Proc. Nati. Acad. Sci. USA Vol. 86, pp. 7701-7705, October 1989 -electroblotting and -microsequencing strategies in generating protein data bases from two-dimensional gels (computerized protein data bases/human genome sequencing) G. BAUW*, J. VAN DAMME*, M. PUYPE*, J. VANDEKERCKHOVE*, B. GESSERt, G. P. RATZt, J. B. LAURIDSENt, AND J. E. CELISt *Laboratorium voor Genetica, Rijksuniversiteit Gent, B-9000 Gent, Belgium; tInstitute for Medical Biochemistry and Bioregulation Research Centre, Aarhus University, DK-8000 Aarhus C, Denmark Communicated by M. Van Montagu, July 3, 1989 (received for review March 8, 1989)

ABSTRACT Coomassie blue-stained, heat-dried, and Here we describe in detail a modified version of the computer-imaged two-dimensional gels used to develop com- blotting/microsequencing procedure that allows sequence prehensive human protein data bases served as the protein analysis of protein spots recovered from Coomassie blue- source to generate partial amino acid sequences. The protein stained, heat-dried 2D gels. The devised protein recovery spots were collected from multiple gels, rehydrated, concen- procedure can be used to concentrate minor protein spots trated by stacking into a new gel, electroblotted onto inert collected from several stained gels. In addition, by making membranes, and in situ-digested with trypsin. Peptides eluting use ofthe information stored in the comprehensive human 2D from the membranes were separated by HPLC and sequenced. gel protein data bases (5, 6), it was possible to analyze Using this procedure, it was possible to generate partial of interest by selecting tissues or cell types where a sequences from 13 human proteins recorded in the amnion cell particular protein was expressed in higher amounts. The protein data base. Eight of these sequences matched those of method was used to generate partial amino acid sequences of proteins stored in data bases, demonstrating that a systematic 13 human proteins: 6 cell growth/transformation-sensitive analysis of proteins by computerized two-dimensional gel elec- markers, 1 epithelial-specific protein, and 6 polypeptides trophoresis can be directly linked to protein microsequencing whose relative degree of expression is not affected signifi- methods. The latter technique offers a unique opportunity to cantly by the growth stage of the cell (ref. 6 and references link information contained in protein data bases derived from therein). The identity of most of these proteins could be the analysis of two-dimensional gels with forthcoming DNA determined because sequences generated from them matched sequence data on the human genome. those stored in protein data bases. Two-dimensional (2D) gel is generally con- MATERIALS AND METHODS sidered the method with the highest resolution for protein separation at the microgram level (1-3). It is, therefore, Cultured Cells and Tissues. Human MOLT-4 cells were regularly used to study phenotypically dependent alterations grown in suspension in Dulbecco's modified Eagle's medium ofprotein expression in total cellular extracts or enriched cell containing 10% (vol/vol) fetal calf serum and antibiotics fractions. The complex protein patterns that may (penicillin at 100 units/ml and streptomycin at 50 ,ug/ml). often Fetal human tissues dissected from a 4-month normal human display up to 2000 spots can be analyzed by computer- male fetus were used in some cases as source of proteins. imaging and the information stored in comprehensive data These experiments have been approved by the Ethical Sci- bases (4-9). This allows a further detailed quantitative com- entific