Electrophoresis 1990, I 1 , 5 1 1 - 5 2 1

Ruedi Aebersold’ John Leavitt2 IThe

University of British Columbia, Vancouver *Institutefor Medical Research, San Jose. CA

Towards an integrated protein database

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Sequence analysis of proteins separated by polyacrylamide gel electrophoresis: Towards an integrated protein database Improved technologies or the synergistic use of complementary methods enhance the efficiency of research and permit the exploration of new approaches for theinvestigation of complex problems: High sensitivity protein sequence analysis and polyacrylamide gel electrophoresis are such complementary methods. Here we summarize the current status of high sensitivity sequence analysis ofproteins separated in polyacrylamide gels and discuss strategies by which this technology can enhance biological research by generating new approaches for the solution of complex, multifacetted problems. Finally, we outline imminent technological advances in the area of high sensitivity protein sequence analysis and argue that further technological developments will ultimately lead to the generation of an integrated protein database (containing structural and functional as well as physiological information in an easily accessible form) of all the proteins separated by high resolution two-dimensional gel electrophoresis.

1 Introduction The develoument and the amlication of recombinant D N A methods have revolutionizedbiological and clinical research in the last fifteen years. Initially, molecular geneticists focused on the analysis of single, selected genes [ 11. Later, improved, subtractive strategies were developed which allowed the analysis of large numbers of cDNA’s identified by subtracting cDNA’s from one cell against an excess of mRNA from a second cell. This method allowed, in principle, to select, isolate and sequence cDNA’s unique to a particular cell type or to a particular physiological or pathologic state [21. In the near future the scope of molecular genetics will be further generalized, by the initiation of attempts to map and sequence entire genomes L3J. Compared to the analysis of DNA, capabilities for the analysis of proteins have lagged behind considerably, mainly because of difficulties in the identification of proteins relevant to a particular experimental or clinical situation, the purification of small amounts of proteins from complex mixtures and because oflimited sensitivity of protein sequence analysis. The combined use of polyacrylamide gel electrophoresis, in particular high resolution two-dimensional gel electrophoretic techniques, and protein sequence analysis has partially alleviated the limitations in protein analysis [4, 51. Protein sequence analysis and polyacrylamide gel electrophoresis are complementary technologies. Given efficient methods for the extraction of separated proteins from the polyacrylamide matrix, the protein chemist can take advantage of the high and tunable separating power of gel electrophoresis for the purification of a protein for partial sequence analysis. Partial sequence information confirms the molecular nature of the protein contained in a gel-band or spot and is the key to gain access to the complete primary structure and the genetic regulatory elements using a variety of well established molecular genetic methods. Recent technical adCorrespondence: Dr. Ruedi Aebersold, Biomedical Research Centre, 2222 Health Sciences Mall, University of British Columbia, Vancouver, B.C. V6T 1W5 Canada Abbreviations: PVDF, polyvinylidene difluoride; RP-HPLC, reverse phase-high performance liquid chromatography; RUBISCO, ribulose biphosphate carboxylase oxygenase; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis

0VCH Verlagsgesellschaft rnhH D-6940 Weinheirn. 1990

vances in both high resolution two-dimensional gel electrophoresis and protein sequence analysis allowed us to combine the two technologies and to explore new approaches for the analysis of proteins and genes which go beyond the reductionist approach of investigating one or a few isolated proteins at a time [ 5 , 411. In this manuscript we summarize thecurrent stateofsequence analysis of proteins isolated from polyacrylamide gels and discuss ways in which two-dimensional gel electrophoresis and protein sequence analysis can form the basis for new approaches to solve complex, multifacetted biological problems. Finally, we extrapolate on the development of going from reductionist to more global strategies and propose the establishment of an integrated protein database, integrating all the structural, functional and physiological information known about the proteins separated in two-dimensional gel patterns. We argue that the integrated protein database is essential and complementary to genome mapping and sequencing efforts and that the systematic protein and cDNA sequence analysis of all the separated species will provide essential information for a wide range ofbiological research projects.

2 The current status of sequence analysis of proteins separated by polyacrylamide gel electrophoresis 2.1 N-Terminal sequence analysis The resolving power of polyacrylamide gel electrophoresis for proteins is superior to other methodsfor protein separation. In addition, several modes of electrophoretic separation with orthogonal separating characteristics are well established and the range of maximal separation can be easily tuned; in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) by choosing an appropriate gel softness, or in carrier ampholyte or Immobiline-based isoelectric focusing gels by choosing an appropriate separating pH range 16-91. The development of the gas-liquid-phase protein sequenator allowed the partial N-terminal sequence analysis of low microgram amounts of protein [ 101. This quantity is well within the loading capacity of polyacrylamide gels. Initial attempts for sequencing proteins separated by polyacrylamide gels aimed 0173-0835/90/0707-0517 $3.50+.25/0

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Electrophoresis 1990,ll. 517-521

R. Aebersold and J. Leavitt

at extracting separated species from the gel matrix for sequence analysis by electrophoretic elution of the proteins into a chamber sealed with a dialysis membrane (electroelution) [ 11, 121. Although this method has been used successfully, it is cumbersome and suffers from a number of drawbacks, including the accumulation of large amounts of contaminants, the potential for N-terminal blocking and low recoveries.

