Proc. Nati. Acad. Sci. USA Vol. 86, pp. 7701-7705, October 1989 Biochemistry Protein-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 computer-imaged two-dimensional gels used to develop com- prehensive human protein data bases served as the protein source to generate partial amino acid sequences. The protein spots were collected from multiple gels, rehydrated, concen- trated by stacking into a new gel, electroblotted onto inert membranes, and in situ-digested with trypsin. Peptides eluting from the membranes were separated by HPLC and sequenced. Using this procedure, it was possible to generate partial sequences from 13 human proteins recorded in the amnion cell protein data base. Eight of these sequences matched those of proteins stored in data bases, demonstrating that a systematic analysis of proteins by computerized two-dimensional gel elec- trophoresis can be directly linked to protein microsequencing methods. The latter technique offers a unique opportunity to link information contained in protein data bases derived from the analysis of two-dimensional gels with forthcoming DNA sequence data on the human genome. Here we describe in detail a modified version of the blotting/microsequencing procedure that allows sequence analysis of protein spots recovered from Coomassie blue- stained, heat-dried 2D gels. The devised protein recovery procedure can be used to concentrate minor protein spots collected from several stained gels. In addition, by making use of the information stored in the comprehensive human 2D gel protein data bases (5, 6), it was possible to analyze proteins of interest by selecting tissues or cell types where a particular protein was expressed in higher amounts. The method was used to generate partial amino acid sequences of 13 human proteins: 6 cell growth/transformation-sensitive markers, 1 epithelial-specific protein, and 6 polypeptides whose relative degree of expression is not affected signifi- cantly by the growth stage of the cell (ref. 6 and references therein). The identity of most of these proteins could be determined because sequences generated from them matched those stored in protein data bases. Two-dimensional (2D) gel electrophoresis is generally con- sidered the method with the highest resolution for protein separation at the microgram level (1-3). It is, therefore, regularly used to study phenotypically dependent alterations of protein expression in total cellular extracts or enriched cell fractions. The complex protein patterns that may often display up to 2000 spots can be analyzed by computer- imaging and the information stored in comprehensive data bases (4-9). This allows a further detailed quantitative com- parison of a large number of gels and a more thorough search for (a) protein(s) whose expression is typically associated with variations in the phenotype, with differentiation, cell cycle, cell lineage, neoplastic transformation, genetic dis- eases, etc. (see, for instance, refs. 4 and 9). Identified marker proteins can then be further characterized by comigration experiments with known proteins or mixtures of proteins derived from isolated cell organelles (nuclei, mitochondria, Golgi, vacuoles, membranes, extracellular spaces, etc.). Alternatively, immunological cross-reactivity with specific antibodies may serve for protein identification. Systematic 2D gel protein analysis has now gained another dimension with the possibility of sequencing (major) protein spots after elution (10, 11) or electroblotting onto inert membranes (12-18). The generated NH2-terminal sequences are generally of sufficient length for a search of protein identity or similarity or for the generation of specific DNA probes for cloning purposes. Proteins that are NH2- terminally blocked (either naturally or artifactually) are cleaved in situ and sequences are obtained from the gener- ated peptides (16, 19, 20). MATERIALS AND METHODS Cultured Cells and Tissues. Human MOLT-4 cells were grown in suspension in Dulbecco's modified Eagle's medium containing 10% (vol/vol) fetal calf serum and antibiotics (penicillin at 100 units/ml and streptomycin at 50 ,ug/ml). Fetal human tissues dissected from a 4-month normal human male fetus were used in some cases as source of proteins. These experiments have been approved by the Ethical Sci- entific Committee of the Aarhus Amtskommune. 