Localization, Annotation, and Comparison of the Escherichia coli K-12 Proteome under Two States of Growth

Here we describe a proteomic analysis of Escherichia coli in which 3,199 protein forms were detected, and of those 2,160 were annotated and assigned to the cytosol, periplasm, inner membrane, and outer membrane by biochemical fractionation followed by two-dimensional gel electrophoresis and tandem mass spectrometry. Represented within this inventory were unique and modified forms corresponding to 575 different ORFs that included 151 proteins whose existence had been predicted from hypothetical ORFs, 76 proteins of completely unknown function, and 222 proteins currently without location assignments in the Swiss-Prot Database. Of the 575 unique proteins identified, 42% were found to exist in multiple forms. Using DIGE, we also examined the relative changes in protein expression when cells were grown in the presence and absence of amino acids. A total of 23 different proteins were identified whose abundance changed significantly between the two conditions. Most of these changes were found to be associated with proteins involved in carbon and amino acid metabolism, transport, and chemotaxis. Detailed information related to all 2,160 protein forms (protein and gene names, accession numbers, subcellular locations, relative abundances, sequence coverage, molecular masses, and isoelectric points) can be obtained upon request in either tabular form or as interactive gel images. Here we describe a proteomic analysis of Escherichia coli in which 3,199 protein forms were detected, and of those 2,160 were annotated and assigned to the cytosol, periplasm, inner membrane, and outer membrane by biochemical fractionation followed by two-dimensional gel electrophoresis and tandem mass spectrometry. Represented within this inventory were unique and modified forms corresponding to 575 different ORFs that included 151 proteins whose existence had been predicted from hypothetical ORFs, 76 proteins of completely unknown function, and 222 proteins currently without location assignments in the Swiss-Prot Database. Of the 575 unique proteins identified, 42% were found to exist in multiple forms. Using DIGE, we also examined the relative changes in protein expression when cells were grown in the presence and absence of amino acids. A total of 23 different proteins were identified whose abundance changed significantly between the two conditions. Most of these changes were found to be associated with proteins involved in carbon and amino acid metabolism, transport, and chemotaxis. Detailed information related to all 2,160 protein forms (protein and gene names, accession numbers, subcellular locations, relative abundances, sequence coverage, molecular masses, and isoelectric points) can be obtained upon request in either tabular form or as interactive gel images. Large scale proteomic analyses of experimental model organisms provide valuable resources to a broad range of investigators working on both general and specific aspects of cell function. Recently there has been renewed interest in the bacterium Escherichia coli as a proof-of-concept model for systems-based approaches (1Holden C. Alliance launched to model E. coli.Science. 2002; 297: 1459-1460Google Scholar, 2Hoyle B. With virtual E. coli, researchers seek to replace the real thing.ASM News. 2002; 68: 481-482Google Scholar, 3Constans A. Pharma companies turn to computer simulations to complement experimentation and trial design.The Scientist. 2004; 18: 33-36Google Scholar). Here we describe a proteomic analysis of E. coli with an eye toward creating a resource of potential value to system approaches as well as problem-based approaches. E. coli is probably the best understood of the simple model organisms and the most amenable to experimental analysis. Its genome has been fully sequenced (4Blattner F.R. Plunkett III, G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. The complete genome sequence of Escherichia coli K-12.Science. 1997; 277: 1453-1462Google Scholar), and the availability of complete genome sequence data bases facilitates the proteomic analysis of E. coli using MS. The proteome of an E. coli cell is estimated to have 4,285 proteins (5Serres M.H. Gopal S. Nahum L.A. Liang P. Gaasterland T. Riley M. A functional update of the Escherichia coli K-12 genome.Genome Biol. 2001; 2 (research0035.1–research0035.7)Google Scholar) with pI values ranging from 3.38 to 13.0 and molecular masses between 1.59 to 248 kDa (6Bjellqvist B. Hughes G.J. Pasquali C. Paquet N. Ravier F. Sanchez J.-C. Frutiger S. Hochstrasser D.F. The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences.Electrophoresis. 1993; 14: 1023-1031Google Scholar, 7Bjellqvist B. Basse B. Olsen E. Celis J.E. Reference points for comparisons of two-dimensional maps of proteins from different human cell types defined in a pH scale where isoelectric points correlate with polypeptide compositions.Electrophoresis. 1994; 15: 529-539Google Scholar, 8Wilkins M.R. Gasteiger E. Bairoch A. Sanchez J.-C. Williams K.L. Appel R.D. Hochstrasser D.F. Link A.J. Protein Identification and Analysis Tools in the ExPASy Server. 