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In 1984 we introduced a novel approach for the isolation of proteins from polyacrylamide gels in a sequenceable form [4, 131. The method consists of the simultaneous electrophoretic transfer “electroblotting” of all proteins in the gel matrix onto a solid support compatible with the harsh chemical conditions in the Edman sequencing chemistry (Fig. 1). We aimed at developing a simple and efficient method which made it possible to transfer the task of protein isolation for sequencing from the laboratory of the specialized protein chemist into the laboratory of the biochemist investigating a protein. With the establishment of protein sequencing core facilities in many major universities and industrial research institutions this concept of despecialization was of particular importance [ 141. The solid support used in the initial electroblotting method was a glass fiber filter covalently modified with quaternary ammonium groups. Subsequently introduced supports included glass fiber filters noncovalently modified with polybases, synthetic polymers based on polyvinylidene difluoride (PVDF) and siliconized, hydrophobic glass fiber filters [ 15- 181 (Table l}. All these supports are compatible with standard gas-liquidphase protein sequence analysis in a variety of commercial and prototype instruments. Two recent developments also made the electroblotting approach compatible with advanced solid-phase sequencing procedures. In the first, proteins are covalently attached during the electroblotting process to glass fiber filters modified withp-phenylene diisothiocyanate [ 191. In the second, proteins are covalently crosslinked onto a PVDF support by a simple chemical reaction after electroblotting and detection of the proteins by staining [201. The advantage ob both these methods lies in the possibility of using advanced solid-phase sequencing methods with the potential for enhanced speed and sensitivity 1211. The covalent attachment of polypeptides for sequence analysis therefore

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SUPPORT Figure 1. Electroblotting. Isolation of proteins from SDS-PAGE gels for N-terminal sequence analysis by electroblotting onto solid supports compatible with the Edman degradation. Proteins are separated by one- or twodimensional PAGE, then the whole gel is placed in contact with a sheet of a suitable support. The gel-filter paper sandwich is placed in a buffer solution (pH 8.2) and an electric field is applied perpendicular to the gel. The SDScoatedproteinsaredrivenoutofthegeltowardtheanodeandaretrappedby ionic and other mechanisms on the support. The blotted proteins are detected by soaking the membrane in a solution of a protein dye. Proteincontaining areas are cut out and inserted directly in the cartridge of a gasphase sequenator without further manipulation.

Name

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QA-GF/F

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Glas fiber filter covalently modified with quaternary ammonium grciupsal Glass fiber filter non covalently modified with polybases”) Polyvinylidene dilluorideal

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Immobilone P 1 I61

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Table 1. Electroblotting supports compatible with protein sequence analysis

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Jenssen (Belgium) Millipore (USA) Applied Biosystems (USA) Biometra (FRG)

a) Compatible with gas-liquid phase sequencing chemistry. b) Compatible with gas-liquidphase sequencing chemistry and advanced solid-phase degradation protocols

Towards an integrated protein database

€lec/rophorcsis 1990.11.5 11-521

provides the key for the development of sequencing chemistries with enhanced sensitivity. The types of blotting membranes described to data are summarized in Table 1. Several of these supports have been commercialized and different varieties of the basic method are widely used. In addition to SDS-PAGE, isoelectric focusing in polyacrylamide gels with immobilized pH gradients and two-dimensional polyacrylamide gel electrophoresis are directly compatible with this procedure [5,221. The relative merits and drawbacks ofeach support have been extensively compared before and will not be discussed further in this manuscript. In an optimized set-up, using quality-controlled chemicals, the electroblotting approach allows the determination of partial N-terminal sequences of protein amounts in the order of 0.5 to a few micrograms, depending on the size of the protein. This amount approximately corresponds to the protein content in a Coomassie Brilliant Blue-detectable band or spot. From that it follows as a rule of thumb that non N-terminally blocked proteins, detectable by Coomassie Blue staining in a polyacrylamide gel, are sequenceable after electroblotting. Limited sensitivity of detection of the phenylthiohydantoins, the final products of the Edman degradation, currently prevents the sequence analysis of even smaller amounts of proteins.

