresis (2DE) and chromatographic separations and impairs with detection and identification of low-abundant proteins. Having this in mind, we have developed the ProteoExtractTM Albumin/IgG removal kit for highly specific chromatographic removal of both serum albumin and immunoglobulines from plasma samples. The ProteoExtractTM Albumin/IgG removal kit was shown to be specific for albumin and immunoglobulines by immunoblotting against marker proteins. More than 90 % of the marker proteins were recovered in the unbound fraction of the provided disposable sample preparation columns, while more than 80 % of both serum albumin and immunoglobulines could be removed. Thus the ProteoExtract‘ Albumin/IgG removal kit was demonstrated to be superior over existing disposable sample preparation strategies for body fluids. Using liquid chromatography and 2-dimensional gel electrophoresis coupled with mass spectrometry, more peptides and proteins could be detected and identified in the protein- depleted plasma samples in comparison with the untreated samples. The presented method thus increases the chance for the detection and indentification of low-abundance proteins in human body fluid samples and provides researchers in the proteomics field with a valuable tool for disease marker identification from human body fluids. POSTER SESSION

P-243 A New, Flexible Assay Format for Quantification of Multiple Proteins in < 20 µL Serum

A. Schmidt, M. Inganäs, A. Eckersten, R. Gunnarsson, A.-K. Honerud, S. Lindman, G. Thorsén, P. Lehtonen, J. Khoshnoodi, M. Ljungström, T. Söderman and Helene Dérand Gyros AB, Uppsala, Sweden

In drug discovery small animal models are used to study the effect of drug candidates on the concentration of representative proteins in serum. Sample availability, typically 20 µl per animal, severely limits the possibility of assaying more proteins in a shorter time. Conventional technologies are poorly designed for handling small volumes. To overcome these difficulties a flexible assay format for protein quantification has been miniaturized and integrated into a CD microlaboratory. A CD contains microstructures in which samples are assayed in parallel as the CD spins. Each microstructure contains a column (10-15 nl) prepacked with streptavidin-coated beads. Biotinylated capturing molecules, selected for the proteins of interest, are bound to the beads, creating protein-specific columns. Samples (200 nl) pass through the columns, followed by complementary, fluorescently-labelled detecting molecules. Specifically-bound proteins are measured on-line by laser induced fluorescence. Parallel processing speeds up handling and improves reproducibility. Picomolar concentrations can be quantified at precision levels ~ 5- 10% CV. Miniaturization reduces volume requirements for sample and reagent. A protein is quantified using <1 µl of sample. Potentially >10 different proteins could be quantified simultaneously from a 20 µl sample, increasing information content and providing results within hours. POSTER SESSION

P-244 Comparison of the new KBiosystems Preptide - A Fully Integrated Proteomic Sample Preparation Robot to Optimized Manual Protocols

S. Kirby1, J. Jones1, and Malcolm Saxton2 1Kbiosystems, Basildon, UK; 2Ludwig Institute for Cancer Research, London, U.K.

The role of high throughput proteomics in disease identification and in drug discovery process continues to be a rapidly expanding area. With an ever-greater demand for the proteomic analysis of samples, the need for rapid and reliable automation in sample handling becomes more pressing . In proteomics, two-dimensional gel electrophoresis is arguably the best technique for the simultaneous separation of thousands of proteins while mass spectrometry is the only technique sensitive, accurate and fast enough for large-scale protein identification. The combination of both methodologies is fundamental to proteomics in many research laboratories. Hence, the aim is to create an integrated platform for gel imaging and cutting as well as in-gel digestion and MALDI sample spotting. Both visible gel stains e.g. coomassie blue and fluorescent gel stains can be imaged using an on-board CCD camera. The gel images can then be analysed off-line using proprietary or third- party software. Spots to be picked can be either imported as a cut file or chosen manually using the platforms software. For the robotic gel spot cutting a polyetheretherketone (PEEK) cutting head has been used. To avoid losing the gel cores vacuum is applied to the cutting head to securely hold gel pieces. These can then be delivered to a 96 well plate and released by an ejection pin. Gels cores have been successfully cut and placed in 96 well plates with a greater than 99.5% success rate. A maximum of 768 excised gel cores can be digested in parallel on the robot using a combination of valve dispensing and syringe pump aspiration. During digestion the fuly enclosed environment is closely regulated keeping both temperature and humidity with in optimized ranges. The accurate and reliable liquid handling and environmental control allows protein digest in the sub-picomole range This is also added by the use of a highly accurate closed loop servo controlled drive system which allows small movements around the gel core, for complete removal of peptides post digestion. Maldi taget loading is achieved by pin loading with hot tool changes between the samples and matrix loaders providing high speed and accuracy. Full data tracking from gel coring to the MALDI target loading, is provided by mounted barcode readers. Producing files which can be easily integration into most commonly used Lims systems. Peptides are collected in a microwell plates for further studies using MS/MS. The poster will show results of the entire automated work flow on the newly developed robotic platform in comparison to our optimised manual procedures POSTER SESSION

P-245 Automated Platform for Immobiline Strip Focusing in Proteomics

A. Posch, C. Obermaier, M. Mast, R. Wildgruber, M. Hauptmann and C. Eckerskorn Tecan Munich GmbH, Proteomics Division, Kirchheim, Germany

Isoelectric focusing using immobilized pH gradients is the key technology in gel-based two- dimensional proteome analysis. Although methodology of IPG focusing has been improved a lot in the past 10 years concerning sample application, IPG-strip rehydration and instrumentation, complaints about the technique´s difficult handling, missing process control and lack of automation are still apparent. Especially for beginners in IPG-focusing, there are numerous pitfalls and drawbacks which seriously influence success rate and process quality. Additionally, when researchers ramp up their proteomics studies, throughput, flexibility and integration of instrumentation becomes increasingly important. Here, we describe a fully automated platform for IPG-focusing leading to less operator errors and higher reproducibility. The key elements of this robotic platform are a new "in-gel- rehydration” procedure, electrode design and the "one-power-supply-per-strip” concept which are compared to current instrumentation for IPG-focusing. POSTER SESSION

P-246 Automated Platform for SDS–PAGE in Proteomics

M. Mast, A. Posch, C. Obermaier, R. Wildgruber, M. Hauptmann and C. Eckerskorn Tecan Munich GmbH, Proteomics Division, Kirchheim , Germany

SDS-PAGE as the second step in two-dimensional electrophoresis is a robust und well- understood technique. However, due to low levels of automation and missing process control, the method is very time-consuming and personal skill influences data quality and reproducibility. To address the current needs of the technique for highest level proteome analysis, w e developed an automated platform for casting and running SDS-PAGE gels. The key elements of the SDS-gel-casting platform are a vacuum degassing unit and individually temperature controlled gel cassettes. Unique process control during gel casting leads to improved reproducibility concerning gel length and thickness, a faster and more homogeneous polymerization step while hands-on-time for assembling, disassembling and cleaning is minimized. The key parameters like voltage/current and temperature to reliably run SDS-gels are individually controlled leading to an unmatched protein separation process in terms of spot positional reproducibility and comparability from run to run. Data from computer assisted gel comparison experiments will be presented and put side by side to results gained with "state- of-the-art” equipment for SDS-PAGE. POSTER SESSION

P-247 Parallel Processing in the Isoelectric Focusing Chip

G.V. Zilberstein, E.M. Baskin and S. Bukshpan Protein Forest Inc., Rabin Science Park, Rehovot, Israel

Classical IEF in immobilized pH-gradients is discussed on theoretical investigation of IEF kinetics. Standard IEF demands co-linearity of the electric field and pH-gradient directions (serial devices). It is shown that IEF separation process based on a continuous, serial pH gradient is incompatible with miniaturization of separation devices. The new realization of IEF device by parallel IEF chip is suggested and analyzed. The main separation tool of the device is a dielectric membrane with conducting channels. Each "i” channel of the membrane is filled by immobiline gel with definite value of pH=pHi . The pH value of the surrounded aqueous solution is not equal to any pHi. In contrast to standard IPG devices, the protein sample does not migrate into the separating gel. The fast particle transport between different channels is accounted for by the convection in the aqueous solution. The new device geometry [1] introduces two new spatial scales to be considered: the scale of transition region from a solution to the gel in a channel and a typical channel size. The corresponding time scales defining the IEF process kinetics are analyzed and scaling laws are obtained. It is shown both theoretically and experimentally that parallel IEF accelerates the fractionation of proteins by their pI down to several minutes and makes possible efficient sample collection and purification.