2D Gel Electrophoresis. The procedures for computerized 2D gel electrophoresis have been described elsewhere (6, 21). Some of the protein preparations were partially enriched by ammonium sulfate fractionation and ion-exchange chroma- tography (22) or by cell organelle fractionation (B.G. and J.E.C., unpublished data), prior to 2D gel electrophoresis (23) (for details, see Table 1). Protein Recovery from Dried 2D Gels. Protein spots from Coomassie blue-stained and heat-dried gels were excised with a minimum of polyacrylamide and submerged in 50 mM boric acid (adjusted to pH 8.0 with NaOH) containing 0.1% SDS (buffer A). The buffer volume (1-2 ml) was not critical as protein losses due to elution were found to be minimal in this particular buffer system. After 2 hr of rehydration, the swollen gel pieces were taken up with tweezers and placed in a gel slot of a new slab gel. This gel was cast using spacers that were 0.5 mm thicker than those of the original 2D gel so as to facilitate loading of the pieces. Here, we used 1- mm-thick 2D gels and 1.5-mm-thick "concentrating" gels. The stacking gel (5% polyacrylamide) extends 2 cm beneath the bottom of the slot, which is 6 mm broad and between 1.5 Abbreviations: IEF, isoelectric focusing; PVDF; polyvinylidene difluoride; NEPHGE, nonequilibrium pH gel electrophoresis; 2D, two-dimensional. 7701 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Transcript
Proc. Nati. Acad. Sci. USAVol. 86, pp. 7701-7705, October 1989Biochemistry
Protein-electroblotting and -microsequencing strategies ingenerating 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, andcomputer-imaged two-dimensional gels used to develop com-prehensive human protein data bases served as the proteinsource to generate partial amino acid sequences. The proteinspots were collected from multiple gels, rehydrated, concen-trated by stacking into a new gel, electroblotted onto inertmembranes, and in situ-digested with trypsin. Peptides elutingfrom the membranes were separated by HPLC and sequenced.Using this procedure, it was possible to generate partialsequences from 13 human proteins recorded in the amnion cellprotein data base. Eight of these sequences matched those ofproteins stored in data bases, demonstrating that a systematicanalysis of proteins by computerized two-dimensional gel elec-trophoresis can be directly linked to protein microsequencingmethods. The latter technique offers a unique opportunity tolink information contained in protein data bases derived fromthe analysis of two-dimensional gels with forthcoming DNAsequence data on the human genome.
Here we describe in detail a modified version of theblotting/microsequencing procedure that allows sequenceanalysis of protein spots recovered from Coomassie blue-stained, heat-dried 2D gels. The devised protein recoveryprocedure can be used to concentrate minor protein spotscollected from several stained gels. In addition, by makinguse ofthe information stored in the comprehensive human 2Dgel protein data bases (5, 6), it was possible to analyzeproteins of interest by selecting tissues or cell types where aparticular protein was expressed in higher amounts. Themethod was used to generate partial amino acid sequences of13 human proteins: 6 cell growth/transformation-sensitivemarkers, 1 epithelial-specific protein, and 6 polypeptideswhose relative degree of expression is not affected signifi-cantly by the growth stage of the cell (ref. 6 and referencestherein). The identity of most of these proteins could bedetermined because sequences generated from them matchedthose stored in protein data bases.
Two-dimensional (2D) gel electrophoresis is generally con-sidered the method with the highest resolution for proteinseparation at the microgram level (1-3). It is, therefore,regularly used to study phenotypically dependent alterationsofprotein expression in total cellular extracts or enriched cellfractions. The complex protein patterns that may oftendisplay up to 2000 spots can be analyzed by computer-imaging and the information stored in comprehensive databases (4-9). This allows a further detailed quantitative com-parison of a large number of gels and a more thorough searchfor (a) protein(s) whose expression is typically associatedwith variations in the phenotype, with differentiation, cellcycle, cell lineage, neoplastic transformation, genetic dis-eases, etc. (see, for instance, refs. 4 and 9). Identified markerproteins can then be further characterized by comigrationexperiments with known proteins or mixtures of proteinsderived from isolated cell organelles (nuclei, mitochondria,Golgi, vacuoles, membranes, extracellular spaces, etc.).Alternatively, immunological cross-reactivity with specificantibodies may serve for protein identification.
Systematic 2D gel protein analysis has now gained anotherdimension with the possibility of sequencing (major) proteinspots after elution (10, 11) or electroblotting onto inertmembranes (12-18). The generated NH2-terminal sequencesare generally of sufficient length for a search of proteinidentity or similarity or for the generation of specific DNAprobes for cloning purposes. Proteins that are NH2-terminally blocked (either naturally or artifactually) arecleaved in situ and sequences are obtained from the gener-ated peptides (16, 19, 20).