2.2 Internal sequence analysis of proteins separated by polyacrylamide gel electrophoresis Unfortunately, many proteins are not accessible to the Edman degradation because they lack a free amino terminus. Modifications of the a-amino group frequently occur post- or cotranslationally within the cell and, less frequently, during initial purification steps. While the artifactual N-terminal blocking can be efficiently controlled by the use of high purity, quality-controlled reagents and by avoiding conditions known to be amino reactive [231, enzymatically modified amino groups create a more serious problem. Generally, the type of co- or

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posttranslational N-terminal modification of a protein is not known and is difficult to determine. Even if the type of N-terminal modification has been chemically characterized, it is difficult to reverse because chemical or enzymatic deblocking methods compatible with microgram or submicrogram amounts of proteins have not yet been developed. Therefore, the most efficient way to generate sequence information ofNterminally blocked proteins is to chemically or proteolytically cleave the intact polypeptide chain and to isolate and sequence individual cleavage fragments. In addition to overcoming N-terminal blockage there are further reasons for the determination of internal stretches of sequence. Molecular cloning of genes through the use of synthetic oligodeoxynucleotides, reverse translated from the peptide sequences, can be made more efficient and accurate by using an appropriate set of multiple, independent probes. Alternatively, two independent oligonucleotide probes derived from peptide sequences can be used to amplify the intervening segment of D N A by the polymerase chain reaction [241. This enzymatically synthesized segment is a faithful, nondegenerate copy of the mRNA template and therefore provides an ideal probe for the efficient screening of cDNA or genomic libraries L2.51. The availability of multiple, independent stretches of peptide sequences increases the accuracy and significance of homology searches of sequence data bases for related proteins. Methods for the internal sequence analysis also allow the isolation and sequence analysis of specifically labeled proteolytic fragments such as peptides containing posttranslational modifications or peptides containing the active sites of enzymes. Three efficient methods for the determination of internal protein sequences at the low picomole level have been developed 126-281. All these procedures are based on the separation of the intact polypeptide chain by polyacrylamide gel electrophoresis. First, we developed a method which relies on reverse phase-high performance liquid chromatography (RP-HPLC)

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sequencing. Proteins are separated by oneor two-dimensional PAGE, then the whole gel is placed in contact with a sheet of nitrocellulose. The gel-nitrocellulose sandwich is placed in a slightly basic buffer solution and an electric field is applied perpendicular to the gel. The SDS-coated proteins are driven out of the gel and trapped on the nitrocellulose membrane (electroblotting). Electroblotted proteins are detected by protein staining. Protein-containing areas are cut out and substrates are enzymatically or chemically cleaved on the matrix. Resulting cleavage fragments are released into the supernatant, separated by RP-HPLC. Individual peptide fragments are collected for sequence analysis.

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Electrophoresis 1990,lI. 5 1 7 - 5 2 1

Figure 3. Isolation of peptides for internal sequence analysis. Proteins from a whole-cell lysate of the human lymphoblastoid cell line CCRF-CEM (ATCC No. C C L 1 19). (a)Two-dimensional electrophoresis(isoe1ectricfocusing followed by SDS-PAGE) ofproteins metabolically labeled with [35S]methionine. Proteins used as examples are circled. (b) HPLC map of peptides released after in situ tryptic digestion of the protein #7 (Fig. 6) after electroblotting onto nitrocellulose. (c) HPLC map of peptides released after in silu tryptic digestion of the P-subunit of mitochondria1 FZC-ATPase (protein #6) after electroblotting onto nitrocellulose. (d) Blank digest with trypsin. Asterisks (*) indicate peptides for which sequence analysis was carried out. AU, absorbance unit.

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Generated cleavage fragments are efficiently released into the supernatant, separated by narrow-bore RP-HPLC and collected for sequence analysis (Figs. 3, 4). In a modification Cycle 1 :Ala A of this method, proteins transferred to PVDF membranes were chemically fragmented by cyanogen bromide and released into the supernatant for further electrophoretic separation 4 :Ala 3 2 ;:Ile Glu of the cleavage fragments 1271. The third method was recently - * u developed by Kennedy and co-workers 1281.It is based on the A" A A 5 : Leu method of Cleveland and related methods for the peptide map6 :Gly ping by partial enzymatic or chemical cleavage 1291. Intact 7:Iie . n polypeptides are separated by one- or two-dimensional poly8:Tyr acrylamide gel electrophoresis, detected by protein staining, and slices containing individual proteins are cut out. Pro9:Pro A teins in the slice are subjected to partial chemical or enzymatic 10: Ala A n cleavage. The resulting cleavage fragments are separated in a Il:Val second polyacrylamide gel and isolated for sequence analysis A A. I2:Asp by electroblotting onto a suitable support. All three methods A fL. I3:Pro have been successfully usedin anumber ofprojects. In the first A, > . n,. 14: Leu and second method proteins are completely digested, in most cases leading to fragments of comparable size irrespective of I5:Asp Figure 4 . Internal sequence analysis. Sequence analysis of tryptic peptide the nature of the protein. Therefore, the same conditions can be used for most proteins and generally no optimization ofthe from a peptide from protein #6 (Fig. 3, Fig. 6) clf F,-ATPase P-subunit. method for a specific protein is required. This is of particular Peptide peak was collected from narrow-bore RP-IHPLC and applied to the importance for low abundance proteins. cartridge of a gas-phase protein sequenator. HLPLC analysis of PTH

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residues in cycles 1- 15 are shown. (Top lane), Mixture of PTH standards, 10 pmol each. PTH derivatives are designated by the single letter code of corresponding amino acids.

for the separation of proteolytic cleavage fragments for sequence analysis 1261 (Fig. 2). Proteins separated by one-or two-dimensional polyacrylamide gel electrophoresis are transferred to nitrocellulose by electroblotting and detected by protein staining. Individual protein bands are then cut out and the proteins are enzymatically cleaved on the membrane.