Reference: [1] G. V. Zilberstein, S. Bukshpan, Patent application WO 03/0089777 A2 POSTER SESSION

P-248 2D-LC-MS/MS Analysis of Gradiflow Fractionated Native Human Plasma

D. Rothemund1, A. Liew1, I. Bate1, M. Raftery2, M. Guilhaus2 , V. Wasinger2, V. Locke3 1Gradipore Ltd., Sydney, NSW, Australia; 2Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney, NSW, Australia; 3Gradipore Inc., Hawthorne, NY, USA

A major challenge in the ability to identify the whole proteome of any sample is the dynamic range of the protein expression. Plasma has some highly abundant proteins, such as albumin, present in amounts that vary up to 8 orders of magnitude in comparison to the least abundant species. This difference is too great for current technology and limits the success of identification of lower abundance proteins therefore sample fractionation is necessary. The Gradiflow BF400 was used to fractionate each human plasma sample into four fractions; a pI>5.25 fraction, an albumin fraction, a MW > albumin fraction (>125 kDa nominal molecular weight cut-off, MWCO) and a MW < albumin fraction (<45 kDa nominal MWCO). The broad fractionation was performed to isolate albumin and reduce sample complexity, whilst limiting the protein cross-over between fractions. The Gradiflow BF400 separations were performed under native conditions. Protein migration in the Gradiflow BF400 was different amongst the anticoagulants, citrate, EDTA and heparin, as well as compared to serum alone. Gradiflow fractions were initially analyzed by 1D PAGE to demonstrate effective separation. Subsequently, each fraction was digested using trypsin and peptides separated by 2 dimensional liquid chromatography (2D-LC). Salt step elutions from strong cation exchange, followed by elution from reverse phase were used prior to MS/MS analysis (QStar/LC Packings System). Proteins ranging from MW 1.7 kDa to greater than 500 kDa with pIs of 4.5 to 9.4 were identified. Several novel proteins were found and the preliminary results show evidence of differing protein populations in the various anticoagulant plasma samples. POSTER SESSION

P-249 Rehydration or Cup Loading onto Alkaline IPG’s, The Choice is Yours

A.R. Goodall and B. Herbert Proteome Systems, North Ryde, Sydney, Australia

Although the development of IPGs has simplified the analysis of samples via 2-D, problems still persist in the separation at alkaline pH, such as in the use of IPGs of pH 6-11 or more narrow alkaline ranges. Other investigators have found that osmotic flow during the focusing phase is a major factor influencing the quality of the separation generated. Because a typical cell lysate contains predominantly acidic proteins, conventional in-gel rehydration loading often produces poor results because the acidic proteins must migrate out of the strip, which leads to streaky poorly resolved gels. Cup loading at the anode is usually reported to be the solution because the acidic proteins never enter the IPG, however, the load capacity of cup loading is limited. Paper bridge loading also forces the proteins to enter the IPG through a single point. Here we show that rehydration loaded whole lysates can be focused in alkaline gradients producing excellent results. Rehydration loading, focusing gel side down and the use of slow voltage gradient ramping during the IEF step reduces the impact of electro-endo-osmotic flow during the focusing phase thus allowing for the equilibrium focusing of most samples yielding well resolved arrays. In addition, when cup loading is used, long voltage gradients result in minimal or no precipitation at the application site. POSTER SESSION

P-250 Innovative Sample Preparation Technology Enabling High Sensitivity MALDI MS Analysis

W. Chen, P.J. Lee, S. Vazquez, J.W. Finch and J.C. Gebler Life Sciences R&D, Waters Corporation, Milford, MA, USA

Identifications and detailed characterizations of protein complexes separated by polyacrylamide gel electrophoresis (PAGE) require simplified and robust sample preparation methods for interfacing electrophoretic techniques to mass spectrometry. These methods should be routine, reliable, and easily adapted to any number of digests, large or small. Current employed technology often requires considerable user intervention and is difficult to handle with low concentrations of protein digest without sample loss. We present a simple and robust sample preparation technology for MALDI-MS analysis that enables sample preparations directly on the MALDI-MS target plate. A large volume (up to 10 µL) of diluted sample is placed on the MALDI target plate, dried down with focusing to a confined region coated by a membrane having peptide binding property. The region is then subsequently washed to remove contaminates that are contained in sample and may interfere with MALDI analysis, whereas analytes are selectively adsorbed onto the membrane and enriched. The validity of the technology was successfully investigated by direct processing in-gel digest samples of faint 2D gel spots from yeast cytosol. It is demonstrated that the on-target sample clean-up and enrichment of peptides can facilitate identification of proteins from gel separations. As a result, the sensitivity of analysis is significantly improved (with limit of detection at about 100 attomol). The method is also compared with alternative sample preparation methods and the results indicate that the cleanup is at least as efficient as other methods. Additional benefits of the current technology are demonstrated by on-target cleanup for micro-scale chemical reaction mixtures. Data from direct on-target cleanup of several peptide derivatization reaction mixtures such as guanidination clearly prove the general utility of this technology as a sample preparation method for MALDI MS analysis. POSTER SESSION

P-251 Optimization of In-Gel Digestion using Solid Phase Extraction Microplate

M. Nissum1, U. Schneider1, S. Kuhfuss1, C. Obermaier1, R. Wildgruber1, A. Posch1, U. Knecht2, N. Ingenhoven2, and C. Eckerskorn1 1Tecan Munich GmbH, Proteomics Division, Kirchheim, Germany; 2Tecan Switzerland AG, Männedorf, Switzerland

Introduction: Improved 2D-gel protocols and increasingly sensitive staining techniques make less abundant proteins visual on 2D-gels. However, the identities of these proteins are difficult to access with current techniques. The in-gel digestion process is often carried out in large solvent volumes. Large amount of peptides may be lost through inefficient purification steps. This leads to loss of sensitivity. We present a new way of processing 2D-gel plugs using a solid phase extraction plate optimized for in-gel digestion. With this plate unparalleled sensitivity is obtained compared to present methods. In addition, the plate is ideal for high throughput since 96 samples can be processed at the same time. Methods and Instrumentation: 2D-gel spots were excised with a spot picking device and placed directly in the solid phase extraction plate. The gel plugs were processed in the solid phase extraction plate and resulting peptides were eluted directly onto a MALDI-TOF target ready for analysis. The entire process was carried out on a robotic platform. Proteins were identified through database search. 1D gels with known quantities of protein and in-solution digest were used for comparison with existing methods. Preliminary Data: The processing of the solid phase extraction plate is integrated in a fully automated system. It includes the automated transfer of the solid phase extraction plates loaded with gel plugs from the spot picking device to the robotic platform, as well as the transfer of the MALDI-TOF target to the mass spectrometer. With minimum user intervention a high throughput can be achieved mounting up to approximately 3000 samples per 24 hours. The used solid phase extraction system showed an increase in sensitivity up to two orders of magnitude compared to currently available systems. Weak spots from 2D gels were excised and processed with the system leading to identification of low abundant proteins. POSTER SESSION

P-252 Application of Automated Integrated Sample Preparation and MALDI QIT ToF MS for Structural Elucidation of Proteins

R.L. Martin1, K. Tanaka2, M. Resch3 1Shimadzu Biotech, Manchester, UK; 2Mass Spectrometry Research Lab, Shimadzu Corporation, Kyoto, Japan; 3Shimadzu Biotech, Duisburg, Germany

Introduction: MALDI ToF MS has become widely accepted in the field of protein determination. High sensitivity, resolution and mass accuracy combined with rapid automated analysis for high throughput are all vital factors in this area, the union of which provides simple, rapid protein identification. Peptide mass fingerprints may be generated for first pass protein assignment by database searching. The confidence in protein identification can be improved by the use of post source decay (PSD). A curved field reflectron significantly increases the speed and sensitivity of the PSD analysis by enabling the acquisition of a fully focussed seamless PSD spectrum in a single measurement (sPSD). However, it has become apparent that this approach does not provide a universal solution to proteomics problems as it relies on the protein being present in a database. A new generation MALDI ToF MS equipped with a quadrupole ion trap (QIT), provides unprecedented levels of structural information on protein sequence through MSn analysis. The MALDI QIT ToF MS results in high mass accuracy and resolution irrelevant of mode of operation, combined with the unique sequencing ability of a radical new approach in ion trap design and operation. Methods: Protein samples were separated by 2D-PAGE electrophoresis and robotically processed to generate tryptic digests on the Xcise™ integrated gel processing platform (Shimadzu Biotech). This system is capable of scanning and imaging a gel, excising protein spots of interest, performing a trypsin digest, subsequently desalting the samples using C18 Ziptips (Millipore) and automatically spotting them on a standard 384-position stainless steel MALDI target. Digests were consequently analysed automatically by the AXIMA-CFR™, a curved field reflectron MALDI MS (Shimadzu Biotech), generating peptide mass fingerprints and seamless post source decay (sPSD) spectra. Additional analyses were carried out on the AXIMA-QIT™ (Shimadzu Biotech), a MALDI QIT ToF mass spectrometer. Results: Software specifically developed for proteomics (IntelliMarque™, Shimadzu Biotech) was employed to automatically acquire, process and database search protein digests. Initial database searches from peptide mass fingerprints using Mascot™ (Matrix Science) provided first pass identification of a number of proteins. These results are considered automatically and ions that have been matched to a protein sequence are subjected to data dependent automatic sPSD, imroving the confidence in protein assignment. In addition, automatic sPSD is performed on a number of ions that do not have a positive match with the suggested protein and subsequent secondary automated database searches are carried out using the fragmentation data. A number of peptide peaks evident in the MALDI ToF MS spectrum were selected for MS2 analysis on the MALDI QIT ToF MS, a system capable of MSn. A significant increase in fragmentation was observed when compared with post source decay.