Committee of the Aarhus Amtskommune. parison of a large number of gels and a more thorough search 2D . The procedures for computerized for (a) protein(s) whose expression is typically associated 2D gel electrophoresis have been described elsewhere (6, 21). with variations in the phenotype, with differentiation, cell Some of the protein preparations were partially enriched by cycle, cell lineage, neoplastic transformation, genetic dis- ammonium sulfate fractionation and ion-exchange chroma- eases, etc. (see, for instance, refs. 4 and 9). Identified marker tography (22) or by cell organelle fractionation (B.G. and proteins can then be further characterized by comigration J.E.C., unpublished data), prior to 2D gel electrophoresis (23) experiments with known proteins or mixtures of proteins (for details, see Table 1). derived from isolated cell organelles (nuclei, mitochondria, Protein Recovery from Dried 2D Gels. Protein spots from Golgi, vacuoles, membranes, extracellular spaces, etc.). Coomassie blue-stained and heat-dried gels were excised Alternatively, immunological cross-reactivity with specific with a minimum of polyacrylamide and submerged in 50 mM may serve for protein identification. boric acid (adjusted to pH 8.0 with NaOH) containing 0.1% Systematic 2D gel protein analysis has now gained another SDS (buffer A). The buffer volume (1-2 ml) was not critical dimension with the possibility of sequencing (major) protein as protein losses due to elution were found to be minimal in spots after elution (10, 11) or electroblotting onto inert this particular buffer system. After 2 hr of rehydration, the membranes (12-18). The generated NH2-terminal sequences swollen gel pieces were taken up with tweezers and placed in are generally of sufficient length for a search of protein a gel slot of a new slab gel. This gel was cast using spacers identity or similarity or for the generation of specific DNA that were 0.5 mm thicker than those of the original 2D gel so probes for cloning purposes. Proteins that are NH2- as to facilitate loading of the pieces. Here, we used 1- terminally blocked (either naturally or artifactually) are mm-thick 2D gels and 1.5-mm-thick "concentrating" gels. cleaved in situ and sequences are obtained from the gener- The stacking gel (5% polyacrylamide) extends 2 cm beneath ated peptides (16, 19, 20). the bottom of the slot, which is 6 mm broad and between 1.5

The publication costs of this article were defrayed in part by page charge Abbreviations: IEF, ; PVDF; polyvinylidene payment. This article must therefore be hereby marked "advertisement" difluoride; NEPHGE, nonequilibrium pH gel electrophoresis; 2D, in accordance with 18 U.S.C. §1734 solely to indicate this fact. two-dimensional.

7701 Downloaded by guest on September 23, 2021 7702 Biochemistry: Bauw et al. Proc. Natl. Acad. Sci. USA 86 (1989) cm and 3 cm deep (depending on the number of gel pieces to submerge the membrane pieces (between 100 and 150 ,ul). collected in the slot). The separation gel was as described by To this was added 1 ,ul of a freshly prepared solution of Laemmli (24). The gel pieces (up to 15 pieces could be trypsin at 1 mg/ml in buffer B. The digestion proceeded for combined in a single slot) were then overlayed with gel 4 hr at 37°C. The supernatant was then transferred into a sample buffer [1% SDS/10% (vol/vol) glycerol/0.1% bro- second Eppendorf tube and the membrane pieces were mophenol blue/50 mM dithiothreitol/13 mM Tris HCl, pH further washed once with 100,ul of 80% (vol/vol) formic acid 6.8) and occasionally trapped air bubbles were removed. Gel and four times with 100 ,ul