MATERIALS AND METHODSCultured Cells and Tissues. Human MOLT-4 cells were
grown in suspension in Dulbecco's modified Eagle's mediumcontaining 10% (vol/vol) fetal calf serum and antibiotics(penicillin at 100 units/ml and streptomycin at 50 ,ug/ml).Fetal human tissues dissected from a 4-month normal humanmale fetus were used in some cases as source of proteins.These experiments have been approved by the Ethical Sci-entific Committee of the Aarhus Amtskommune.2D Gel Electrophoresis. The procedures for computerized
2D gel electrophoresis have been described elsewhere (6, 21).Some of the protein preparations were partially enriched byammonium sulfate fractionation and ion-exchange chroma-tography (22) or by cell organelle fractionation (B.G. andJ.E.C., unpublished data), prior to 2D gel electrophoresis (23)(for details, see Table 1).
Protein Recovery from Dried 2D Gels. Protein spots fromCoomassie blue-stained and heat-dried gels were excisedwith a minimum of polyacrylamide and submerged in 50 mMboric acid (adjusted to pH 8.0 with NaOH) containing 0.1%SDS (buffer A). The buffer volume (1-2 ml) was not criticalas protein losses due to elution were found to be minimal inthis particular buffer system. After 2 hr of rehydration, theswollen gel pieces were taken up with tweezers and placed ina gel slot of a new slab gel. This gel was cast using spacersthat were 0.5 mm thicker than those of the original 2D gel soas to facilitate loading of the pieces. Here, we used 1-mm-thick 2D gels and 1.5-mm-thick "concentrating" gels.The stacking gel (5% polyacrylamide) extends 2 cm beneaththe 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 chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Proc. Natl. Acad. Sci. USA 86 (1989)
cm and 3 cm deep (depending on the number of gel piecescollected in the slot). The separation gel was as described byLaemmli (24). The gel pieces (up to 15 pieces could becombined in a single slot) were then overlayed with gelsample buffer [1% SDS/10% (vol/vol) glycerol/0.1% bro-mophenol blue/50 mM dithiothreitol/13 mM Tris HCl, pH6.8) and occasionally trapped air bubbles were removed. Gelelectrophoresis was carried out until the dye reached thebottom of the separation gel.
Protein Electrotransfer. Proteins were electroblotted ontoglassfiber sheets coated with poly(4-vinyl-N-methylpyri-dinium iodide) or polyvinylidene difluoride (PVDF) mem-branes as described (15) using 50 mM Tris/50 mM boric acid(pH 8.3) as transfer buffer. The transfer was carried out forat least 8 hr at 35 V by using a Bio-Rad Transblot apparatus.Gels containing >12.5% polyacrylamide were equilibratedfor 2 hr in bufferA (see above) before transfer to minimize geldistortion during the blotting process.PVDF-membrane- and glassfiber-membrane-bound pro-
teins were visualized by staining with Amido black andfluorescamine, respectively (12, 15).Membrane in Situ Protease Cleavage. Proteolytic digestion
was carried out as described (19). Briefly, the membranepiece carrying the protein was excised, cut into pieces ofapproximately 3 by 3 mm, and collected in an Eppendorftube. They were then immersed in 200-500 Al of a 0.2%polyvinylpyrrolidone (30 kDa) solution in methanol. After 30min, the quenching mixture was diluted with an equal volumeof distilled water and further incubated for 5-10 min. Thesupernatant was then discarded and the membrane pieceswere washed four times with 200-500 ,ul water and once with500 ttl 0.1 M Tris HCl (pH 8.5, buffer B). The buffer wasremoved and replaced by a volume of the same buffer enough
IEF -a,¢|;;asS~~~~~~~~~~~~~~~~~~~o __.t ....
Cl) iF i5 c
to submerge the membrane pieces (between 100 and 150 ,ul).To this was added 1 ,ul of a freshly prepared solution oftrypsin at 1 mg/ml in buffer B. The digestion proceeded for4 hr at 37°C. The supernatant was then transferred into asecond Eppendorf tube and the membrane pieces werefurther washed once with 100,ul of80% (vol/vol) formic acidand four times with 100 ,ul of distilled water. All washingsolutions were added to the digestion mixture in the secondEppendorf tube. In some cases, the peptide solution wasstored at -20°C until HPLC analysis.In situ digestion on coated glassfiber sheets was carried out
as for PVDF membranes, except that quenching of remainingprotein-absorbing sites was done with 0.2% polyvinylpyrroli-done/50% (vol/vol) methanol/50% (vol/vol) H20.