Conclusions: Automated MALDI peptide mass fingerprinting and database searching of tryptically digested gel spots has clearly become a high throughput option for protein identification. The confidence in the protein identity suggested by the automated database search may be improved by performing automated seamless PSD followed by a secondary search. An added advantage of this system is the ability to automatically acquire a second series of seamless PSD experiments in order to attempt to identify minor components within the protein spot. The MS2 spectra, generated on the AXIMA-QIT, of tryptic peptides show extensive fragmentation allowing either database searching or de novo interpretation. This extra information can enhance the specificity of database searching and protein identification. POSTER SESSION

P-253 Improving MALDI-MS Analysis using a Micro-Fluidic Sample Preparation Platform

A. Schmidt, S. Wallenborg, M. Gustafsson, B. Ek, M. Holmqvist and P. Andersson Gyros AB, Uppsala, Sweden

Sample preparation before MALDI MS analysis is an essential step to achieve highest possible sensitivity. We describe a CD-based microfluidic sample preparation platform which could offer a range of preparation solutions. Steps can include desalting, enzymatic digestion, reaction for chemically assisted fragmentation (CAF) and sample capture on immobilized metal affinity chromatography (IMAC) and/or combinations of these. Capture beds of 10nL minimize the area available for irreversible binding of peptides. A combination of capillary and centrifugal force is used to load liquids, run reactions or elute packed beds. Typically, >96 microstructures work in parallel. Each microstructure integrates steps such as injection, volume definition and elution. An important design feature is evaporation as samples crystallize on target areas during a specifically-developed spin cycle. Precise control of liquid flow uses a valving solution compatible with many solvents, solvent mixtures, pH and MALDI matrixes and can handle protein solutions and biological samples such as serum. For example, during desalting, 50% acetonitrile, 1-10 mg/mL HCCA and additives, such as fucose can be used and for IMAC applications, DHB with additives has been successful. The desalting application generates high quality spectra in the 50 attomole region. In one run 96 samples are crystallized on MALDI target areas on the CD. Using a 5 fmol in-solution BSA digest, success rate was ≥90%. In the CD, samples are processed in parallel through one or more methods, e.g. sample can be digested with different enzymes in separate microstructures on the same CD. By combining MALDI MS peak lists generated for the different enzymes, high sequence coverage is obtained. Using trypsin, Asp-N and Glu-C, sequence coverage was 21%, 24% and 15% respectively, but combining peak lists gave sequence coverage of 55%. Precise control of liquid flow rates by spin programs ensures sufficient time for reactions. The same approach is used in an IMAC application. Sample is added to two microstructures on the same CD. In one structure phosphorylated peptides are captured and eluted onto the target area. The other sample is dephosphorylated on-column using alkaline phosphatase followed by elution onto the target area. The presence of phosphorylation is confirmed by comparing peak lists. This was demonstrated by loading a mixture of a BSA tryptic digest, _-casein and a tyrosine phosphorylated synthetic peptide, onto two columns. Phosphorylated peptides were detected after the IMAC procedure and the corresponding dephosphorylated peptides were detected after the dephosphorylation reaction. POSTER SESSION

P-254 About Stringency Parameters in Automatic Sample Processing for Protein Identifications

P. Hufnagel, U. Schweiger-Hufnagel, M. Lubeck and U. Rapp Bruker Daltonik GmbH, Bremen, Germany

Introduction: The identification of all proteins visible on a protein gel should involve not only MALDI TOF fingerprinting methods but also MALDI TOF/TOF experiments and LC-ESI-MS/MS runs. The information yield of these technologies is linked to the single-experiment time consumption in a reciprocal way. Therefore, an intelligent and integrated use of different mass spectrometric methods is required. Only those samples are submitted to a more powerful yet slower MS mode, that need more clarification. Samples that gave satisfying results already are not further analyzed. Thus, the workload of the slower instruments can be reduced without compromizing identification yields. Methods: Samples of human HEK cells are separated using a standard 2D gel electrophoresis system and colloidal coomassie stained. protein spots are excised from the gel and digested using commercial robotic devices in combination with a chemical kit based on porcine trypsin. Three kinds of MS experiments are performed: (1) MALDI TOF MS, (2) Lift MS/MS on the same MALDI target while it is still in the instrument, (3) LC-MS/MS runs on a nano-LC system coupled to an ESI ion trap. All digest solutions are prepared onto a MALDI target by using a small portion of every sample, only. The remaining solution is kept in the microtiter plate until MALDI TOF and TOF/TOF analysis is finished. Then, the remaining samples for LC-MS/MS have been determined, and can be performed. Results: One crucial prerequisite for this integrated approach is to avoid sample loss during waiting times. Freezing or drying should be avoided since such phase transitions always lead to a certain degree of sample loss. We have placed an unsealed plate with in-gel digests on a 100 fmol level, after MALDI preparation, onto an autosampler and tried to identify all samples by LC-MS/MS. Preliminary results show that we could increase the time that samples can be analyzed in a row without a decrease in identification score to at least 80 hours. 88 selected spots were automatically processed and by applying the MS and MS/MS methodologies spectra recorded. For the identification of the proteins different scoring levels were set (e.g. 100 and 300 on a MASCOT based scale) and compared as identification criteria. In several cases the peptide mass fingerprint (PMF) spectrum is sufficient for identification because of wealth of peptide information. However, in other cases the PMF and TOF/TOF spectra only together with nanoHPLC-MS/MS data could result in an identification. At hand of several spot identifications the value and stringency of the individual or combined spectra is discussed. POSTER SESSION

P-255 Tandem Mass Tag1 as a Novel Approach for Relative quantification Based on MS/MS Experiments

C. Hamon, J. Schaefer,* A. Thompson,* K. Kuhn, S. Kienle, H. Legner, P. Schmid, J. Schwarz and T. Neumann Proteome Sciences plc, Coveham House, Cobham, Surrey, UK * Authors contributed equally to the work

Since the beginning of the endeavour of proteomics in the 90s, two-dimentional gel electrophoresis (2DE) in combination with MS or MS/MS identification of stained spots has been the gold standard method [2]. Nethertheless, the 2DE approach has limitations in respect of some proteins classes which are not well represented such as hydrophobic proteins, proteins with small and large molecular weights and proteins with extrem pI values. As a result, an intensive effort has been made over the last few years to develop methods for gel-free solution proteomics having the potential to address all kind of proteins. A new approach to quantification techniques that has emerged from this challenge is the combined use of stable-isotope labeling of proteins and mass spectrometric detection. For example, the ICAT technology [3] combines chromatography based method and mass spectrometry to get a quantitative profile of a complex protein digest based on isotope-coded affinity tags. This technique uses "heavy" and "light" isotope tags to label peptides from corresponding protein in pairs of samples under comparison. Although the ICAT approach is sophisticated and promising, it is associated with several problems, such as the variation with time in ESI-MS of ionization efficiency of peptide pairs [4]. The Proteome Sciences proprietary technology, Tandem Mass Tag (TMT®), is a promising new approach. This technique is based on quantitative differential MS/MS analysis of a complex peptide mixture using a novel class of reagents for protein profiling technologies such as Protein Sequence Tag procedures [5]. Beside the features shared with the other peptide isotope labelling techniques, the TMT® approach also possesses some great additional advantages. First, the pair of the isotopic TMT® units has the same mass and each tag of the pair is differentiated only by the sequence of its isotopic distribution in the structure. These confer to the TMT® labelled peptides the same coelution profile in the chromatographic separation of the peptide mixture. Therefore, they act as more precise reciprocal internal standards, which leads to a more accurate quantification. The quantitative analysis relies on the detection of an MS/MS generated tag fragment from each TMT® unit in the pair. The fragment are differentiated from each other by an isotopic mass. High signal to noise is achieved with the use of MS/MS to perform a very accurate quantification. In addition, this allows untagged material to be ignored, greatly enhance the quality data. Finally, the MS/MS data still provides good information about the TMT® labelled peptide sequence making the new approach very attractive. Quantification of the peptide and determination of its peptide sequence is performed both in the same MS/MS experiment. The concept of this new quantification technique and preliminary results will be presented.