Fragments generated by complete enzymatic or chemical cleavage of a protein tend to be too small to be separable by SDS-PAGE. An ideal method to separate and recover small fragments for sequence analysis is RP-HPLC. If SDS-PAGE is used for the separation ofpeptides, cleavage fragments in the size range above 5000-8000 D a need to be generated. This is best achieved by partial cleavage. Methods based on partial cleavage therefore require the establishment of idiotypic cleavage conditions, strong enough to fragment most of the intact polypeptide, but weak enough to prevent complete break-

Electrophoresis 1990, 1 1 . 5 11-521

down into small fragments. This makes such procedures more difficult to use for a novel protein and may require a number of trials to optimize cleavage conditions. Compared to the isolation of proteins for N-terminal sequence analysis by electroblotting, methods for the determination of internal sequence require additional handling steps and are therefore less efficient. Typically, two- to threefold larger amounts of proteins are required to obtain internal sequence information than for N-terminal sequences.

3 Strategies for the sequence analysis of proteins isolated from polyacrylamide gels

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3 11. (ii) The lack of the possibility for cross-referencing: The same protein species is often characterized simultaneously from different angles. The reductionist approach does not allow the integration of seemingly unrelated efforts until the molecular identity of proteins is established. This is exemplified by our discovery (by protein sequence analysis) that the cholinergic neuronal differentiation factor is identical to the leukemia inhibitory factor [321. (iii) The reductionist approach makes the distribution of information difficult. Published information is typically targeted to a segment of the research community studying a problem from a similar point of view and reaches specialists in different areas of research only in special cases.

3.1 The reductionist approach

3.2 The subtractive approach

The reductionist approach is defined here as the analysis of one or several proteins at a time by a research team. Most biological research projects are carried out that way. Typically, a project will involve the establishment of an assay to test for the presence of a particular protein. This protein is then purified by classical biochemical methods for detailed structural and functional analysis. Partial protein sequence information is used to confirm the molecular nature of the protein or provides the key to the determination of the complete primary structure and genetic regulatory elements by recombinant D N A methods. In the reductionist approach, PAGE is used as just another, albeit powerful purification step. In many cases the use of PAGE as the final purification step can replace a whole series of conventional biochemical separation methods, thus making the isolation procedure much more efficient. In some special situations high resolution two-dimensional gel electrophoresis is sufficient to purify a protein from a total cell lysate for sequence analysis in a single operation and therefore completely eliminates low efficiency, classical purification steps [51. In the reductionist approach, the combinationofgel electrophoresis and protein sequence analysis does not offer principally new avenues, but it enhances the efficiency and speed of research. In fact, many low abundance proteins can only be isolated for sequence analysis by the use of polyacrylamide gel electrophoretic methods. Thanks to the despecialization of protein isolation for sequence analysis, this task can now be performed in any typical biochemistry laboratory. The actual sequence analysis is often performed on a cost recovery basis in microchemical core facilities or in the form of collaborative projects with groups specializing in sequence analysis. The reductionist approach is typical for small research groups. By focusing the effort on a restricted research area, progress is virtually assured and the competitiveness of the group is maintained.

In analogy to the situation in molecular genetics, the development of improved protein isolation/protein sequencing methods has opened up new approaches for the analysis of biological problems. Improved methods have made it possible to move from a reductionist type of research to more global strategies, thus making investigations on the protein level of more complex problems feasible. In particular, the possibility to determine partial sequences of proteins separated by twodimensional gel electrophoresis has enabled us to eliminate some of the shortcomings of the reductionist approach. High resolution two-dimensional polyacrylamide electrophoresis is the protein analytical method with the highest resolving power 191. Protein separation, essentially still performed according to O’Farrell, is now supported with digital pattern storage and analysis systems [33-401, so that significant differences in related two-dimensional patterns can be quantitatively and qualitatively determined by a subtractive analysis [4 1,421.

