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Poster Session POSTER SESSION POSTER SESSION 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
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