References: [1] Thompson, A.; Schäfer, J.; Kuhn, K.; Kienle, S.; Schmidt, G.; Neumann, T.; Hamon, C. Anal. Chem. 2003, 78: 1895-1904. [2] Huber L.A.; Pfaller K.; Vietor I. Circ. Res. 2003, 92: 962-968. [3] Gygi, S.P.; Rist, B.; Gerber, S.A., Turecek, F.; Gelb, M.H.; Aebersold, R. Nature Biotech., 1999, 17: 994-999. [4] Zhang, R.; Regnier, F. E. J. Prot Res. 2002, 1: 139-147. [5] Kuhn, K.; Thompson, A.; Prinz, T.; Mueller, J.; Baumann, C.; Schmid, G.; Neumann, T.; Hamon, C. J. Prot Res. 2003, 2: in press. POSTER SESSION

P-256 E2 Tag – New Powerful Tool for Protein Tagging

R. Kurg, T. Mandel, K. Samuel, T. Talpsep, S. Tobi, M. Ustav, M. Vaher, M. Vahter Quattromed Ltd, Tartu, Estonia, and Tartu University, Tartu, Estonia

Epitope tagging is a recombinant DNA technique by which a protein is made immunoreactive to a pre-existing antibody. Epitope tagging can be effectively used to express, detect, characterize and purify recombinant proteins. Epitope tags are useful to study the protein- protein or protein-nucleic acid interactions. Unfortunately the available tags on the market have several disadvantages like low affinity and/or non-specific binding of the anti-tag antibody, influence on the biological activity of the tagged protein etc. We have developed a new epitope tagging system that uses an epitope of Bovine Pappillomavirus type-1 transactivator protein E2 as tag. E2Tag is a noncharged (the sum of positively and negatively charged amino acid is equal) peptide, which consists of 10 amino acids (SSTSSDFRDR). A mouse monoclonal anti-E2Tag antibody recognizes the E2Tag as well as recombinant E2-tagged proteins with high specificity and affinity. The purified antibody – epitope complex tolerates high salt concentrations up to 2 M and produces a strong signal with minimal non-specific staining on Western blot, when used with Anti-Mouse IgG Alkaline Phosphatase or Horseradish peroxidase. E2Tag system has been useful in immunofluorescence microscopy (when used with Anti-Mouse IgG FITC conjugate) to study the localization of the expressed protein in cells. The E2Tag has minimal effects to the biological properties of tagged protein. E2Tag is applicable in mammalian, bacterial, yeast and plant cells. We have demonstrated the advantages of the new tag in different applications: purification and characterization of bacterial transcription factor Xyl S, molecular cloning and expression of the new cyclooxygenase from arctic coral, identification of many new gene products from mammalian cells etc. In combination with other tags the E2Tag has been very useful to study protein-protein and protein-nucleic acid interactions. Due to its biological properties the E2Tag is a good candidate for protein chip development and for TAP (tandem antibody purification). POSTER SESSION

P-257 Integrating a New Peptide De-Novo Sequencing Tool for Sophisticated Data Analysis

D. Wunderlich, U. Schweiger-Hufnagel, M. Lubeck, D. Suckau, A. Ingendoh and C. Baessmann Bruker Daltonik GmbH, Bremen, Germany

During a MS database search in Proteomic research several MS-peaks can not be identified due to post-translational and other modifications. Also sequence errors in the database can be a reason for it. In order to identify those peaks, a de-novo sequencing software was implemented into a proteom software solution. Here we present the application of the de-novo software module for the identification of modified peptides in a protein sequence. Enzymatic digests of proteins from 2-D gels of various organisms were measured with an electrospray ion trap and MALDI TOF/TOF mass spectrometer by generating MS- and MS(n) spectra. A peaklist was created automatically and used by the de-novo sequencing software for generating peptide sequence proposals, considering all provided hints including possible modifications. The resulting peptide sequences were scored against the experimental spectrum or used for homology searches. The sequence information delivered by the de-novo tool was used for homology searches in order to explain non-identified MS peaks. Two strategies were used: (1) A sequence tag, obtained from the de-novo software, delivered sequence information about the major part of the sequence. (2) Multiple sequence proposals were obtained, which altogether were automatically used for the further analysis. Using the BLAST technology, public and locally available protein sequence databases were screened with both, sequence tags as well as sequence proposal collection. An amino acid transition and an unusual modification (double oxidized tryptophane) were easily localized applying this procedure. POSTER SESSION

P-258 A Novel High Capacity Trap for Faster, Even More Sensitive Proteomics Applications

M. Lubeck, U. Schweiger-Hufnagel, G. Zurek, M. Schubert, A. Brekenfeld, and C. Baessmann Bruker Daltonik GmbH, Bremen, Germany

Three dimensional ion traps with their capability of fast MS to MS/MS switching are widely used as working horses for protein identification. A new instrument with an about 10x higher ion storage capacity shows a dramatically improved performance for proteomics applications in terms of sensitivity, speed and mass accuracy. The improved sensitivity allows reliable protein identification in the sub-fmol range with good data quality. As ion traps are usually directly coupled to an HPLC system, time for MSn experiments is limited to the peak width of the chromatography. New column types enable faster peptide separation with only a few minutes for an entire LC-MS/MS run. The chromatographic peak widths in the range of about 3-4 seconds require a mass spectrometer with a very short duty cycle. The higher capacity of the ion trap with improved geometry results in very good ion statistics for single scans, so that there is no need for averaging. The new geometry also makes a very high scan speed (26 000 amu/s) possible with good mass resolution. The short duty cycle results in fragmentation of more precursor ions per time for fast analysis of complex mixtures. Example data demonstrating the performance for various proteomics applications will be shown. POSTER SESSION

P-259 The Application of a High-field Asymmetric Waveform Ion Mobility Spectrometer (FAIMS) Coupled to a Q-Tof Mass Spectrometer for the LC-MS Analyses of Complex Peptide Mixtures

J. I. Langridge1, J. Wildgoose1, J. B Hoyes1, A. Millar1, R. W. Purves2 and D. A. Barnett2 1Waters Corporation, Micromass MS Technologies Centre, Manchester, UK; 2Ionalytics Corporation, Ottawa, Ontario, Canada

With the decoding of the genes that compose the human genome, emphasis has switched to the identification of the translated gene products that comprise the proteome. Mass spectrometry has firmly established itself as the primary technique for identifying proteins due to its unparalleled speed, sensitivity and specificity. Currently, the most commonly used mass spectrometric technique for protein identification and characterisation is electrospray ionisation (ESI) interfaced to a tandem mass spectrometer allowing fragmentation studies by low energy MS/MS. Separation of a complex digest mixture is often achieved by microcapillary liquid chromatography with on-line mass spectral detection using automated acquisition modes whereby conventional MS and MS/MS spectra are collected in a data dependant manner. This information can be used directly to search databases for matching sequences leading to identification of the parent protein. However, often the limiting factor for identification of the protein is not the quality of the MS/MS spectrum produced, but is the initial identification of the multiply charged peptide precursor ion in the MS mode of operation. This is due to the level of background chemical noise, which may be produced in the ion source of the mass spectrometer. Therefore, a method whereby the chemical noise is reduced allowing the mass spectrometer to specifically target peptide related ions, would be highly advantageous for the study of protein digests. All data has been acquired on a Q-Tof micro mass spectrometer equipped with the Ionalytics Selectra, a FAIMS system, (Ionalytics, Ottawa, Canada) fitted to the regular electrospray source. Standard peptide mixtures and tryptic digest samples were separated by nanoscale HPLC using a Waters CapLC configured with a C18 trapping column and a 75 micron id C18 analytical column. The mass spectrometer was operated in both the LC-MS and LC-MS/MS mode of operation. Data has been acquired from standard peptides and tryptic digests of proteins, to allow optimal tuning of the FAIMS parameters with regard to gas flow rate, gas composition, and compensation voltage (CV). The analysis of these peptides showed significantly increased signal to noise ratios, at their optimal CV, in comparison to operation of the mass spectrometer without the FAIMS device fitted. Both direct infusion and nanoscale LC separations showed reduced background without significant losses in absolute ion current. Examples will be presented showing the advantage of FAIMS in combination with ESI-MS/MS for the identification and characterisation of complex tryptic peptide mixtures. POSTER SESSION

P-260 Identification of Sub-femtomole Level Peptides and Protein Digests Using Tandem MS Data by Matrix-Assisted Laser Desorption Ionization/ Linear Ion Trap Mass Spectrometer