However, the reductionist approach has a number of drawbacks for solving complex biological problems, including. (i) The elimination of complex associations :By reducing acomplex problem to the analysis of one or a few proteins, related or associated phenomena easily elude perception. An example is the sequence analysis of a single polypeptide which is functionally associated with other proteins to form a multi protein cluster. In this situation, the separation and characterization of all components of the protein complex is of particular importance. A powerful way to analyze protein clusters in their full complexity is the immune precipitation of the intact complex with antisera specific for one component followed by the separation of individual polypeptide chains by PAGE [30,

Subtractive, quantitative two-dimensional PAGE, combined with protein sequence analysis, allows the simultaneous, quantitative analysis of the thousands of proteins resolved in a single operation, the determination of significant differences between the patterns and, in principle, the determination ofthe molecular identity of the resolved protein species by partial sequence anlysis. The power and feasibility of this technology is illustrated in the sequence analysis of plastins. Plastins are a family of differentially expressed cytoplasmic protein isoforms. L-plastin was originally identified as an abundant transformation-induced polypeptide of neoplastic human fibroblasts by subtractive analysis of the two-dimensional protein pattern from normal and transformed human fibroblasts [41,431 (Fig. 5 ) . Initial attempts to determine the N-terminal protein sequence of L-plastin failed, most likely due to N-terminal blocking. For that reason, multiple internal stretches of peptide sequence were determined using a method described above 151. Plastin was electroblotted from analytical two-dimensional gels onto nitrocellulose and cleaved on the matrix with trypsin. Resulting cleavage fragments were separated by narrow-bore RP-HPLC and sequenced. The resulting protein sequence information enabled us to isolate, clone and sequence the gene coding for plastin by the use of degenerate synthetic oligonucleotides. The elucidation of the complete L-plastin primary structure and the isolation and analysis of closely related genes led to significant insights into structure, function and regulation of plastin expression [44,451. The major findings included (i) The discovery of additional plastin isoforms:

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Elecrrophoresis 1990. 11, 5 1 7 - 5 2 1

Figure 5. Identification of L-plastin by subtractive two-dimensional pattern analysis. High resolution two-dimensional protein patterns from whole cell lysates from normal (A) and neoplastic, transformed human fibroblasts (B). tpl, T-plastin isoform; Ipl, L-plastin identified by subtractive analysis. Other labeled proteins include the marker proteins: T, tropomyosins; A, actin; V, vimentin.

Plastins are a group of protein isoforms differentially expressed in a variety of tissues [441. Furthermore we established the homology between plastin and other actin-binding proteins isolated from microvilli [45, 461. (ii) The determination of specific sequence motifs in the plastin primary structure and their functional consequences: We demonstrated that Lplastin contains a sequence motif typical for the binding site for C a + + as found in calmodulin or troponin C [47]. We furthermore determined that plastins contain an actin binding sequence related to the actin binding sequences in dystrophin and alpha actinin [48, 491. The presence of these specific sequence motifs in plastins, together with the correlation of the expression of the L-plastin isoform with a non-adherent phenotype suggests a functional involvement for plastins in the organization and regulation of cytosk,eletal microfilament bundling [501. (iii) The elucidation of the evolutionary pathway of plastins: A pattern of internal duplications in the plastin sequences suggested an evolutionary pathway for the formation of these proteins from a small precursor domain [45J.(iv)The determination ofthemolecular basis for differential expression of the plastin isoforms: A.nalysis of the 5’ untranslated regions of genomic DNA clones isolated from a variety of cell types will lead to the characterization of the tissue-specific, genetic regulatory elements. The described example illustrates several advantages of the subtractive approach. First, proteins iinvolved in complex biological processes such as transformation, differentiation or regulation of physiological activities can be identified and characterized without the availability of ,a specific assay for a

protein. Second, the subtractive approach allows the discrimination between primary and secondary events in the analysis of complex regulatory networks [421. Third, in many cases, the subtractive approach permits progressing from the primary structure of a protein to its function. The subtractive approach shows its biggest promise in the investigation of complex biological problems. It is most successful ifclose control patterns can be established which allow the filtering out of relevant changes by subtracting the experimental pattern from control pattern. The use of closely related experimental and control patterns and standardized sample preparation methods essentially eliminates artifacts such as polymorphisms, species differences and pattern differences due to artifactual covalent protein modifications. Limitations in protein sequencing sensitivity in many cases prevent the determination of the molecular nature of proteins identified by the subtractive approach. There are many more proteins resolved in a high-resolution two-dimensional gel than can be sequenced. Imminent significant improvements in the sensitivity of the protein sequencing process will, however, at least in part, alleviate these restrictions [211. The technical capability to perform subtractive analyses and protein sequence analysis of identified protein spots greatly enhances the scope and complexity of problems which can be successfully approached. But can these rather complex technologies be easily acquired and mastered by a typical research group? The availability of cheaper, reliable, user-friendly and standardized methods and equipment in the near future will certainly give an affirmative answer to this question.