of distilled water. All washing electrophoresis was carried out until the dye reached the solutions were added to the digestion mixture in the second bottom of the separation gel. Eppendorf tube. In some cases, the peptide solution was Protein Electrotransfer. Proteins were electroblotted onto stored at -20°C until HPLC analysis. glassfiber sheets coated with poly(4-vinyl-N-methylpyri- In situ digestion on coated glassfiber sheets was carried out dinium iodide) or polyvinylidene difluoride (PVDF) mem- as for PVDF membranes, except that quenching ofremaining branes as described (15) using 50 mM Tris/50 mM boric acid protein-absorbing sites was done with 0.2% polyvinylpyrroli- (pH 8.3) as transfer buffer. The transfer was carried out for done/50% (vol/vol) methanol/50% (vol/vol) H20. at least 8 hr at 35 V by using a Bio-Rad Transblot apparatus. Peptide Separation by Reversed-Phase HPLC. The com- Gels containing >12.5% polyacrylamide were equilibrated bined washing solutions (±600 ,l) were loaded on a C4 for 2 hr in buffer A (see above) before transfer to minimize gel reversed-phase column (0.46 x 25 cm; Vydac Separations distortion during the blotting process. Group) and the peptides were eluted with a linearly increas- PVDF-membrane- and glassfiber-membrane-bound pro- ing gradient of acetonitrile in 0.1% trifluoroacetic acid. The teins were visualized by staining with Amido black and column was equilibrated in 0.1% trifluoroacetic acid; the fluorescamine, respectively (12, 15). gradient was started 5 min after injection and reached 70% Membrane in Situ Protease Cleavage. Proteolytic digestion (vol/vol) acetonitrile after an additional 70 min. Eluting was carried out as described (19). Briefly, the membrane peptides were detected by UV absorbance at 214 nm (absor- piece carrying the protein was excised, cut into pieces of bance units full scale: 0.1 or 0.2) and collected by hand in approximately 3 by 3 mm, and collected in an Eppendorf Eppendorf tubes. A Waters-Millipore HPLC apparatus con- tube. They were then immersed in 200-500 Al of a 0.2% sisting of two pumps (model 510), a gradient controller, and polyvinylpyrrolidone (30 kDa) solution in methanol. After 30 a model 481 variable wavelength detector was used for these min, the quenching mixture was diluted with an equal volume separations. The major peptides were further dried in a of distilled water and further incubated for 5-10 min. The SpeedVac (Savant) concentrator and stored at -20°C prior to supernatant was then discarded and the membrane pieces sequence analysis. were washed four times with 200-500 ,ul water and once with Amino Acid Sequencing. Peptides were selected for amino 500 ttl 0.1 M Tris HCl (pH 8.5, buffer B). The buffer was acid sequence analysis on the basis of peak height and peak removed and replaced by a volume ofthe same buffer enough resolution and were redissolved in 30 ,l4 of0.1% trifluoracetic IEF - a,¢|;;asS~~~~~~~~~~~~~~~~~~~o __.t .... Cl) iF i5 c 110

_ _;. -68

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_ x o .. ... _ o, -43

v - W_*m$ ...... : I ,__ _ ...... ,:: -36

f _' '.:.: 9 -30

FIG. 1. Fraction of a synthetic image of an IEF fluorogram of [35S]methionine-labeled proteins from amnion cells (for the complete 2D gel pattern, see ref. 5). Proteins that have been microsequenced are indicated with their corresponding number in the comprehensive human amnion protein data base (5). MW, Mr. Downloaded by guest on September 23, 2021 Biochemistry: Bauw et al. Proc. Natl. Acad. Sci. USA 86 (1989) 7703