2-D Proteome Analysis Protocols. Humana Press, Totowa, NJ1998: 531-552Google Scholar). These proteins are distributed among four well defined subcellular compartments: 1) the cytosol (2,885 known and predicted species), 2) the inner membrane (670 known and predicted species), 3) the outer membrane (87 known and predicted species), and 4) the periplasm, which separates the two membranes (138 known and predicted species). Recent “gel-free” proteomic approaches have coupled orthogonal chromatographic approaches with MS/MS (9Chen J. Lee C.S. Shen Y. Smith R.D. Baehrecke E.H. Integration of capillary isoelectric focusing with capillary reversed-phase liquid chromatography for two-dimensional proteomics separation.Electrophoresis. 2002; 23: 3143-3148Google Scholar) where one or more peptide tags serve as proxies for the identity, state, and abundance of a given protein. In the present work, we used the conventional and established method of 2D 1The abbreviation used is: 2D, two-dimensional. 1The abbreviation used is: 2D, two-dimensional.-PAGE (10O’Farrel P.H. High resolution two-dimensional electrophoresis of proteins.J. Biol. Chem. 1975; 250: 4007-4021Google Scholar) for protein separation and analysis based on several considerations. 1) The approach is both accepted and accessible. 2) With the ability to separate thousands of proteins on a single gel, the resolution of 2D-PAGE remains unchallenged. 3) With 2D-PAGE, protein mobility is highly sensitive to modification, making it possible to assess both the integrity and modification state of individual species. 4) The method lends itself readily to either absolute or relative quantification of intact protein species using several complementary approaches. 5) Gel visualization as the initial phase of analysis provides an immediate qualitative evaluation of the quality and global outcome of an experiment. In the present study we biochemically fractionated E. coli into its subcellular components and created high resolution annotated two-dimensional electrophoresis protein gels of the whole cell, inner membrane, outer membrane, and the intervening periplasmic space at proteomic scale. From these gels, we collated the identity, location, abundance, modification state, apparent isoelectric point, and molecular mass for 2,160 protein spots corresponding to 424 known and 151 putative genes. Building on these results, we used DIGE to determine the major differences in protein expression that occur when cells are grown in the presence or absence of amino acids. Shown in Fig. 1 and Supplemental Fig. 1 are two-dimensional electrophoresis gels of E. coli proteins derived from either whole cells or cells that had been biochemically fractionated into their periplasm, inner membrane, and outer membrane components. Circled spots correspond to protein species identified by tryptic digestion and LC/MS/MS analysis resulting in an average sequence coverage of ∼20% for each polypeptide. The results of this analysis have been statistically summarized in Supplemental Tables 1 and 2. Detailed information for each entry is listed in Supplemental Table 3 and includes: protein and gene name, National Center for Biotechnology Information (NCBI) accession number, subcellular location (Swiss-Prot), subcellular location, and abundance (this work). All the information provided herein for each protein entry and their isoforms has been made available at www.projectcybercell.ca, which also includes the sequence coverage for all identified spots and their predicted and measured molecular mass and pI. The data can be accessed interactively in either tabular form or by selecting a given protein spot from any of the six displayed gels. In all, 2,160 species were identified representing 575 different ORFs. Among these are 151 proteins (and their locations) whose existence had been predicted from hypothetical ORFs and 76 proteins (and their locations) of unknown function. In the case of the whole cell component, focusing the first dimension over the narrow pH ranges 4.5–5.5 and 5.5–6.7 significantly increased spot resolution, resulting in the identification of 113 protein entries not found at the broader pH range (Supplemental Table 1). Overall the efficiency of compartmental fractionation achieved here is evident from the dramatically different gel patterns observed for each compartment. An estimate of the purity of each compartment (inner membrane, 94%; periplasm, 87%; outer membrane, 87%) is derived from the sum of spot intensities corresponding to known or predicted members of a given compartment relative to the total spot intensity of the gel. These values are likely underestimates based on two considerations. 1) A small but significant fraction of inner and outer membrane proteins are known to co-localize at Bayer adhesion sites, points of contact between both membranes (11Danese P.N. Silhavy T.J. Targeting and assembly of periplasmic and outer membrane proteins in Escherichia coli.Annu. Rev. Genet. 1998; 32: 59-94Google Scholar). 