H. Tran, V. Kovtoun, G. Stafford and K. Miller Thermo Electron, San Jose, CA; USA

Sub-femtomole levels of several peptides and protein digests were detected using a novel, low pressure MALDI/Linear Ion Trap (MALDI-LT) instrument. This configuration combines the simplicity of MALDI with high capacity ion storage, fast scan rates, and tandem analysis capabilities of a linear ion trap (LT), to create an ideal system for sensitive, high throughput analysis and identification of protein digests. In this experiment, samples of standard peptides and protein digests, at low femtomole to attomole levels, were prepared in acetonitrile/water and mixed with a diluted α-cyano-4-hydroxycinnamic acid solution (1mg/mL). The digest/matrix mixture was placed on the MALDI plates as 1 mL spots. An MS method was created, using one full MS scan followed by several data-dependent MS/MS scans with dynamic exclusion. Peptide/protein identifications were accomplished with TurboSEQUEST using an indexed bovine/horse database. Results show successful identification of peptides and protein digests at 1 femtomole to 500 attomoles or less. MALDI-LT offers several significant advantages in terms of high throughput and sensitivity. Fast mass scan rate of 16,000 m/z per second yields improved experimental rate and data coverage. Searchable MS/MS data at 1 femtomole levels were acquired with only 5 micro-scans (approximately 1 s total scan time). Efficient fragmentation of peptides resulted in strong y and b ion series, giving library searchable spectra and resulting in better X correlation values in a SEQUEST database search. MALDI-LT can achieve attomole level detection of peptides and proteins from biological samples using full scan tandem MS data. POSTER SESSION

P-261 Deisotoping and Deconvolution of Liquid Chromatography Electrospray Ionisation Mass Spectra

U. Bauer, R. Moraga, B. Spira, C. Baumann and J. Schwarz Proteome Sciences plc, Coveham House, Cobham, Surrey, UK

Gel-free proteomics techniques rely on the combination of liquid chromatography (LC) and electrospray ionisation mass spectrometry ESI-MS/MS e.g. MudPIT [1], ICAT [2] or PST®. Therefore the gel-free approach basically overcomes many technical limitations of the conventional 2D electrophoresis, but bears multiple challenges for bioinformatics. Standard procedures to analyse complex protein mixtures include sample pre-fractionation and HPLC separation, before extensive data-dependent tandem MS fragmentation of peptides is performed. The correlation of MS/MS fragmentation patterns with amino acid sequences in a database [3] is currently the most prominent method to interpret the data. However, the number of tandem MS spectra which can be obtained from an LC-MS/MS run is limited, hence imposing natural limits to the approach. Deisotoping and deconvolution of ESI-MS profile mass spectra is computationally expensive, because electrospray ionisation generates multiply charged ions, usually present in an envelope of distinct charge states. Several methods have been proposed to deconvolve spectra containing multiply charged ions [4-8]. However, none of these methods meet our key requirements: (i) applicable to whole LC-MS runs consisting of several thousand MS profile scans (ii) resolution of co-eluting peptides and perhaps overlapping signals (iii) sensitivity to detect signals close to signal-to-noise level, and (iv) preservation of quantitative information on the abundance of individual peptides in the mixture. The algorithm has been tested on different HPLC-ESI-MS profile spectra derived from peptide mixtures of different complexity using the PST®technology e.g. synthetic mixture of 11 proteins, yeast cell lysate, human cell lines. We will present detailed algorthmic outline, parallel implementation of the algorithm using PVM, data export based on XML, and different application examples.

References: [1] Gygi S. P., et. al. (1999), Nature Biotechnolgy,17, p. 994-999. [2] Washburn M. P., Wolters D., Yates III J. R. (2001), Nature Biotechnology, 19, p. 242-247. [3] Eng J. K., et al. (1994), Am. Soc. Mass Spectrom., 5, p. 976-989. [4] Mann M. et al. (1989), Anal. Chem., 61, 1702-1708. [5] Ferrige A. G. et al. (1992), Rapid Com in Mass Spectrom., 6, 707-711. [6] Zang Z., Marshall A. G.(1998), Am Soc Mass Spectrom, 9, p. 255-233. [7] Wehofsky M., Hoffmann(2002), J. Mass Spectrom., 37, p. 223-229. [8] Zheng M. et al.(2003), Rapid Commun. Mass Spectrom., 17, p. 429-436. POSTER SESSION

P-262 Target Validation Via CALI: Mass Spectrometric Analysis of Induced Photo- oxidation of Peptides and Proteins

N.K. Scheffler and E. Horstkotte Xerion Pharmaceuticals AG, Martinsried, Germany

Chromophore-assisted laser inactivation (CALI) can specifically inactivate protein function by targeted induction of photo-oxidation of amino acids at functional sites of the protein. The implementation of process automation, combined with the use of target-specific scFv antibodies selected by phage display technologies, has transformed CALI from an academic tool to an industrial scale protein target validation technology that provides rapid information about protein function in a cellular process or a diseased state. However, the underlying photochemical mechanism of CALI is still subject of discussion. In an effort to study CALI induced modifications on the amino acid level we analyzed a series of seven peptides containing one or more amino acids known to be amenable to oxidation which are tryptophan, histidine, tyrosine as well the sulphur containing derivates cysteine and methionine. These peptides were irradiated in the presence of a chromophore suitable for CALI and modifications on the amino acid side chains were monitored by MS analysis. In all cases we obtained a pattern of new and induced peaks with mass differences of +14, +16 or multiples thereof compared to the unmodified peptide peak. The mass shift of +32 and +16 indicated the incorporation of oxygen upon irradiation. The exact sites of modification were determined by sequencing of the modified peptides using MALDI-PSD and MS-MS experiments on electrospray and Tof-Tof type instruments. Information regarding the efficiency of the photo-oxidation of the different amino acids was yielded based on the kinetic data obtained from samples containing different dye concentrations. Based on these results all photo- oxidation susceptible amino acids could be lined up according to the efficiency of their photo- oxidation: Trp>His>Cys>Tyr>Met. In order to investigate a possible competition effect we analyzed a mixture of only two peptides as well as several tryptic digests of proteins after sensitized irradiation. We found that only intramolecular but no intermolecular competition effects play a role. As a next step we analyzed an intact protein after sensitized irradiation to evaluate sterical and structural influences on the photo-oxidation process. A mass shift of the intact protein was observed corresponding to roughly 3-4 incorporated oxygens per molecule. Modification sites were analyzed by MALDI-MS of the tryptic digest of the modified protein. Spectra were compared with the ones obtained from the unmodified protein. Modifications were assigned to specific surface accessible amino acids, but not all surface accessible Trp residues were found to be oxidized. Correlation of our data with the 3D crystal structure leads to the conclusion that this is caused by shielding effects of surrounding amino acids. With these studies we have gained valuable information towards a better understanding of the mechanism of the CALI process. We are ultimately aiming at the reliable prediction of inactivation efficiency of any possible target candidate based on its primary sequence combined with tertiary structure information. POSTER SESSION

P-263 An Integrated Platform for the Analysis of Complex Protein Mixtures

S. Hahner, A. Resemann, W. Jabs, D. Suckau and M. Lubeck Bruker Daltonik GmbH, Bremen, Germany

The coupling of liquid chromatography (LC) with mass spectrometry has proven to be a powerful tool for comprehensive proteome analysis. Until recently, ESI has been used online with LC for the analysis of complex protein mixtures. With the introduction of the new MALDI- TOF/TOF mass spectrometer with MS/MS capabilities, the coupling with LC has become a promising option in protein analysis. Advantageous in offline-LC-MALDI is that there are no temporal constraints on MS or MS/MS measurements. In an even more advanced approach the two MS techniques were used here in a parallel setup in which the LC flow of the separated peptides is split allowing for both online analysis with ESI and subsequent offline MALDI analysis. A new high capacity ion trap capable of ultrafast scans in the range of 1 MS + 4 MS/MS spectra in 3 sec provides highest MS/MS throughput. Subsequent analysis of the same LC separated peptide sample deposited onto a MALDI target is performed first of all in MS-mode followed by precursor ion selection for MALDI-MS/MS measurements which have not been selected previously during online-ESI-MS/MS. This combined ‚ESI-MALDI‘ approach provides an increased information readout from a single LC run due to the complementary MS/MS data set obtained from advanced interaction of both ionization techniques. This approach clearly benefits from the online coupled LC separation with ESI capable for MS/MS data acquisition with high-throughput as well as of the subsequent temporal decoupled offline-MALDI MS/MS analysis with intelligent precursor ion selection for MS/MS measurements. POSTER SESSION

P-264 Integrated System for Identifying Low Abundant Proteins from Gels

A. Dedeo1, W. Kopaciewicz1, P.Clark1, M.Emerick1, A. Tomlinson2, C. E. Murphy2, E. Chernokalskaya1, and M. Hornberger3 1Millipore, Life Science Division, Danvers, MA USA; 2Applied Biosystems, Framingham, MA USA; 3Millipore GmbH, Germany