acid/30% acetonitrile before loading on a precycled Poly- for microsequencing included six proliferation-sensitive and/ brene-coated glass filter. The sequence analysis was carried or transformation-sensitive polypeptides (IEFs 6318, 7205, out with a gas-phase sequencer (model 470A, Applied Bio- 8214, 8505, and 9109 and NEPHGE 3004) (7), an epithelial systems) equipped with an on-line phenylthiohydantoin marker (IEF 9105), and six polypeptides (IEFs 8502, 8704, amino acid derivative analyzer (model 120A) and run with the 9105, 9205, 9209, and 9806) whose rate of synthesis is not sequencing program recommended by the manufacturer. affected significantly by changes in growth rate and/or trans- Computer Search for Identity or Similarity. The amino acid formation (NEPHGE is nonequilibrium pH gel electropho- sequence comparisons were carried out using the FASTDB resis). To facilitate microsequencing, these proteins were cut computer program of Intelligenetics. Two protein data bases out from Coomassie blue-stained gels of (i) partially purified were screened, the Protein Identification Resources (release protein fractions of human MOLT-4 cells (IEFs 6318, 7205, 18) from the National Biochemical Research Foundation and 8502, 8505, 8704, 9205, 9209, 9109, and 9806 and NEPHGE the Swiss-Prot data base (release 9) from EMBL. 3004; Table 1) or (it) total protein extracts of fetal human tissues (IEFs 5206, 8214, and 9105; see Table 1). With the RESULTS exception of NEPHGE 3004, all other microsequenced pro- teins are indicated with their corresponding number in the Fig. 1 shows the imaged 2D gel isoelectric focusing (IEF) master AMA protein data base (Fig. 1) (5). protein pattern of a [35S]methionine-labeled total extract of Gel pieces excised from 3 to 15 dry gels depending on their transformed human amnion cells (AMA). Proteins selected abundance were re-eluted and concentrated in a one- Table 1. Partial amino acid sequences generated from proteins isolated from two-dimensional gels Ref. number in Molecular AMA protein mass, Residues data base* kDa Source Sequence Protein sequenced Ref(s). IEF 9806 110.9 MOLT-4*t IP?PEAVKPDD?(D)E?APAKIP ?VPPMANNPSYQGI ?TDAPQ(P)(K) EIEDPEDRKPED IEF 8704 93.6 MOLT 4t SGTSEFLNK Endoplasmin 168-176 25, 26 (F)AFQAEV 75-81 GLFDEYGSK 395-403 IEF 8505 56.3 MOLT-4t IKPHLMSQELPED?(D)KQPVK 3-PHase or PDI 351-370 27, 28 LITLEEEMTK 317-326 QLAPI?DKLGETYKD(H)EN 402-419 IEF 8502 52.8 MOLT-4t TIFTGHTAVVEDVS?(H)LL?E DFSIHR (T)PSSDVLVFDYTKHPSK IEF 6318 37.3 MOLT-4t MTDQEAIQDL Human B23 52-61 ADKDYHFKVDNDENEHQLSL Xl B23 homology 24-46 29, 30 IEF 7205 36.7 MOLT-4t ?FAFVQYVNE(R) hnRNP protein C 51-61 31 SAAEMYGS?FDLDYDFQ(R) 100-117 IEF 5206 35.5 Fetal human lung§ IVADKDYSVTANSK LDH H chain 77-90 32 YLMAEK 172-177 IEF 9205 34.3 MOLT-40 L?TDGDKAFVDFLSDEIKEE EV(S)FQ(S)TGER IEF 8214 33.0 Fetal human lung§ QVYEEEYGSSLEDDVVG Lipocortin V 126-143 33 GTVTDFPGFDER 6-18 VLTEIIASR 108-117 ?GTDEEKFITIFGT(R) 187-201 IEF 9209 31.9 MOLT-4 ?YNHIK ?FGDLR IQADGLV?GS(S)K IEF 9109 31.1 MOLT-4 YSEKEDKYEEEIK TM ,3 chain homology 177-189 34 EENVGLHQTLDQTLNELN?I 228-247 IEF 9105 31.0 Fetal human skin§ QTF?EAMA?L?TL(S)E ENLTL?TA?NA?(E)(E)GGE?PQEPQ VFYLK SAYQEAMDISK NEPHGE 3004 18.2 MOLT-4 VSFEL Human cyclophilin 20-24 35 TE?LDG 118-123 SIYGEKFEDENF 77-88 ,B-PHase, f subunit of prolyl-4-hydroxylase; PDI, protein disuffide isomerase; Xl, Xenopus laevis; hnRNP, heterogeneous nuclear ribonucleoprotein; LDH, lactate dehydrogenase; H, heavy; TM, horse platelet tropomyosin. *From Celis et al. (5). tIsolated from gels of partially purified cyclin/proliferating cell nuclear antigen preparations from human MOLT-4 cells. Steps in purification involved 40-80% ammonium sulfate