2) The location of a significant number of proteins that contribute to the total gel intensities have not been reported or predicted previously (Supplemental Table 2). The assignment of proteins to compartments was determined by a conservative estimate of their relative enrichment from gel to gel. A total of 459 protein entries were found to be unique to one gel or another and were therefore immediately assigned to a corresponding compartment (Supplemental Table 1). Another 112 proteins partitioned sufficiently to a given compartment to warrant assignment (Supplemental Figs. 2–4). Of the protein entries in Swiss-Prot that presently have no designated location, 222 proteins have been assigned here. Other location assignments made here are in generally good agreement with those reported by Swiss-Prot (Supplemental Table 2) but not entirely free of discrepancy. The artifactual association of proteins and/or membrane during cell lysis and fractionation probably account in part for some of these discrepancies. For example, the ribosomal protein RplO and the recombination protein RecA are clearly cytosolic in light of their functions yet appear to co-localize with the outer membrane. Interestingly both proteins are known to form polymers (12Yu X. Egelman E.H. Image analysis reveals that Escherichia coli RecA protein consists of two domains.Biophys. J. 1990; 57: 555-566Google Scholar, 13Molloy M.P. Phadke N.D. Chen H. Tyldesley R. Garfin D.E. Maddock J.R. Andrews P.C. Profiling the alkaline membrane proteome of Caulobacter crescentus with two-dimensional electrophoresis and mass spectrometry.Proteomics. 2002; 2: 899-910Google Scholar), a reasonable explanation for their (and possibly other’s) co-elution with the denser outer membrane fraction. On the other hand, it is equally likely that some of the seemingly controversial assignments made here have biological meaning. There are for example 12 proteins that Swiss-Prot has classified as cytosolic that we assigned to the inner membrane (AceE, AceF, FabZ, FruB, GlpD, ManX, Rne, RpoE, SdaB, ThrA, TrpD, and UspA). These discrepancies can be reasonably reconciled if these proteins were peripherally bound to the inner membrane on the cytoplasmic side. Similarly there are four proteins that are classified as periplasmic by Swiss-Prot that we assigned to either the inner or outer membrane (DacB, DcrB, HybA, and YraP). These apparent differences could be reasonably accommodated if these proteins were peripherally associated with the side of either the inner or outer membrane that faces the periplasmic space. Our search of the available literature indicates that the assignments made here are likely correct for at least eight of these proteins (AceE and AceF (14Guest J.R. Angier S.J. Russell G.C. Structure, expression, and protein engineering of the pyruvate dehydrogenase complex of Escherichia coli..Ann. N. Y. Acad. Sci. 1989; 573: 76-99Google Scholar), FabZ (15Heath R.J. Rock C.O. Roles of the FabA and FabZ β-hydroxyacyl-acyl carrier protein dehydratases in Escherichia coli fatty acid biosynthesis.J. Biol. Chem. 1996; 271: 27795-27801Google Scholar), FruB (16Reizer J. Reizer A. Kornberg H.L. Saier Jr., M.H. Sequence of the fruB gene of Escherichia coli encoding the diphosphoryl transfer protein (DTP) of the phosphoenolpyruvate: sugar phosphotransferase system.FEMS Microbiol. Lett. 1994; 118: 159-162Google Scholar), GlpD (17Schryvers A. Lohmeier E. Weiner J.H. Chemical and functional properties of the native and reconstituted forms of the membrane-bound, aerobic glycerol-3-phosphate dehydrogenase of Escherichia coli..J. Biol. Chem. 1978; 253: 783-788Google Scholar), SdaB (18Shao Z. Newman E.B. Sequencing and characterization of the sdaB gene from Escherichia coli K-12.Eur. J. Biochem. 1993; 212: 777-784Google Scholar), DcrB (19Samsonov V.V. Samsonov V.V. Sineoky S.P. DcrA and dcrB Escherichia coli genes can control DNA injection by phages specific for BtuB and FhuA receptors.Res. Microbiol. 2002; 153: 639-646Google Scholar), and HybA (20Menon N.K. Chatelus C.Y. Dervartanian M. Wendt J.C. Shanmugam K.T. Peck Jr., H.D. Przybyla A.E. Cloning, sequencing, and mutational analysis of the hyb operon encoding Escherichia coli hydrogenase 2.J. Bacteriol. 1994; 176: 4416-4423Google Scholar)). Deviations between the observed and predicted Mr and/or pI of any given protein are often indicative of modifications such as chain cleavage or the covalent modification of amino acids. Supplemental Fig. 5 (A and B) illustrates the overall extent of protein modification as a correlation of the observed and expected Mr and pI. Of the 575 different protein entries compiled from the six reference gels (Fig. 1 and Supplemental Fig. 1), 241 (42%) were found to exist in more than one form at an average of 7.5 forms (spots) per entry (or 3.5 forms when all 575 entries are considered). For the 241 entries that were subject to modification, 70% of the modified forms varied only in their pI, whereas 22% varied only in their Mr. Only 8% of the variation could be attributed to changes in both pI and Mr. We conclude from these results that the majority of these forms arise from the modification of individual amino acids with particularly dramatic examples being OppA (distributed between 15 spots ranging over 7.4 pH units), AtpA (12 spots over 1.1 pH units), and GapA (14 spots over 2.8 pH units). A more comprehensive analysis of this type of modification reveals that they result from the deamidation of asparagine and glutamine to their acidic counterparts, aspartate and glutamate. 