Proteomic Analysis of an Extreme Halophilic Archaeon, Halobacterium sp. NRC-1
2003; Elsevier BV; Volume: 2; Issue: 8 Linguagem: Inglês
10.1074/mcp.m300044-mcp200
ISSN1535-9484
AutoresYoung Ah Goo, Eugene C. Yi, Nitin S. Baliga, W. Andy Tao, Min Pan, Ruedi Aebersold, David R. Goodlett, Leroy Hood, Wailap Victor Ng,
Tópico(s)Mass Spectrometry Techniques and Applications
ResumoHalobacterium sp. NRC-1 insoluble membrane and soluble cytoplasmic proteins were isolated by ultracentrifugation of whole cell lysate. Using an ion trap mass spectrometer equipped with a C18 trap electrospray ionization emitter/micro-liquid chromatography column, a number of trypsin-generated peptide tags from 426 unique proteins were identified. This represents approximately one-fifth of the theoretical proteome of Halobacterium. Of these, 232 proteins were found only in the soluble fraction, 165 were only in the insoluble membrane fraction, and 29 were in both fractions. There were 72 and 61% previously annotated proteins identified in the soluble and membrane protein fractions, respectively. Interestingly, 57 of previously unannotated proteins found only in Halobacterium NRC-1 were identified. Such proteins could be interesting targets for understanding unique physiology of Halobacterium NRC-1. A group of proteins involved in various metabolic pathways were identified among the expressed proteins, suggesting these pathways were active at the time the cells were collected. This data containing a list of expressed proteins, their cellular locations, and biological functions could be used in future studies to investigate the interaction of the genes and proteins in relation to genetic or environmental perturbations. Halobacterium sp. NRC-1 insoluble membrane and soluble cytoplasmic proteins were isolated by ultracentrifugation of whole cell lysate. Using an ion trap mass spectrometer equipped with a C18 trap electrospray ionization emitter/micro-liquid chromatography column, a number of trypsin-generated peptide tags from 426 unique proteins were identified. This represents approximately one-fifth of the theoretical proteome of Halobacterium. Of these, 232 proteins were found only in the soluble fraction, 165 were only in the insoluble membrane fraction, and 29 were in both fractions. There were 72 and 61% previously annotated proteins identified in the soluble and membrane protein fractions, respectively. Interestingly, 57 of previously unannotated proteins found only in Halobacterium NRC-1 were identified. Such proteins could be interesting targets for understanding unique physiology of Halobacterium NRC-1. A group of proteins involved in various metabolic pathways were identified among the expressed proteins, suggesting these pathways were active at the time the cells were collected. This data containing a list of expressed proteins, their cellular locations, and biological functions could be used in future studies to investigate the interaction of the genes and proteins in relation to genetic or environmental perturbations. Since the completion of the first bacterial genome, Haemophilus influenzae (1Fleischmann R.D. Adams M.D. White O. Clayton R.A. Kirkness E.F. Kerlavage A.R. Bult C.J. Tomb J.F. Dougherty B.A. Merrick J.M. McKenney K. Sutton G. Shirley R. Liu L. Glodek A. Kelley J.M. Weidman J.F. Phillips C.A. Spriggs T. Hedblom E. Cotton M.D. Utterback T.R. Hanna M.C. Nguyen D.T. Saudek D.M. Brandon R.C. Fine L.D. Fritchman J.L. Fuhrmann J.L. Geoghagen N.S.M. Gnehm C.L. McDonald L.A. Small K.V. Fraser C.M. Smith H.O. Venter J.C. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd.Science. 1995; 269: 496-512Google Scholar), more than 100 microbial genome sequences including Halobacterium sp. NRC-1 (2Ng W.V. Ciufo S.A. Smith T.M. Bumgarner R.E. Baskin D. Faust J. Hall B. Loretz C. Seto J. Slagel J. Hood L. DasSarma S. Snapshot of a large dynamic replicon in a halophilic archaeon: Megaplasmid or minichromosome? Genome Res. 1998; 8: 1131-1141Google Scholar, 3Ng W.V. Kennedy S.P. Mahairas G.G. Berquist B. Pan M. Shukla H.D. Lasky S.R. Baliga N.S. Thorsson V. Sbrogna J. Swartzell S. Weir D. Hall J. Dahl T.A. Welti R. Goo Y.A. Leithauser B. Keller K. Cruz R. Danson M.J. Hough D.W. Maddocks D.G. Jablonski P.E. Krebs M.P. Angevine C.M. Dale H. Isenbarger T.A. Peck R.F. Pohlschroder M. Spudich J.L. Jung K.W. Alam M. Freitas T. Hou S. Daniels C.J. Dennis P.P. Omer A.D. Ebhardt H. Lowe T.M. Liang P. Riley M. Hood L. DasSarma S. Genome sequence of Halobacterium species NRC-1. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12176-12181Google Scholar) have been determined (www.ncbi.nlm.nih.gov/PMGifs/Genomes/micr.html). These sequences constitute the primary digital information for global understanding of physiology, pathogenicity, and molecular machineries essential for the survival or adaptation of the organisms in different environmental conditions. The Halobacterium genome consists of a 2,014-kb large chromosome and two smaller chromosomes (191 kb and 365 kb) encoding ∼2,630 putative protein genes (2Ng W.V. Ciufo S.A. Smith T.M. Bumgarner R.E. Baskin D. Faust J. Hall B. Loretz C. Seto J. Slagel J. Hood L. DasSarma S. Snapshot of a large dynamic replicon in a halophilic archaeon: Megaplasmid or minichromosome? Genome Res. 1998; 8: 1131-1141Google Scholar, 3Ng W.V. Kennedy S.P. Mahairas G.G. Berquist B. Pan M. Shukla H.D. Lasky S.R. Baliga N.S. Thorsson V. Sbrogna J. Swartzell S. Weir D. Hall J. Dahl T.A. Welti R. Goo Y.A. Leithauser B. Keller K. Cruz R. Danson M.J. Hough D.W. Maddocks D.G. Jablonski P.E. Krebs M.P. Angevine C.M. Dale H. Isenbarger T.A. Peck R.F. Pohlschroder M. Spudich J.L. Jung K.W. Alam M. Freitas T. Hou S. Daniels C.J. Dennis P.P. Omer A.D. Ebhardt H. Lowe T.M. Liang P. Riley M. Hood L. DasSarma S. Genome sequence of Halobacterium species NRC-1. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12176-12181Google Scholar). Among these, 41% matched to genes of known function in public databases. The predicted proteome is highly acidic with a median isoelectric point of 4.9 (4Kennedy S.P. Ng W.V. Salzberg S.L. Hood L. DasSarma S. Understanding the adaptation of Halobacterium species NRC-1 to its extreme environment through computational analysis of its genome sequence. Genome Res. 2001; 11: 1641-1650Google Scholar). The high negative surface charge of predicted proteins provides stability to the proteins in nearly saturated intracellular salinity where other conventional proteins would become denatured (4Kennedy S.P. Ng W.V. Salzberg S.L. Hood L. DasSarma S. Understanding the adaptation of Halobacterium species NRC-1 to its extreme environment through computational analysis of its genome sequence. Genome Res. 2001; 11: 1641-1650Google Scholar, 5Lanyi J.K. Salt-dependent properties of proteins from extremely halophilic bacteria. Bacteriol. Rev. 1974; 38: 272-274Google Scholar). The archaeon Halobacterium sp. NRC-1 provides a relatively simple model for understanding a complex system of how cells adjust to various environmental stimuli. Halobacterium flourishes in extremely saline environments (>4 m salts), and its metabolism is subject to fluctuations in sunlight, oxygen, temperature, nutrients, and salinity. Halobacterium thrives in this harsh environment by appropriately tuning its extraordinary physiology in response to different environmental stimuli. For example, it can relocate, in search of favorable environments, using sensors that can discriminate beneficial and detrimental spectra of light (6Bogomolni R.A. Spudich J.L. Identification of a third rhodopsin-like pigment in phototactic Halobacterium halobium.Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6250-6254Google Scholar, 7Spudich J.L. Bogomolni R.A. Mechanism of colour discrimination by a bacterial sensory rhodopsin. Nature. 1984; 312: 509-513Google Scholar, 8Spudich J.L. Color sensing in the Archaea: A eukaryotic-like receptor coupled to a prokaryotic transducer. J. Bacteriol. 1993; 175: 7755-7761Google Scholar), aerotaxis transducer (HtrVIII) (9Brooun A. Bell J. Freitas T. Larsen R.W. Alam M. An archaeal aerotaxis transducer combines subunit I core structures of eukaryotic cytochrome c oxidase and eubacterial methyl-accepting chemotaxis proteins. J. Bacteriol. 1998; 180: 1642-1646Google Scholar), and buoyant gas-filled vesicles (10DasSarma S. Damerval T. Jones J.G. Tandeau de Marsac N. A plasmid-encoded gas vesicle protein gene in a halophilic archaebacterium. Mol. Microbiol. 1987; 1: 365-370Google Scholar, 11DasSarma S. Arora P. Lin F. Molinari E. Yin L.R. Wild-type gas vesicle formation requires at least ten genes in the gvp gene cluster of Halobacterium halobium plasmid pNRC100. J. Bacteriol. 1994; 176: 7646-7652Google Scholar). The Halobacterium transducer, HtrVIII, combines subunit I core structures of eukaryotic cytochrome c oxidase and eubacterial methyl-accepting chemotaxis proteins to mediate aerotaxis (9Brooun A. Bell J. Freitas T. Larsen R.W. Alam M. An archaeal aerotaxis transducer combines subunit I core structures of eukaryotic cytochrome c oxidase and eubacterial methyl-accepting chemotaxis proteins. J. Bacteriol. 