fractionation, DEAE-Sephacel chromatography (fraction eluted with 0.3 M KCI), and HPLC (TSK DEAE-SPW) chromatography (23). The fraction eluted with 0.45-0.8 M sodium acetate was applied to the gel. tIsolated from gels of 0.6 M NaCl extracts of nuclear pellets from human MOLT-4 cells. The extracts were further purified by hydroxyapatite. Proteins eluting with 0.3 M potassium phosphate (pH 7) were applied to the gel (IEF or NEPHGE) (B.G. and J.E.C., unpublished data). §Cut from gels of total extracts. Question marks in the sequences indicate the positions where residues could not be identified unambiguously; residues in parentheses are the most probable assignment. Downloaded by guest on September 23, 2021 7704 Biochemistry: Bauw et al. Proc. Natl. Acad. Sci. USA 86 (1989)

dimensional gel using the protein-stacking properties of the Protein IEF 9 8 0 6 ( 1 0.9 kDa) discontinuous Laemmli gel system (24). Proteins were then A electroblotted on membranes and digested in situ with tryp- sin. The peptides released to the supernatant were further separated by reversed-phase HPLC and sequenced. Compared to similar previously published procedures (15, 16, 20), this approach shows many improvements. (i) Gels can be handled by conventional methods involving Coomassie blue-staining (dried, stored, and/or scanned for synthetic imaging) and no special precautions are necessary to guarantee subsequent amino acid sequence determination. The standard use of Coomassie blue-stained, dried gels as a protein source increase considerably the versatility of the method. Indeed, it is now possible to use gels that are B routinely generated during the development of the compre- hensive human protein data bases (5, 6). Furthermore, the information from these data bases allowed us to select gels from tissues or cells in which the protein of interest is most abundantly present (here we used fetal human tissues as a better source of some proteins; Table 1). In this study we

often used gels that have been stored in a dried state for >6 -0 -I- -* -1: months at room temperature. As will become evident from 20 30 40 the sequence analyses (Table 1) and much to our surprise, we never noticed any deamidation at amide residues or oxidation Protein IEF 8214 (33 kDa) C AUFS of methionine residues. (ii) This protein recovery technique 2 1 4 is easily used to concentrate spots from multiple gels to study

less abundant polypeptides. The internal sequencing __ (iii) -e_____| strategy employed here also avoids situations where proteins |I,J, 2 -0 II are NH2-terminally blocked either as a result of co-II or 20 ~~30 40 post-translational modification or due to spurious artifactual reactions with components of the gel matrix and/or chemi- Sequence analysis of cals used in the procedure. (iv) The membrane in situ digestion procedure used in this study is similar to that originally described by Aebersold et al. (16) but has been adapted to fit with the PVDF- or polybase-coated glassfiber blotting procedure (19). Compared to other in situ cleavage methods, this approach combines several advantages that are illustrated for proteins IEFs 9806, 8502, and 8214 in Fig. 2. First, the number of peptides released from the membrane into the supernatant is generally much smaller than expected from the number of potential cleavage sites. As a conse- quence, peptide HPLC chromatograms are extremely simple and most peptides are obtained in pure form (compare the peptide patterns of IEF 9806, a 110-kDa protein, and IEF 8502, a 53-kDa protein). Such a situation is not encountered when proteins are exhaustively digested in the gel matrix (36). In this