Even with the most sophisticated technology, the depth of proteome coverage is usually low with abundant proteins dominating the analysis. There are likely to be many more proteins in the gel than are clearly visualized by commercially available stains. These proteins are often still accessible to mass spectral analysis. In this paper, we describe an approach for improved analysis of low abundant proteins. Gel pieces were excised from weakly stained regions of a 2-D gel and processed using a system composed of an integrated 96 well sample preparation and presentation system (ZipPlate™) that transfers digested proteins directly onto compatible MALDI targets. The sample preparation system is a 96 well solid phase extraction device containing 300 nl of C18 membrane that can be operated by vacuum or centrifugation. Gel pieces are added to the wells where they are destained and digested with trypsin. The resulting peptides are then desalted and concentrated on the C18 media. Upon elution from the ZipPlateC18, samples can be collected for off-line spotting or LC/MS/MS or directly captured on a Voyager DE™ MALDI-TOF-MS compatible target. A new target system has been engineered such that 4 targets fit neatly under the ZipPlate device, which is dimensioned to SBS standards. POSTER SESSION

P-265 Hydrophilic Interaction Solid Phase Extraction (HILI-SP) in Proteome Analysis: Sample Preparation for 2D-PAGE and HPLC Separation

U. Schneider, A. Posch, G. Weber, P. Weber, M. Nissum, C. Obermaier, R. Wildgruber, S. Kuhfuss and C. Eckerskorn Tecan Munich GmbH, Kirchheim, Germany

Hydrophilic interaction chromatography (HILIC) is a long standing variant of normal phase chromatography, which binds proteins to a strongly hydrophilic support based on interaction of hydrophilic parts of the proteins with the support. Binding occurs in highly concentrated organic solvent, whereas elution occurs, when the support is flushed with aqueous solutions. Free Flow Electrophoresis is a continuous electrophoretic technology which allows the prefractionation of proteomes according to the isoelectric points of the proteins (FFE-IEF), thereby dividing the proteome into discrete pI ranges. Fractions from the FFE are usually not easily amendable to further analytical steps, unless additives like glycerol and the electroendoosmosis inhibitor HPMC are removed from the sample. Here we show the utility of a solid phase extraction (SPE-) protocol utilizing poly-(2- hydroxyethyl)-aspartamide silica (PolyLC inc.), for hydrophilic interaction SPE with complex protein mixtures, termed HILI-SP. We have integrated HILI-SP post-FFE sample processing into the concept of proteome prefractionation by demonstrating the concept of "compatible recovery” which allows quantitative recovery of proteins bound to the HILI-SP support by elution with the downstream analytics buffer, e.g. if 2D-PAGE to be required as downstream analytics, elution will take place with 2D-PAGE sample buffer. POSTER SESSION

P-266 Evaluation of Off-line Strong Cation Exchange Chromatography in Combination with LC-MS/MS for the Analysis of Complex Protein Digest Samples on a Hybrid Quadrupole Orthogonal Acceleration Time-of Flight (Q-Tof) Mass Spectrometer

J. P.C Vissers1, C. Hughes2, I. Campuzano2, T. McKenna2 and J.I. Langridge2 1Waters Corporation, Almere, Holland; Micromass UK Ltd, Manchester. 2Waters Corporation, MS Technologies Centre, Wythenshawe, Manchester, UK

Advances in both HPLC and mass spectrometry instrumentation have allowed the analysis of protein complexes which have not been separated on a two dimensional gel. These experiments involve separation of the complex digest mixture by microcapillary liquid chromatography connected to an instrument capable of data directed switching between the MS and MS/MS modes. Protein identification is then achieved via databank searching of the ESI-MS/MS, providing qualitative information on the proteins that are present. Hundreds of MS/MS spectra can be acquired in a fully automated fashion, resulting in the identification of significant numbers of proteins, including low copy number proteins, from a single LC-MS/MS experiment. If, however, a complex protein mixture is to be investigated then a fractionation step prior to separation of the peptides on the basis of their hydrophobicity is advantageous. This has resulted in 2D HPLC approaches being adopted for the analysis of extremely complex tryptic digest samples. By placing a strong cation exchange (SCX) cartridge followed by a C18 trap cartridge, or by using a bi-phasic analytical column it is possible to pre-fractionate the peptides on-line before separation and analysis by reverse phase LC-MS/MS. In this paper we describe the optimisation of an off-line SCX fractionation step, in combination with automated fraction collection, prior to analysis by reverse phase LC-MS/MS. We will compare this to off-line reverse phase fractionation of the sample, followed by automated nanospray and on-line 2D-LC. Data will be presented on a global tryptic digest of a Saccharomyces cerevisiae cell lysate. POSTER SESSION

P-267 Evaluation and Optimisation of Reverse Phase Column Material for the Analyses of Complex Tryptic Peptide Mixtures by LC-MS and LC-MS/MS

I. Campuzano, T. McKenna, and J. Langridge Waters Corporation, MS Technologies Centre, Wythenshawe, Manchester, UK

Electrospray mass spectrometry coupled to nanobore reverse phase chromatography has rapidly become the chosen method for the separation and characterisation of enzymatically digested protein samples. A key component in the MS analysis is the complexity of the sample that elutes into the mass spectrometer, from the HPLC column, at any given moment in time. This can have profound influence on the number of peptides and hence proteins identified, and also the dynamic range of the identified components. Therefore, the peak capacity of the chromatographic system is often an important attribute. Additional chromatographic attributes such as retention time reproducibility and injection methods can have profound effect on the results, especially if relative quantification is desired. In particular, the analysis of post translationally modified peptides can be problematic, due to their chromatographic behavior on standard C18 reverse phase chromatography used for peptide mapping. A classic example is the case of phosphopeptides, which can often be extremely hydrophilic in nature, and as such are poorly bound by many C18 stationary phases. In this paper we investigate different stationary phases for peptide mapping and in particular we will focus on how to improve chromatographic separations over those methods currently in use. Additionally, the use of a novel online peptide trapping procedure in combination with reverse phase nanoscale HPLC, will be discussed. Data will be presented showing the improved peptide recovery, particularly of small, hydrophilic peptides from a protein digest, resulting in an increased level of protein coverage. POSTER SESSION

P-268 Increase of Resolution for Complex Protein Digests by SCX Gradient Optimization in Offline 2-Dimensional LC-MS/MS

M. Vollmer and P. Hörth Agilent Technologies, Waldbronn, Germany

In contrast to on-line 2D-HPLC in which salt steps are injected onto the SCX (strong cation exchange) column, off-line 2D-HPLC with a continuous salt gradient increases the resolution of complex proteome samples significantly. In addition off-line 2D-HPLC is even more advantageous in providing more flexibility for SCX column dimensions and in the number of collected fractions. To achieve optimal resolution there is a strong need to optimize the first separation step in order to achieve a considerable increase in overall peak capacity. This is especially important if scaling up is necessary for the detection of low abundant proteins and if the workflow includes the modification of fractions. Therefore a defined 10 protein mix and an E.coli total lysate was chosen as model system to demonstrate the importance of the above mentioned issues. The fractions obtained from a continous gradient SCX chromatography using a novel cation exchange material (prototype), were collected with a microfraction collector which is able to collect fractions in the low microliter range. Subsequently the fractions were re-injected to the nanoflow reversed phase dimension which was coupled to nano ESI-MS/MS. The obtained MS/MS data of the eluted peptides were used for database search with Spectrum Mill software for protein identification. Results clearly demonstrate that long shallow SCX gradients significantly increase the number of detected proteins. Additionally, the provided data emphasizes the importance that the column dimensions of a 2 D LC system need to fit properly together in order to maximize overall peak capacity of the separation system. POSTER SESSION

P-269 Comparison of Online and Off-line 2D HPLC as Tool for Proteomic Research

R. Moritz, E. Nägele and M. Vollmer Agilent Technologies Deutschland GmbH, Waldbronn, Germany

Automated on-line 2D-HPLC of digested proteins is nowadays a well-established technology for proteome characterization. Typically, peptide fractions are stepwise eluted from a strong cation exchange (SCX) column and further separated by reversed-phase (RP) chromatography. The peptides are separated in an alternating fashion by short period salt steps followed by shallow gradient RP-HPLC. Methods have been described with or without intermediate enrichment of the SCX fractions on top of a short enrichment column. Finally, proteins are identified by peptide Nanospray iontrap/MS-MS analysis and subsequent database search. Although such an approach offers the advantage of fully unattended operation it is limited in chromatographic resolution and flexibility. As an alternative technique we present herein off-line 2D-HPLC for proteome characterisation using a newly developed microfraction collector and compared it to online 2D-HPLC. For a low complexity ten protein standard mixture the number of identified peptides was comparable for both techniques. However, the analysis of a highly complex protein sample like total yeast extract, revealed clearly a significant increase in identified peptides by the off-line method. The improvement obtained by off-line 2D-HPLC was attributed to higher resolution in gradient SCX chromatography which finally gives the MS more time for precursor selection and peptide fragmentation. Furthermore off-line 2D-HPLC provides more flexibility for the investigator since fractions from the first dimension can be stored, reanalyzed, or chemically and enzymatically tagged or modified prior to the final separation step and MS analysis. We therefore conclude that off-line 2D-HPLC has the potential to provide additional information to the classical SCX-RP on-line approach. POSTER SESSION