Peptide Separation by Reversed-Phase HPLC. The com-bined washing solutions (±600 ,l) were loaded on a C4reversed-phase column (0.46 x 25 cm; Vydac SeparationsGroup) and the peptides were eluted with a linearly increas-ing gradient of acetonitrile in 0.1% trifluoroacetic acid. Thecolumn was equilibrated in 0.1% trifluoroacetic acid; thegradient was started 5 min after injection and reached 70%(vol/vol) acetonitrile after an additional 70 min. Elutingpeptides were detected by UV absorbance at 214 nm (absor-bance units full scale: 0.1 or 0.2) and collected by hand inEppendorf tubes. A Waters-Millipore HPLC apparatus con-sisting of two pumps (model 510), a gradient controller, anda model 481 variable wavelength detector was used for theseseparations. The major peptides were further dried in aSpeedVac (Savant) concentrator and stored at -20°C prior tosequence analysis.Amino Acid Sequencing. Peptides were selected for amino
acid sequence analysis on the basis of peak height and peakresolution and were redissolved in 30 ,l4 of 0.1% trifluoracetic
FIG. 1. Fraction of a synthetic image of an IEF fluorogram of [35S]methionine-labeled proteins from amnion cells (for the complete 2D gelpattern, see ref. 5). Proteins that have been microsequenced are indicated with their corresponding number in the comprehensive human amnionprotein data base (5). MW, Mr.
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Proc. Natl. Acad. Sci. USA 86 (1989) 7703
acid/30% acetonitrile before loading on a precycled Poly-brene-coated glass filter. The sequence analysis was carriedout with a gas-phase sequencer (model 470A, Applied Bio-systems) equipped with an on-line phenylthiohydantoinamino acid derivative analyzer (model 120A) and run with thesequencing program recommended by the manufacturer.Computer Search for Identity or Similarity. The amino acid
sequence comparisons were carried out using the FASTDBcomputer program of Intelligenetics. Two protein data baseswere screened, the Protein Identification Resources (release18) from the National Biochemical Research Foundation andthe Swiss-Prot data base (release 9) from EMBL.
RESULTS
Fig. 1 shows the imaged 2D gel isoelectric focusing (IEF)protein pattern of a [35S]methionine-labeled total extract oftransformed human amnion cells (AMA). Proteins selected
for microsequencing included six proliferation-sensitive and/or transformation-sensitive polypeptides (IEFs 6318, 7205,8214, 8505, and 9109 and NEPHGE 3004) (7), an epithelialmarker (IEF 9105), and six polypeptides (IEFs 8502, 8704,9105, 9205, 9209, and 9806) whose rate of synthesis is notaffected significantly by changes in growth rate and/or trans-formation (NEPHGE is nonequilibrium pH gel electropho-resis). To facilitate microsequencing, these proteins were cutout from Coomassie blue-stained gels of (i) partially purifiedprotein fractions of human MOLT-4 cells (IEFs 6318, 7205,8502, 8505, 8704, 9205, 9209, 9109, and 9806 and NEPHGE3004; Table 1) or (it) total protein extracts of fetal humantissues (IEFs 5206, 8214, and 9105; see Table 1). With theexception of NEPHGE 3004, all other microsequenced pro-teins are indicated with their corresponding number in themaster AMA protein data base (Fig. 1) (5).Gel pieces excised from 3 to 15 dry gels depending on their
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 MolecularAMA protein mass, Residuesdata base* kDa Source Sequence Protein sequenced Ref(s).
,B-PHase, f subunit of prolyl-4-hydroxylase; PDI, protein disuffide isomerase; Xl, Xenopus laevis; hnRNP, heterogeneous nuclearribonucleoprotein; 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 purificationinvolved 40-80% ammonium sulfate fractionation, DEAE-Sephacel chromatography (fraction eluted with 0.3 M KCI), and HPLC (TSKDEAE-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.
Biochemistry: Bauw et al.