case, the complexity of the peptide pattern is I generally directly related to the size of the digested protein so I that large proteins are difficult to analyze. In addition, peptide chromatograms from such digests are seldom free of artifactual peaks due to gel contaminants or protein dyes. Our modified in situ cleavage procedure was also found to be FIG. 2. HPLC traces of peptides released from some blotted advantageous compared to the gel in situ partial digest proteins after in situ trypsin digestion (A-C). AUFS, absorbance unit protocol used to obtain internal protein sequences (20). full scale. (D) HPLC traces of the phenylthiohydantoin amino acid Indeed, the latter is difficult to reproduce and the large residues in cycles 1-13 of peptide 2 of protein IEF 8214. Identified fragments obtained are often derived from the same region in amino acids are indicated by the one-letter amino acid code. the sequence. As a result, fragments are recovered in low yield and often display the same NH2-terminal sequence. of prolyl-4-hydroxylase (IEF 8505), nuclear ribonucleopro- Another major advantage of this procedure resides in the tein particle C protein (IEF 7205), lactate dehydrogenase H unusually high sequencing efficiencies encountered with pep- chain (IEF 5206), lipocortin V (IEF 8214), and cyclophilin tides released from membrane-bound proteins (initial se- (NEPHGE 3004) (for references, see Table 1). The peptides quencing yields often exceed 80%). The latter point is illus- of protein IEF 8704 matched those found in the sequence of trated in Fig. 2D showing the traces of the phenylthiohydan- the 108-kDa chicken heat shock protein, a protein that is very toin amino acid derivative chromatograms of cycles 1-12 of homologous to murine endoplasmin. The two peptides of a peptide from protein IEF 8214 (Fig. 2C). Clearly, <0.002 protein IEF 9109 are identical to sequences of horse platelet adsorbance units (<10 pmol) can be reliably sequenced. tropomyosin. Our analyses of protein IEF 6318 cover two Eight out of 13 proteins could be recognized on the basis peptides. One is identical to a sequence located in the of identity or homology between generated peptide se- COOH-terminal end of a sequence encoded by a partial quences (Table 1) and protein sequences stored in data bases. cDNA clone of human nuclear phosphoprotein B23 and is This allowed a straight forward identification of the P subunit very similar to the corresponding sequence derived from a Downloaded by guest on September 23, 2021 Biochemistry: Bauw et al. Proc. Natl. Acad. Sci. USA 86 (1989) 7705 full-length cDNA sequence of the Xenopus laevis B23 pro- Bravo, R. (Academic, New York), pp. 445-476. tein. The second peptide is located very near the NH2 4. Celis, J. E., Madsen, P., Gesser, B., Kwee, S., Nielsen, H. V., that is Rasmussen, H. H., Honore, B., Leffers, H., Ratz, G. P., Basse, B., terminus of the protein sequence currently only Lauridsen, J. B. & Celis, A. (1989) in Advances in Electrophoresis, documented for the X. laevis cDNA sequence. Based on the ed. Chrambach, A. (VCH, Weinheim, F.R.G.), in press. observed homologies, polypeptide IEF 6318 is identified as 5. Celis, J. E., Ratz, G. P., Celis, A., Madsen, P., Gesser, B., Kwee, the human B23 nuclear phosphoprotein (for references on S., Madsen, P. S., Nielsen, H. V., Yde, H., Lauridsen, J. B. & protein similarities, see Table 1). The identity oflipocortin V, Basse, B. (1988) Leukemia 9, 561-601. protein B23, and endoplasmin has been verified by cross- 6. Celis, J. E., Ratz, G. P., Madsen, P., Gesser, B., Lauridsen, J. B., Brogaard-Hansen, K. P., Kwee, S., Ramussen, H. H., Nielsen, reactivity with specific antibodies (33, 37, 38). H. V., Cruger, D., Basse, B., Leffers, H., Honore, B., M0oler, D. Peptides from five other proteins (IEF 8502, 9105, 9205, & Celis, A. (1989) Electrophoresis 10, 76-115. 