P-270 Biomarker Identification from Human Blood by Differential Peptide DisplayTM

H. Tammen1, T. Möhring1, M. Kellmann1, A. Pich2, H.H. Kreipe2 and Rüdiger Hess1 1BioVisioN AG, Hannover, Germany; 2Institute of Pathology, Medizinische Hochschule Hannover, Germany

Differential Peptide Display (DPD) is a technique to generate comprehensive peptide maps of about 3,000 peptides from blood serum covering a mass range of 750 to 15,000 Da. After peptide extraction the sample is separated by means of RP-HPLC (Reversed phase high pressure liquid chromatography) and the peptides eluting from the HPLC column are collected into 96 fractions. Each individual fraction is subjected to MALDI-TOF-MS and the mass spectra of all 96 fractions are combined resulting in a two-dimensional display of peptide masses, where the abscissa displays the mass to charge ratio, the ordinate is determined by the retention time on the RP-HPLC and the signal intensity is depicted by the color saturation. The underlying software allows the processing and analysis of vast amounts of data. Peptide maps of individual samples can be superimposed, facilitating the detection of differences in the resultant subtractive peptide maps. The selected peptides of interest are identified by subjecting the respective HPLC-fractions to ESI-qTOF-MS/MS resulting in peptide fragment spectra. Saved in MASCOT generic files, these spectra serve to identify the corresponding peptide sequence by remote database searching. Applications like analysis of changes during coagulation in the peptide pattern or mass spectrometric phenotyping of individuals are presented. POSTER SESSION

P-271 An Integrated Software Platform for the Acquisition and Analysis of Process and Result Data in Proteomics

K. Rein1, B. Götzelmann2, S. Losko3, P. Ohl4 and C. Eckerskorn1 1Tecan Munich GmbH, Proteomics Division, Kirchheim, Germany; 2Avarto Systems GmbH - Bertelsmann, Gütersloh, Germany; 3Biomax Informatics AG, Martinsried, Germany; 4MGM EDV Beratung GmbH, München, Germany

Software to support Proteomics processes today comes in two flavours: LIMS systems support sample tracking in the process and result databases allow to store and visualize result data. These systems often lack 1) the acquisition of process parameters as required for a process to be reproducible and 2) a high degree on flexibility to combine different experiment steps to a variety of Proteomics processes and 3) a single framework for a unified data analysis of all different processes. Here we show a workflow driven software design for the acquisition and analysis of process and result data in Proteomics experiments, the Integrated Proteomics Environment (IPE). The IPE allows to set-up and track Proteomics experiments, control the experiment process, steer instrument controls, do data analysis and document the results. The IPE covers several important Proteomics experiment steps, from which standardized processes can be created on the fly. Data analysis steps can be combined with any compatible experiment steps. All process and result data can be visualized and analysed in a unified way. Several advanced data analysis tools (e.g. statistics and data mining) are integrated either bi- directionally as an experiment step or uni-directionally as an export interface. POSTER SESSION

P-272 Intelligent and Automated MALDI MS/MS Acquisition

S. Bailey1, A. Wattenberg1, G. Körting1, K.-O. Kräuter2, H.E. Meyer1, M. Blüggel1 1Protagen AG, Dortmund, Germany; 2Bruker Daltonik GmbH, Bremen, Germany

There is an increasing impact in high throughput technologies in protein analytics. Besides the processing of high number of samples the intelligent data handling is of high importance. We set up an automated feedback for the acquisition of additional mass spectra based on the protein identification results from primarily acquired MS data.

For identifying proteins by MALDI mass spectrometry two different types of spectra can be used in principle: (1) Peptide mass fingerprint (PMF): represents the specific pattern of enzymatic cleaved peptides of a protein. (2) Peptide fragmentation fingerprint (PFF): represents the specific fragmentation pattern of a peptide.

Following high resolution 2D gel electrophoresis the peptide mass fingerprint is significant for identifying a protein of a well known organism. For a sample from an organism with partly sequenced genome, for proteins with unknown posttranslational modifications or for a protein mixture the additional information derived from the fragmentation spectra is necessary.

To realise an intelligent feedback of the results of the peptide mass fingerprints to the acquisition of fragmentation spectra a closely linkage of sample processing and analysing the results via a database is required. The peptide masses of a fingerprint spectrum which are suited for fragmentation basically are selected by an evaluation scheme. The intelligent feedback is based on filtering this mass list depending on the protein identification result and the chosen strategy: (1) Identification Strategy: no protein identified by peptide mass fingerprint Æ acquisition of as much fragmentation spectra as possible. (2) Verification Strategy: protein identified by peptide mass fingerprint Æ acquisition of some fragmentation spectra of peptides belonging to the identified protein. (3) Further Elucidation Strategy: protein identified by peptide mass fingerprint Æ acquisition of some fragmentation spectra of peptides which do not belong to the identified protein as the bigger part of the fingerprint spectrum is unexplained yet.

The performance of this method concerning an increased success rate at lower expense of acquisition is demonstrated with a mixture of five proteins. POSTER SESSION

P-273 A new Software Environment for Comprehensive Review of MS Data from Proteomics Experiments

C.A. Miller, B.D. Miller, J. Roark, C. Sauber and F. Mandel Agilent Technologies, Santa Clara, CA, USA

Mass Spectrometry has become a core technology for proteomics research and the increasing interest in large-scale proteome characterization, e.g. shotgun proteomics, can readily lead to a bottleneck in data interpretation and review. An advanced informatics tool is introduced which provides an environment for the rapid, comprehensive review of data from large-scale proteomics experiments. Within this environment, protein database search, result review and validation of database search results can be performed. In addition, large datasets can be compared across multiple experiments and the results can be readily summarized at the protein level. For those peptides not identified through database search, an automated de novo sequencing tool is available and the results from this tool are integrated in the informatics environment to enable cross correlation with database search results. The software also offers an open platform for analysis from multiple MS techniques and vendors. This work will demonstrate the features and performance of the new informatics software for analyzing data from typical proteomics experiments. POSTER SESSION

P-274 A Fully Integrated Proteomics Laboratory Information Management System

E. Brzezinski1, T. Stevenson1, M. McDowell1, G. Kilby1, D. Baker1, J. Rogers1,2 1Pfizer Global Research and Development, Ann Arbor, MI, USA; 2Present address: Abbott Laboratories, Abbott Park, IL, USA

Proteomics is data intensive in its application to drug discovery and is capable of generating large quantities of samples, raw data, results and supplemental information. In order for proteomics projects to progress in a rapid timeframe to successfully impact drug discovery, a systematic approach to handling the underlying scientific data elements is critical. Working with two outside companies, Cimarron and Scimagix, we have developed an integrated Laboratory Information Management System for managing proteomics data. The Cimarron component of the system tracks samples from submission through 2D gel electrophoresis. Gel images are uploaded and stored within Scimagix’s ProteinMine application. From ProteinMine images can either be searched, analyzed or exported to a third party image analysis package. Within ProteinMine spot sets can be selected and cut lists generated for our spot cutting robot, a Genomic Solutions Propic. The spotlist is exported to the mass spectrometry workflow. The MS workflow tracks the gel plugs through MALDI and/or LC MS/MS. Once MS analysis is completed, the system allows the protein ID to be efficiently linked to the original spot on the gel image within ProteinMine. POSTER SESSION

P-275 Towards an Integrated Bioinformatics Platform for Gel-Based and Gel-Free Protein Profiling Techniques

J. Müller, M. Schärfke, R. Joubert, B. Spira, R. Moraga and U. Bauer Proteome Sciences plc, Coveham House, Cobham, Surrey, UK