Proc. Natl. Acad. Sci. USA 86 (1989)
dimensional gel using the protein-stacking properties of thediscontinuous Laemmli gel system (24). Proteins were thenelectroblotted on membranes and digested in situ with tryp-sin. The peptides released to the supernatant were furtherseparated by reversed-phase HPLC and sequenced.Compared to similar previously published procedures (15,
16, 20), this approach shows many improvements. (i) Gelscan be handled by conventional methods involvingCoomassie blue-staining (dried, stored, and/or scanned forsynthetic imaging) and no special precautions are necessaryto guarantee subsequent amino acid sequence determination.The standard use of Coomassie blue-stained, dried gels as a
protein source increase considerably the versatility of themethod. Indeed, it is now possible to use gels that areroutinely generated during the development of the compre-hensive human protein data bases (5, 6). Furthermore, theinformation from these data bases allowed us to select gelsfrom tissues or cells in which the protein of interest is mostabundantly present (here we used fetal human tissues as abetter source of some proteins; Table 1). In this study weoften used gels that have been stored in a dried state for >6months at room temperature. As will become evident fromthe sequence analyses (Table 1) and much to our surprise, wenever noticed any deamidation at amide residues or oxidationof methionine residues. (ii) This protein recovery techniqueis easily used to concentrate spots from multiple gels to studyless abundant polypeptides. (iii) The internal sequencingstrategy employed here also avoids situations where proteinsare NH2-terminally blocked either as a result of co-II orpost-translational modification or due to spurious artifactualreactions with components of the gel matrix and/or chemi-cals used in the procedure. (iv) The membrane in situdigestion procedure used in this study is similar to thatoriginally described by Aebersold et al. (16) but has beenadapted to fit with the PVDF- or polybase-coated glassfiberblotting procedure (19). Compared to other in situ cleavagemethods, this approach combines several advantages that areillustrated for proteins IEFs 9806, 8502, and 8214 in Fig. 2.First, the number of peptides released from the membraneinto the supernatant is generally much smaller than expectedfrom the number of potential cleavage sites. As a conse-quence, peptide HPLC chromatograms are extremely simpleand most peptides are obtained in pure form (compare thepeptide patterns of IEF 9806, a 110-kDa protein, and IEF8502, a 53-kDa protein). Such a situation is not encounteredwhen proteins are exhaustively digested in the gel matrix(36). In this case, the complexity of the peptide pattern isgenerally directly related to the size of the digested protein sothat large proteins are difficult to analyze. In addition,peptide chromatograms from such digests are seldom free ofartifactual peaks due to gel contaminants or protein dyes. Ourmodified in situ cleavage procedure was also found to beadvantageous compared to the gel in situ partial digestprotocol used to obtain internal protein sequences (20).Indeed, the latter is difficult to reproduce and the largefragments obtained are often derived from the same region inthe sequence. As a result, fragments are recovered in lowyield and often display the same NH2-terminal sequence.Another major advantage of this procedure resides in theunusually high sequencing efficiencies encountered with pep-tides released from membrane-bound proteins (initial se-quencing yields often exceed 80%). The latter point is illus-trated in Fig. 2D showing the traces of the phenylthiohydan-toin amino acid derivative chromatograms of cycles 1-12 ofa peptide from protein IEF 8214 (Fig. 2C). Clearly, <0.002adsorbance units (<10 pmol) can be reliably sequenced.
Eight out of 13 proteins could be recognized on the basisof identity or homology between generated peptide se-quences (Table 1) and protein sequences stored in data bases.This allowed a straight forward identification of the P subunit
A
B
Protein IEF 98 0 6 ( 1 0.9 kDa)
-020 30
-I- -*-1:40
Sequence analysis of
FIG. 2. HPLC traces of peptides released from some blottedproteins after in situ trypsin digestion (A-C). AUFS, absorbance unitfull scale. (D) HPLC traces of the phenylthiohydantoin amino acidresidues in cycles 1-13 of peptide 2 of protein IEF 8214. Identifiedamino acids are indicated by the one-letter amino acid code.