2A. Lopez-Campistrous, P. Semchuk, L. Burke, T. Palmer-Stone, S. J. Brokx, G. Broderick, D. Bottorff, S. Bolch, J. H. Weiner, and M. J. Ellison, manuscript in preparation. Eighteen forms were identified that exhibited significant differences from the expected mass (Supplemental Fig. 2A, not labeled). Of these, four showed a higher than expected mass, whereas 14 exhibited a lower than expected mass. The nature of the higher than expected masses remains unresolved. Of the smaller mass variants, six were identified as truncated versions of full-length entries based on the extent of protein sequence covered by their tryptic fragments. Forms of GyrB, PrsA, and YacE carried C-terminal deletions, whereas Lon and AlaS carried N-terminal deletions, and Ptr appeared to be deleted from both ends of the predicted protein sequence. Although it is reasonable to conclude from these findings that these fragments arise from proteolytic cleavage, we note that in no instance has the sequence of the predicted protein been confirmed experimentally. 3Swiss-Model Repository, a database for theoretical protein models (swissmodel.expasy.org/repository/). When taken globally, the results of the present study reveal that the extent of E. coli protein modification is considerably greater than reported previously. Using a similar approach to that described here, Link et al. (22Link A.J. Robison K. Church G.M. Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12.Electrophoresis. 1997; 18: 1259-1313Google Scholar) reported that 18% of 223 unique proteins identified existed as modified forms compared with the 42% of 575 proteins identified in the present a of of the in differences in and can also account for this discrepancy. We examined the of amino acid on protein expression by Protein derived from whole cells grown in the presence and absence of amino acids were first modified with that exhibited different properties with amino without amino and on the gel (Supplemental Fig. The relative of protein to each spot was determined by of the of each A total of spots were identified that exhibited significant differences in abundance between the two corresponding to 23 different proteins (Supplemental Table most of these proteins (18Shao Z. Newman E.B. Sequencing and characterization of the sdaB gene from Escherichia coli K-12.Eur. J. Biochem. 1993; 212: 777-784Google Scholar) showed of expression in the absence of amino with the greater of that be for amino acid and Fig. 2 is a resolution that positions the major changes in protein to Most of the proteins in the absence of amino acids are involved in acid and included in this are proteins involved in and and proteins of the system and has that these proteins are during of Escherichia coli..J. Bacteriol. 1975; Scholar, M. T. and changes in proteome and properties in a of Escherichia coli during from to in and Microbiol. 2001; therefore their in the absence of amino acids a similar for On the other hand, of the system is for the of amino acids separation of and for amino acids in Escherichia coli Bacteriol. 1978; Scholar), clearly an to where amino acids are with of expression in the absence of amino acids and are and the of global and expected from cells that and more In to the present there have been to the E. coli proteome that account for unique protein of the known E. coli ORFs (4Blattner F.R. Plunkett III, G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. The complete genome sequence of Escherichia coli K-12.Science. 1997; 277: 1453-1462Google Scholar, M.H. Gopal S. Nahum L.A. Liang P. Gaasterland T. Riley M. A functional update of the Escherichia coli K-12 genome.Genome Biol. 2001; 2 (research0035.1–research0035.7)Google Scholar). The results of all four have been statistically summarized in Table In to this two of these used 2D-PAGE and are therefore more Link et al. (22Link A.J. Robison K. Church G.M. Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12.Electrophoresis. 1997; 18: 1259-1313Google Scholar) identified and assigned different proteins to the four compartments examined here, whereas the 2D-PAGE data L. Sanchez J.C. K. M.R. M. Frutiger S. Appel R.D. Bairoch A. C. Hughes G.J. Williams K.L. Hochstrasser D.F. Escherichia coli database 1998; Scholar, L. C. Appel R.D. Hochstrasser D.F. Sanchez J.C. in the Escherichia coli proteome 2001; Scholar, R. T. S. S. two-dimensional gel electrophoresis and mass based proteomic analysis of Escherichia 2002; 2: Scholar) identified different proteins from whole both of these data with by The most study a high and a lower of unique proteins by followed by tandem of tryptic derived from either or total membrane of E. coli F. J. M. Jr., K. J. S. E. D.F. a protein of Escherichia to its Acad. Sci. S. A. Scholar). These data with study than the 2D-PAGE approaches for the high for the lower of four proteomic et et and by only one amino acid and be by they are as a single et et al. et al. and by only one amino acid and be by they are as a single in a is apparent from these comparisons that 2D-PAGE both in of and of protein 2D-PAGE is for other of information such as protein modification state, absolute and relative abundances, pI, and Mr. The that these approaches complementary the two by all of proteomic the for significant in and with

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.