9209, and 9806) could not be correlated with known protein 7. Garrets, J. I. (1989) J. Biol. Chem. 264, 5269-5282. sequences. The obtained partial sequence data, however, are 8. Garrets, J. I. & Franza, B. R. (1989) J. Biol. Chem. 264, 5283-5298. of sufficient length for the synthesis of specific DNA probes 9. Garrets, J. I. & Franza, B. R. (1989) J. Biol. Chem. 264, 5299-5312. 10. Weber, K. & Osborn, M. (1975) in The Proteins I, ed. Neurath, H. for isolating and sequencing cDNA clones. & Hill, R. L. (Academic, New York), pp. 179-223. 11. Hunkapiller, M. W., Lujan, E., Ostrander, F. & Hood, L. E. (1983) Methods Enzymol. 91, 227-236. DISCUSSION 12. Vandekerckhove, J., Bauw, G., Puype, M., Van Damme, J. & Van The systematic use of protein microsequencing methods in Montagu, M. (1985) Eur. J. Biochem. 152, 9-19. 13. Aebersold, R. H., Teplow, D. B., Hood, L. E. & Kent, S. B. H. conjunction with the 2D gel protein separation and data (1986) J. Biol. Chem. 261, 4229-4238. acquisition system extends considerably the possibilities of 14. Matsudaira, P. (1987) J. Biol. Chem. 262, 10035-10038. the latter. Indeed, proteins from 2D gels can now be identified 15. Bauw, G., De Loose, M., Inze, D., Van Montagu, M. & Vande- by direct partial sequence comparison rather than by indirect kerckhove, J. (1987) Proc. Natl. Acad. Sci. USA 84, 4806-4810. methods such as comigration with selected markers or im- 16. Aebersold, R. H., Leavitt, J., Saavedra, R. A., Hood, L. E. & Kent, S. B. H. (1987) Proc. Natl. Acad. Sci. USA 84, 6970-6974. munological cross-reactivity with specific antibodies. This 17. Eckerskorn, C., Mewes, W., Goretzki, H. & Lottspeich, F. (1988) may largely extend the identification possibilities for sets of Eur. J. Biochem. 176, 509-519. proteins whose post-translational modification or relative 18. Walsh, M. J., McDougall, J. & Wittmann-Liebold, B. (1988) Bio- rate of synthesis is altered as a result of various cell stimuli. chemistry 27, 6867-6876. In addition, the obtained partial amino acid sequence infor- 19. Bauw, G., Van den Bulcke, M., Van Damme, J., Puype, M., Van Montagu, M. & Vandekerckhove, J. (1988) J. Prot. Chem. 7, mation is in most cases sufficient to clone and sequence 194-196. previously unidentified proteins. 20. Kennedy, T. E., Gaminovicz, M. A., Barzilai, A., Kandel, E. R. & Even though this study describes a first attempt to combine Sweath, J. D. (1988) Proc. Natd. Acad. Sci. USA 85, 7008-7012. 2D gel data bases with protein microsequencing, the results 21. Garrets, J. I., Farrar, J. T. & Burwell, I. C. B. (1984) in Two- are encouraging. For example, the direct identification of Dimensional Gel Electrophoresis ofProteins: Methods and Appli- cations, ed. Celis, J. E. & Bravo, R. (Academic, New York), pp. proliferation- and/or transformation-sensitive proteins B23, 37-91. heterogeneous nuclear ribonucleoprotein, lipocortin V, 22. Ogata, K., Ogata, Y., Nakamura, R. & Tan, E. M. (1986) J. tropomyosin, cyclophilin, and 3-prolyl-4-hydroxylase add to Immunol. 