Electrophoresis 1990,lI. 511-521

Although the subtractive approach is extremely powerful for the analysis of complex biological problems within one research group, it suffers from serious limitations with respect to the exchange of information. There is no vehicle for the communication of two-dimensional protein patterns. This is not primarily a logistic problem but rather a principal one, resulting from the lack of standardization of two-dimensional protein patterns established in different laboratories. This has the significant consequence that the exchange of data is reductionist. Because two-dimensional gel patterns cannot generally be compared between laboratories, results obtained by using the subtractive approach need to be stripped down for communication to a reductionist format, thus eliminating the possibility to relate the full complexity of the gained information. Provided the two-dimensional gel patterns are compatible, this problem can be overcome by electronically communicating the total qualitative and quantitative information gained by the subtractive approach to interested researchers. In addition, we envision the integration of the structural, functional and biological information generated from any protein in the two-dimensional gel pattern in a single, centralized data base. We therefore call this database the “integrated protein database”.

4 The integrated protein database The single most important prerequisite for the generation of a protein database based on two-dimensional gel electrophoretic patterns is the technology to reproducibly generate two-dimensional gel patterns in different laboratories over long periods of time and the ability to efficiently and unambiguously communicate this information. As early as the late 70’s highly reproducible two-dimensional gel electrophoretic patterns could be generated in specialized laboratories. Unfortunately, the patterns obtained in one laboratory were not directly comparable to the ones obtained in another laboratory. Nevertheless, Anderson and Anderson [ 5 11 proposed the concept of a “human protein index”, a database containing structural, functional and clinical information in the form of annotation files to the protein spots resolved in two-dimensional polyacrylamide gels. Several such catalogs were subsequently attempted. The most elaborate databases were prepared by Bravo and Celis from human HeLa cells (52, 531, by Neidhardt and Phillips for E . coli [54, 551, by McLaughlin [561 and Boucherie 1571 for yeast, by Garrels and Franza from normal and transformed rat fibroblasts [42,581 and by Celis and co-workers 1591. It rapidly became evident that it is essential to move beyond the purely descriptive dimension of two-dimensional protein catalogs. An integrated protein database requires the inclusion of additional aspects such as sequence information, subcellular localization, posttranslational modifications and more, of all the separated proteins. Ofparticular importance is the determination of the sequence of separated proteins. Knowledge of the primary structure not only unambiguously identifies a protein and allows its classification, but also provides the key for elaborate functional and physiological studies such as gene targeting by site-directed recombination in culturedorembryonicstemcells [601orbytheestablishment of transgenic animals [6 11. In cell types backed up by detailed genetic maps such as E. colior yeast, many proteins spots can be structurally identified by mutational genetic analysis. In cells from genetically more complex and less investigated

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organisms the most efficient way to the characterization of protein spots is direct protein sequence analysis. Once the sequence of a protein is established in one cell type, the use of different radiolabeled amino acids for the metabolic labeling of proteins can be used to cross-match patterns using microdensitometry [621. The establishment of an integrated protein database requires the fulfillment of a number of postulates. They fall into two categories, technical and structural.

4.1 Technical postulates for the establishment of an integrated protein database Technological postulates for the establishment of an integrated protein database concern the compatibility and performance of the individual methods, including high-resolution two-dimensional gel electrophoresis, quantitative pattern analysis, information exchange and protein sequence analysis. Current technology is not sufficient nor are individual methods necessarily compatible. Therefore improved methods and improved equipment need to be developed.

4.1.1 Two-dimensional polyacrylamide gel electrophoresis Specialized laboratories are capable of reproducibly performing high resolution two-dimensional gel electrophoresis, so that patterns over a long period of time can be quantitatively and qualitatively compared. The transfer of this ability to nonspecialized laboratories and the possibility to compare twodimensional gel patterns between laboratories require the availability of standardized, commercially available gel running hardware and reproducible electrophoresis conditions as well as quality-controlled gel chemicals and standardized sample preparation methods. Two electrophoresis systems, the Iso-Dalt [631 and a system released by the Millipore Corporation, essentially fulfill these requirements. High resolution two-dimensional gel electrophoresis is the method with the highest resolving power for proteins. The separation of up to 7000 proteins in a single operation have been reported [64, 651. However, even this number of detected spots only represents a fraction of the proteins present in a typical eukaryotic cell. Strategies for higher resolution and higher detection sensitivity are therefore essential. In addition, a centralized repository of antibodies for the characterization of protein spots by two-dimensional Western analysis would be a valuable asset. 4.1.2 Computerized pattern analysis and information

exchange Several increasingly powerful and elaborate software systems for the analysis of two-dimensional gel patterns have been described in recent years [33-401. In addition to fast, reliable and automated pattern acquisition and matching capabilities, the establishment of an integrated protein database requires provisions for an extensive database structure and the ability to easily access and transfer data. These aspects have been extensively discussed before [65, 661.