Conventional 2D gel electrophoresis (2DE) serves as gold standard for protein profiling. High- throughput application of the 2DE technology became feasible due to robotics spot handling platforms and improvements in peptide mass fingerprint analysis (PMF). In parallel, gel-free techniques based on the combination of liquid chromatography (LC) and tandem-MS maturate rapidly. Hence, to conduct drug target identification projects, a bioinformatics platform to analyze, maintain and search proteomics data is indispensable for high-throughput analysis. Our software platform, which has been developed in-house, supports the two complementary experimental methods: (i) the conventional 2DE/PMF approach and (ii) the gel-free Protein Sequence Tag (PST®) technology. The PSS® (Proteomics Software System) engine offers (i) a fully automated 2D gel image analysis which is tightly integrated with the robotic Ettan Spot Handling Platform to treat excised gel plugs and (ii) an automated PMF data analysis. It contains three technical components: the PSS® input module to register the experimental data, the PSS® Staging Area to schedule and perform computation tasks on the data and the PSS® Database to store, query and combine processed results. Ongoing work includes the automation of our gel-free PST® approach. It comprise the submission of LC-MS and LC-MS/MS data via the PST® input module to the repository. The input data is then subjected to a deisotoping/deconvolution algorithm and a pipeline based on the Sequest® algorithm, respectively. Protein identifications are generated on the ground of a specially compiled PST® peptide database.Finally proteins identified using either the conventional or the gel-free PST® technique are annotated in terms of function, biological process and cellular localisation in a semi-automatic manner. A so-called "Proteomics Engine” which automatically delivers protein annotations is currently under development. The software system is designed as a client server-application including an oracle database, several proprietary algorithms and a variety of visualization tools implemented in C/C++ and JAVA. It currently runs in an UNIX environment consisting of a SGI Origin 2000, a SUN 250 and several linux workstations. POSTER SESSION

P-276 Latest Software Developments for Automated Image Analysis of Pre- Fractionated Samples and Narrow Range 2D-Gels

R. Wildgruber1, M. Soegtrop2, G. Schmidt2, S. Fater2, R. Humberg2, K. Rein1, P.J.A. Weber1, G. Weber1, C. Obermaier1, M. Mast1, A. Posch1 and C. Eckerskorn1 1Tecan Munich GmbH, München, Germany; 2Definiens AG, München, Germany

To exploit the full resolving power of 2D-Electrophoresis it is nowadays state of the art to use overlapping narrow range IPGs (0.5 to 1.5 pH units per 18 or 24 cm separation distance) and sample prefractionation, for example by Free Flow Electrophoresis (FFE), in combination. This again leads to a drastically increased number of gels per analyzed pH unit but on the other hand a large number of novel proteins can be separated and visualized. This increased amount of data has to be handled by image analysis systems which have to cope with replicates of one sample (and/or gradient respectievly), and with small regions of overlapping spots inbetween fractions and gradients. Software solutions are urgently needed thus to accomplish these novel and extended tasks from gel puzzling to statistic analysis sets. Here we demonstrate how to analyze (FFE-) prefractionated samples of mouse liver proteins on narrow pH range IPGs using TECAN's Pro Team IEF and how to exploit statistically relevant data using Definiens‘ Proteomweaver. POSTER SESSION

P-277 A New Double Chip Format to Overcome Cross-Reations in Multiplexed Sandwich Immuno-Assays

H. Clausen-Schaumann Nanotype GmbH, Gräfelfing, Germany

Protein assays provide direct access to biologically and pharmacologically relevant information, and are widely used in pharma R&D and bio-medical diagnostics. To obtain a maximum of information from smallest amounts of complex biological samples, there is a growing need for highly multiplexed protein assays. In conventional single marker assays, pairs of capture and detection antibodies are used, to increase the specificity and precision of the assay. However, when used in a multiplexed assay, both capture, and detection antibodies are plagued by cross-reactions and non-specific binding. This leads to large numbers of false positives, and a high level of non-specific background signal, and thus limits the level of multiplexing which can be achieved. Therefore, eliminating cross-reactions and reducing non-specific binding are key factors, for successfully developing highly multiplexed protein assays. Nanotype has developed a new double chip format, consisting of a capture array and a reference array, with the fluorescently labeled detection antibodies coupled to the reference array via molecular force sensors. For each spot of capture antibodies on the capture array, there is a corresponding spot on the reference array, containing the respective detection antibodies. After incubation of the capture surface with the sample solution, the two chip surfaces are brought into contact, to allow for binding of the detection antibodies to the captured antigens. The two surfaces are then separated again, and only where the detection antibodies could bind specifically to their respective antigens, the force sensors yield, and the fluorescently labeled detection antibodies are transferred to the capture array. The force sensors enable discrimination between specific and non-specific binding and increase the sensitivity of our assay. At the same time they allow for the local application of detection antibodies from the reference surface. This second chip surface therefore provides a second dimension for specific encoding in our assay. False positive results caused by cross-reactive antibodies and non-specific binding can be avoided. Consequently, this new double chip format allows for highly specific multiplexed protein assays, without costly and time consuming optimization of antibodies. It reduces the complexity of a multi marker assays to the simplicity of a single marker ELISA. POSTER SESSION

P-278 Practical Proteomics: Transitioning of ELISA Assays to a Quantitative Protein Microarray Format

J.L. Tonkinson, W. Zhao, J. Beator and D. Osborn Schleicher & Schuell BioScience, Inc., Keene, NH, USA

The development of protein microarray technology promises to greatly increase the power of diagnostic tests. With microspot immunoassays, small volumes of patient or research samples can be screened for multiple analytes simultaneously, rather than for a single protein as in current ELISA tests. This ability would provide a profile as well as relative levels of relevant antigens. Protein profiling in a research or clinical setting provides the researcher or clinician with greater insight into biological processes and disease than does simple measurement of the levels of a few antigens independently. Although protein microarrays hold a tremendous amount of promise, the technology is still in its infancy. In this study, we report on our efforts to adapt traditional cytokine ELISA assays to a microarray format. This resulted in an antibody chip where a single sample could be quantitatively assayed for 9-16 cytokines simultaneously. We used multi-well nitrocellulose coated microscope slides as the substrate onto which monoclonal capture antibodies were arrayed, along with a biotinylated-antibody/streptavidin- Cy5 fluorescent detection scheme. Standard immunoassay parameters such as minimum detectable dose, dose response linearity and statistical reproducibility were found to be outstanding relative to bulk ELISA. POSTER SESSION

P-279 Quantitative Immunodetection using Infrared Technology

H. Wohlgemuth LI-COR® Biosciences GmbH, Bad Homburg, Germany

LI-COR® Biosciences offers superior instrument systems for biotechnology. The company pioneered the development of highly sensitive and reliable infrared fluorescence labeling and detection systems for applications in the Proteomics and Genomics area. Recently, we released a new technology for sensitive and quantitative 2-color immunodetection: the Odyssey® Infrared Imaging System. Customer data comparing a serial dilution of AMPK alpha 1 processed with the Odyssey® system to ECL/film/Kodak CCD camera results show that the system combines unparalleled sensitivity in fluorescence detection with precise quantification of each signal. It thus opens new perspectives for quantitative analysis of immuno-stained samples. The Odyssey® utilizes two independent laser/detection systems in the infrared region, consecutively two targets can be labeled simultaneously. This enables for example optimal phosphorylation studies: In non-stimulated and EGF-stimulated A431 cell lysates total ERK protein and Tyrosine-phosphorylated ERK protein can be detected at the same time. Furthermore In-Cell Western - a brand new application - allows high throughput detection and quantification of proteins directly in cells, eliminating the bottlenecks of protein extraction, PAGE and membrane transfer. POSTER SESSION

P-280 Novel Multiplexing: Following a Western Signal to the Needle in a Haystack

M. Beaumont, F. Vega, M. Mattison, and C. Hannum DNAX Research, Palo Alto, CA, USA

The Western Blot is a highly sensitive means for detecting the presence of a molecule of interest for which a specific antibody is available. If the target protein is a very minor component of a complicated mix of molecules, however, the western signal alone is not sufficient for guiding the identification of that protein. We describe here a method for multiplexing, or precisely overlapping and matching, a complicated 2D protein pattern and the western signal arising from the specific binding of an antibody to its target protein within that pattern. The example used to demonstrate this method is the identification of a protein that was found to spuriously cross-react, both in immunohistochemistry and on Western Blots, with a specific monoclonal antibody. The method involves the precise matching of fluorescent patterns arising from Cy5-labeled cell lysate proteins and ECL+ Western signal on a single multiplexed membrane. The overlapping images were analyzed using Amersham Decyder software, and the protein responsible for the Western signal was clearly detected within the crowd. The cross-reacting protein was then robotically excised from a parallel Sypro Ruby- stained preparative gel, and its identity was determined by nanospray LC-MS/MS. This is a powerful new method for following the beacon of a Western signal to its source, even within terribly complicated patterns of accompanying proteins. It is a method that should be applicable to all experimental systems in which an antibody is available that works well for western blotting. It is not necessary that the antibody be able to precipitate its target antigen, and consequently it will enable the use of anti-peptide antisera to locate and characterize their matching intact proteins, even within whole cell lysates.