135, 2623-2627. the list of identified proteins that included four components 23. Bravo, R. (1984) in Two-Dimensional Gel Electrophoresis of Pro- of the 40S heterogeneous nuclear ribonucleoprotein particles teins: Methods and Applications, ed. Celis, J. E. & Bravo, R. (heterogeneous nuclear Bla, B2, and (Academic, New York), pp. 3-36. ribonucleoproteins Al, 24. Laemmli, U. K. (1970) Nature (London) 227, 680-685. C4), three tropomyosins (IEFs 9213, 9215, and 9226), two 25. Kulomaa, M. S., Wiegel, N. L., Kleinsek, D. A., Beattie, W. G., heat-shock proteins (hsx70 and hsp83), vimentin, and the Conneely, 0. M., March, C., Zaruchi-Schulz, T., Schrader, W. T. DNA replication protein cyclin/proliferating-cell nuclear an- & O'Malley, B. W. (1986) Biochemistry 25, 6244-6251. tigen (refs. 6, 39, and 40 and references therein). 26. Smith, M. J. & Koch, G. L. E. (1987) J. Mol. Biol. 194, 345-347. Clearly, as more proliferation- and/or transformation- 27. Pihlajaniemi, T., Helaakoski, T., Tasanen, K., Myllyla, R., Hu- htala, M. L., Koivu, J. & Kivirikko, K. I. (1987) EMBO J. 6, sensitive proteins are identified by microsequencing or other 643-649. indirect methods, it should be worthwhile to search for sets 28. Edman, J. C., Ellis, L., Blancher, R. W., Roth, R. A. & Rutter, of coregulated proteins by using computerized 2D gel elec- W. J. (1985) Nature (London) 317, 267-270. trophoresis. Taking into account the rapid progress in gene 29. Chan, P. K., Chan, W. Y., Yung, B. Y. M., Cook, R. G., Aldrich, cloning and sequencing and the possibility of sequencing the M. B., Ku, D., Goldknopf, I. L. & Busch, H. (1986) J. Biol. Chem. human genome, the strategies described and applied in this 261, 14335-14341. 30. Schmidt-Zachmann, M. S., Hugle-Dorr, B. & Franke, W. W. (1987) paper will extend our current understanding of the molecular EMBO J. 6, 1881-1890. dynamics of gene regulation and in particular of cell prolif- 31. Swanson, M. S., Nakagawa, T. Y., LeVan, K. & Dreyfuss, G. eration. (1987) Mol. Cell. Biol. 7, 1713-1739. 32. Sakai, I., Sharief, F. S., Pan, Y.-C. E. & Li, S. S.-L (1987) Bio- The authors acknowledge the skill of Dr. M. De Cock in preparing chem. J. 248, 933-936. the manuscript, the assistance of J. Coppieters during computer 33. Pepinsky, R. B., Tizard, R., Mattaliano, R. J., Sinclair, L. K., searching, and the support of Prof. M. Van Montagu. The work in Miller, G. T., Browning, J. L., Chow, E. P., Burne, C., Huang, Gent was supported by grants from the National Fund for Scientific K. S., Pratt, D., Wachter, L., Hession, C., Frey, A. Z. & Wallner, Research (Belgium) and the Commission of the European Commu- B. P. (1988) J. Biol. Chem. 263, 10799-10811. the was from the 34. Lewis, W. G., Cote, G. P., Mak, A. S. & Smillie, L. B. (1983) nities, whereas work in Aarhus supported by grants FEBS Lett. 156, 269-273. Danish Biotechnology Programme, the Danish Cancer Society, the 35. Haendler, B., Hofer-Warbinek, R. & Hofer, E. (1987) EMBO J. 6, Danish Rheumatoid Society, NOVO Fund, and the Fund for Lae- 947-950. gevidenskabens Fremme. G.B. was indebted to the Instituut tot 36. Elder, J. H., Pickett, R. A., Hampton, J. & Lerner, R. A. (1977) J. Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid Biol. Chem. 252, 6510-6515. en Landbouw for a predoctoral fellowship; J.V. was a Research 37. Feuerstein, N., Spiegel, S. & Mond, J. J. (1988) J. Cell Biol. 107, Associate of the National Fund for Scientific Research (Belgium). 1629-1642. 38. Koch, G. L. E., Booth, C. & Wooding, F. B. P. (1988) J. Cell Sci. 1. O'Farrell, P. H. (1975) J. Biol. Chem. 250, 4007-4021. 91, 511-522. 2. Garrels, J. I. (1979) J. Biol. Chem. 254, 7961-7977. 39. Bravo, R. & Celis, J. E. (1982) Clin. Chem. 28, 949-954. 3. Bravo, R. & Celis, J. E. (1984) in Two-Dimensional Gel Electro- 40. Bravo, R., Frank, R., Blundell, P. A. & McDonald-Bravo, H. (1987) phoresis ofProteins: Methods and Applications, ed. Celis, J. E. & Nature (London) 326, 515-517. Downloaded by guest on September 23, 2021