4.1.3 Protein sequence analysis The sensitivity of the sequencing process is not sufficient to make the sequence analysis of a protein separated by twodimensional gel electrophoresis a general approach. Current-

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ly, the most sensitive sequencing approach is gas-liquid phase Edman degradation [ 101 of polypeptides, non-covalently immobilized on glass fiber filters by a carrier such as polybrene [671. The major limitation of the sequence analysis of noncovalently immobilized substrates is the limited range of degradation chemistries which can be explored because of the possibility of washing out the polypeptide substrates. This substantially inhibits the further development of sequencing protocols. Improved methods are requiried for the optimization of degradation cycles for increased sensitivity, speed and yields, as well as for low backgrounds and extractability of modified compounds such as derivatives of phosphorylated amino acids. One approach which allows the use of a wide range of chemistries is solid-phase sequence analysis in which samples are covalently attached to solid supports (for a review see [687). The major advantages of the solid-phase concept have already been demonstrated in that longer sequencing runs at better repetitive stepwise yields could be obtained at the nanomole level (69,701. The potential of solid-phase sequence analysis for speed and sensitivity has not been fully exploited however, because the covalent immobilization of proteins and peptides tended to be tedious, inefficient, not generally applicable and incompatible with low picomole amounts of substrates. Simple, more generally applicable and lhighly efficient procedures for the covalent coupling of picornole or subpicomole amounts of proteins and peptides to modified glass fiber filter paper and synthetic polymers such as, P V D F have been developed by us and others [19-21, 711. These coupling methods are compatible with state-of-the-art protein and peptide isolation methods. Furthermore., a commercial protein sequenator designed for advanced sdid-phase sequence analysis has been introduced. Efficient methods for the covalent attachment and the availability of a solid-phase sequenator were essential prerequisites for the development of sequencing protocols with enhanced Sensitivity. The gain in sensitivity is realized by the use of modified, Edman-type reagents, yielding thiohydantoins detectable at femtomole or subfemtomole levels. Using chromophoric and fluorescent Edman-type reagents in an experimental solid-phase sequenator we have already achieved femto mole level sensitivity [21]. The addition of optimized detection systems such as photothermal refraction detectors for chromophoric compounds [ 721, laser-induced fluorescence detectors for fluorescent reagents [73] or mass spectrometer:; for the detection of efficiently ionizable derivatives will significantly add to the sensitivity of the sequencing process in the near future.

4.2 Structural postulates for the estabhhment of an integrated protein database Structural postulates concern aspects such as the structure, organization, management, distribution and accessibility of the database. These problems have been extensively discussed in the literature and at conferences andl concrete solutions have been proposed [65,661. However, themodeforthedetermination and integration of protein sequences into the database requires further discussion. One possibility is the sys tematic seq’ience determination of proteins separated by twodimensional gel electrophoresis. One or several laboratories could start to systematically sequence each protein isolated in two-dimensional patterns of a chosen cell or tissue type. This program can be initiated with current telchnology [741. Pro-

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teins of lesser abundance, currently not accessible for direct sequence analysis, would be investigated once sequencing methods with improved sensitivity become available. Alternatively, protein sequences published in the scientific literature could be correlated to corresponding spots in the two-dimensional gel pattern and integrated into the database. Most sequences are determined in reductionist research projects. Therefore, it will be difficult to establish this correlation. The systematic sequence analysis of separated proteins represents a more promising strategy. In particular, the systematic analysis of proteins identified by subtractive analysis of twodimensional gel patterns promises a wealth of biological information. By the subtractive analysis of the two-dimensional gel patterns of normal and transformed fibroblasts, Garrels and co-workers [42, 581 have identified a number of proteins whose expression levels are regulated during transformation in a complex manner. Knowledge of the primary structure of these proteins would be an immense asset for investigations into transformation and growth control at the molecular level. Abundant cellular proteins were often considered uninteresting and of lesser physiological importance. In a pilot study we have partially sequenced some of the more abundant proteins in human lymphocytes. We demonstrate that significant biological information can be gained by a systematic “top-to-bottom” sequencing strategy. The analyzed proteins are highlighted and numbered in Fig. 6. The results from this study are summarized below. Spot #l. Analysis of the N-terminal sequence of this protein in sequence databases identified this protein as RoISS-A antigen, an ubiquitous protein recognized by autoreactive antibodies 175 1 (Fig. 6). The immune response to the RO/SSA antigen has been associated with a number of autoimmune disorders such as systemic lupus erythematosus [761, Sjogren’s syndrome [771, subacute cutaneaous lupus erythematosus 1781. Spot #2. C yclin or P C N A (proliferating cell nuclear antigen): Garrels, Bravo and others determined by subtractive pattern analysis that the appearance of this protein is correlated to the proliferating state of cells 179, 80,8 1I. Molecular cloning and sequencing of the PCNA gene [791 identified the protein as an auxiliary protein for D N A polymerase delta [821. Spot #3. Internal sequence analysis of this protein showed characteristic clusters of acidic amino acid residues (Table 2). Such a motif is typical for a consenseus recognition sequence of casein kinase 11, a protein kinase with specificity for serine and threonine residues. Further analysis suggested that this protein may be phosphorylated in human fibroblasts, making it a possible substrate for casein kinase 11. Spot #4. Internal sequence analysis of this protein confirmed its nature as alpha-tubulin. Spot #5. Internal sequence analysis of this protein confirmed its nature as P-tubulin. Spot #6. Anderson and Anderson suggested, based on circumstantial evidence, that this protein was the P-subunit of mitochondria1 F, ATPase. Sequence analysis of more than 100 amino acid residues confirmed this suggestion.