of prolyl-4-hydroxylase (IEF 8505), nuclear ribonucleopro-tein particle C protein (IEF 7205), lactate dehydrogenase Hchain (IEF 5206), lipocortin V (IEF 8214), and cyclophilin(NEPHGE 3004) (for references, see Table 1). The peptidesof protein IEF 8704 matched those found in the sequence ofthe 108-kDa chicken heat shock protein, a protein that is veryhomologous to murine endoplasmin. The two peptides ofprotein IEF 9109 are identical to sequences of horse platelettropomyosin. Our analyses of protein IEF 6318 cover twopeptides. One is identical to a sequence located in theCOOH-terminal end of a sequence encoded by a partialcDNA clone of human nuclear phosphoprotein B23 and isvery similar to the corresponding sequence derived from a
Protein IEF 8214 (33 kDa)C AUFS 2 1 4
_____ __-e | |I , 2,J-0 II
20 ~~30 40
II
7704 Biochemistry: Bauw et al.
Proc. Natl. Acad. Sci. USA 86 (1989) 7705
full-length cDNA sequence of the Xenopus laevis B23 pro-tein. The second peptide is located very near the NH2terminus of the protein sequence that is currently onlydocumented for the X. laevis cDNA sequence. Based on theobserved homologies, polypeptide IEF 6318 is identified asthe human B23 nuclear phosphoprotein (for references onprotein similarities, see Table 1). The identity of lipocortin V,protein B23, and endoplasmin has been verified by cross-reactivity with specific antibodies (33, 37, 38).
Peptides from five other proteins (IEF 8502, 9105, 9205,9209, and 9806) could not be correlated with known proteinsequences. The obtained partial sequence data, however, areof sufficient length for the synthesis of specific DNA probesfor isolating and sequencing cDNA clones.
DISCUSSIONThe systematic use of protein microsequencing methods inconjunction with the 2D gel protein separation and dataacquisition system extends considerably the possibilities ofthe latter. Indeed, proteins from 2D gels can now be identifiedby direct partial sequence comparison rather than by indirectmethods such as comigration with selected markers or im-munological cross-reactivity with specific antibodies. Thismay largely extend the identification possibilities for sets ofproteins whose post-translational modification or relativerate of synthesis is altered as a result of various cell stimuli.In addition, the obtained partial amino acid sequence infor-mation is in most cases sufficient to clone and sequencepreviously unidentified proteins.Even though this study describes a first attempt to combine
2D gel data bases with protein microsequencing, the resultsare encouraging. For example, the direct identification ofproliferation- and/or transformation-sensitive proteins B23,heterogeneous nuclear ribonucleoprotein, lipocortin V,tropomyosin, cyclophilin, and 3-prolyl-4-hydroxylase add tothe list of identified proteins that included four componentsof the 40S heterogeneous nuclear ribonucleoprotein particles(heterogeneous nuclear ribonucleoproteins Al, Bla, B2, andC4), three tropomyosins (IEFs 9213, 9215, and 9226), twoheat-shock proteins (hsx70 and hsp83), vimentin, and theDNA replication protein cyclin/proliferating-cell nuclear an-tigen (refs. 6, 39, and 40 and references therein).
Clearly, as more proliferation- and/or transformation-sensitive proteins are identified by microsequencing or otherindirect methods, it should be worthwhile to search for setsof coregulated proteins by using computerized 2D gel elec-trophoresis. Taking into account the rapid progress in genecloning and sequencing and the possibility of sequencing thehuman genome, the strategies described and applied in thispaper will extend our current understanding of the moleculardynamics of gene regulation and in particular of cell prolif-eration.
The authors acknowledge the skill of Dr. M. De Cock in preparingthe manuscript, the assistance of J. Coppieters during computersearching, and the support of Prof. M. Van Montagu. The work inGent was supported by grants from the National Fund for ScientificResearch (Belgium) and the Commission of the European Commu-nities, whereas the work in Aarhus was supported by grants from theDanish Biotechnology Programme, the Danish Cancer Society, theDanish Rheumatoid Society, NOVO Fund, and the Fund for Lae-gevidenskabens Fremme. G.B. was indebted to the Instituut totAanmoediging van het Wetenschappelijk Onderzoek in Nijverheiden Landbouw for a predoctoral fellowship; J.V. was a ResearchAssociate of the National Fund for Scientific Research (Belgium).
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