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Figure 6. Identification of partially sequenced proteins. Proteins from whole cell lysate of the human lymphoblastoid cell line CCRF-CEM were separated by high resolution two-dimensional electrophoresis (isoelectric focusing/SDS-PAGE). Numbered spots were partially N-terminally and/or internally sequenced. Sequences and interpretations are given in the text. A, actin. Table 2. Partial amino acid sequences of protein #3a) Peptide

Sequence

NH,-S-L-I-I-V-X-K-K-L-X-G-D-COOH Vl NH,-T-L-L-G-D-G-P-V-COOH Tl NH,-M-V-G-S-Y GP-X-P-E-E-V-E-F-L-COOH T2 NH,-V-E-E-D-D-D-D-E-L-D-X-K-LN-Y-K-COOH T3 a) Internal stretches of protein sequence from the protein in spot # 3 (Fig. 6). V,, peptide derived by cleavage with staphylococcal V8 protease; T,, T,, T,, tryptic peptides; X, undetermined amino acid residue. Amino acids are denoted in the single-letter code.

Spot #7. This 60-66 kDa mitochondria1 protein was originally called actin-related protein, based on the cross reactivity of actin-specific antibody in Western blot analysis 1831. The same protein was named p24 by Bravo and co-workers 1531 and shown to be moderately responsive to heat shock. N-terminal and internal sequence information showed that the protein is strongly related to the gro EL gene product in E . coli and ribulose biphosphate carboxylase oxygenase (Rubisco) binding protein. This group of proteins is collectively termed chaperonins 1841. These proteins are thought to be involved in the assembly of multichain protein complexes such as phage heads or the enzyme Rubisco. Recently, the complete sequence of the mitochondria1 protein in Spot #7 has been determined by recombinant DNA methods [SSl.

Spot #8. Plastin. The biology of plastin has been summarized above. Plastin was detected by subtractive pattern analysis as a protein expressed in transformed, but not in normal human fibroblasts [4 1,431. Sequence analysis and subsequent studies demonstrated that plastins are afamily of Ca++andactin-binding phosphoproteins possibly involved in the bundling of cytoskeletal actin filaments [44,451. Spot #9.Thisproteinwastermedp13 by Bravoet al. [531.Internal sequence analysis identified this protein as a member of the heat stock protein family [861. Further analysis demonstrated that the expression of this protein is upregulated by heat stock, but not by the application ofchemical stress as imposed by acetoactidine treatment. The protein is difficult to extract with detergents and therefore is possibly associated with the cytoskeleton. Spot #lo. Only a short stretch of sequence containing the seven N-terminal amino acids were determined. The determined sequence stretch is identical with the N-terminal sequence of the 90 kDa protein of the heme regulated eIF-2 alpha kinase [87], an enzyme system responsible for the phosphorylation and inhibition of eIF-2 alpha, the smallest subunit of eukaryotic peptide initiation factor 2.

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5 Conclusions In molecular genetic research the last decade has witnessed a trend to move from the analysis of single genes or small gene families to programs to map and sequeince entire genomes. The feasibility of attempting research projects with broader scopes was preceded by the development of improved technologies. In recent years significant and complementary technological advances in the analysis of proteins have lbeen achieved. They include improved methods for the identification of proteins relevant to specific biological processes by subtractive twodimensional polyacrylamide gel electrophoresis, methods for the N-terminal and internal sequence analysis of small amounts of proteins and techniques to rapidly move from partial protein sequences to the isolation and complete sequence determination of the corresponding geine, using strategies based on the polymerase chain reaction [ 2 5 , 881 and rapid automatedDNA sequence analysis [89]. As aconsequenceof these developments, biological investigations of complex problems can now be attempted on the protein level. In analogy to the situation in molecular glenetics, this will ultimately lead to the determination of the complete primary structure of all the proteins expressed in a given cell type. A catalogue of the expressed gene sequences, annotated to high resolution two-dimensional protein patt erns will provide a powerful route for the targeted and efficient use of the wealth of information contained in genome sequence databases.

W e thank Dr. Rodrigo Bravo, Squibb Imtitute for Medical Research, Princeton, N.J., for providing proteins for crossreferencing spots in different gel systems and for helpful discussions. Received October 30, 1989

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