1998; 180: 1642-1646Google Scholar). One of interesting features of Halobacterium is its ability to survive aerobically as a chemoheterotroph and anaerobically using light and/or arginine as energy sources. Halobacterium sp. derives energy from light by its retinal-containing light-driven ion transporters, bacteriorhodopsin and halorhodopsin (12Oesterhelt D. Meentzen M. Schuhmann L. Reversible dissociation of the purple complex in bacteriorhodopsin and identification of 13-cis and all-trans-retinal as its chromophores. Eur. J. Biochem. 1973; 40: 453-463Google Scholar, 13Dunn R. McCoy J. Simsek M. Majumdar A. Chang S.H. Rajbhandary U.L. Khorana H.G. The bacteriorhodopsin gene. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6744-6748Google Scholar, 14Schobert B. Lanyi J.K. Halorhodopsin is a light-driven chloride pump. J. Biol. Chem. 1982; 257: 10306-10313Google Scholar, 15Krebs M.P. Khorana H.G. Mechanism of light-dependent proton translocation by bacteriorhodopsin. J. Bacteriol. 1993; 175: 1555-1560Google Scholar, 16Kolbe M. Besir H. Essen L.O. Oesterhelt D. Structure of the light-driven chloride pump halorhodopsin at 1.8 A resolution. Science. 2000; 288: 1390-1396Google Scholar). Halobacterium can also ferment arginine via the arginine deiminase pathway to yield 1 mol of ATP for each mole of fermented arginine (17Hartmann R. Sickinger H.D. Oesterhelt D. Anaerobic growth of halobacteria. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3821-3825Google Scholar, 18Ruepp A. Soppa J. Fermentative arginine degradation in Halobacterium salinarium (formerly Halobacterium halobium): Genes, gene products, and transcripts of the arcRACB gene cluster. J. Bacteriol. 1996; 178: 4942-4947Google Scholar). Its intriguing physiology together with the availability of a complete genome sequence led us to catalogue via a simplified shotgun proteomic methodology (19Figeys D. Ducret A. Yates 3rd, J.R. Aebersold R. Protein identification by solid phase microextraction-capillary zone electrophoresis-microelectrospray-tandem mass spectrometry. Nat. Biotechnol. 1996; 14: 1579-1583Google Scholar, 20Link A.J. Eng J. Schieltz D.M. Carmack E. Mize G.J. Morris D.R. Garvik B.M. Yates 3rd, J.R. Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 1999; 17: 676-682Google Scholar) the proteins expressed by Halobacterium sp. NRC-1 in membrane and cytoplasmic compartments. In addition, the proteins involved in metabolic pathways in Halobacterium sp. NRC-1 under standard culture conditions were investigated. Herein we present the results of our initial investigation of the Halobacterium proteome using a simple shotgun proteomic approach that involves bulk digestion of copurified proteins with trypsin followed by a single stage of microcapillary high-pressure liquid chromatography (μLC) 1The abbreviations used are: μLC, microcapillary liquid chromatography; ESI, electrospray ionization; MS/MS, tandem mass spectrometry; MSR, membrane-spanning region; CHP, conserved hypothetical protein; HP, hypothetical protein; CID, collision-induced dissociation. electrospray ionization (ESI) tandem mass spectrometry (MS/MS) analysis of peptides using an ion trap mass spectrometer. The data contains a list of expressed proteins derived by searching peptide tandem mass spectra against the theoretical protein database of Halobacterium sp. NRC-1 (2Ng W.V. Ciufo S.A. Smith T.M. Bumgarner R.E. Baskin D. Faust J. Hall B. Loretz C. Seto J. Slagel J. Hood L. DasSarma S. Snapshot of a large dynamic replicon in a halophilic archaeon: Megaplasmid or minichromosome? Genome Res. 1998; 8: 1131-1141Google Scholar, 3Ng W.V. Kennedy S.P. Mahairas G.G. Berquist B. Pan M. Shukla H.D. Lasky S.R. Baliga N.S. Thorsson V. Sbrogna J. Swartzell S. Weir D. Hall J. Dahl T.A. Welti R. Goo Y.A. Leithauser B. Keller K. Cruz R. Danson M.J. Hough D.W. Maddocks D.G. Jablonski P.E. Krebs M.P. Angevine C.M. Dale H. Isenbarger T.A. Peck R.F. Pohlschroder M. Spudich J.L. Jung K.W. Alam M. Freitas T. Hou S. Daniels C.J. Dennis P.P. Omer A.D. Ebhardt H. Lowe T.M. Liang P. Riley M. Hood L. DasSarma S. Genome sequence of Halobacterium species NRC-1. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12176-12181Google Scholar), their cellular locations (i.e. membrane, cytoplasm, or both) deduced from subcellular fractionation prior to proteome analysis, and the putative biological functions of proteins. Halobacterium sp. NRC-1 (ATCC700922) was cultured at 37 °C in basal salt medium containing 1% peptone (Oxoid, Hampshire, U.K.) and trace metals as previously described (21Oesterhelt D. Stoeckenius W. Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane. Methods Enzymol. 1974; 31: 667-678Google Scholar). Membrane and soluble-cytoplasmic proteins were isolated using a protocol modified from a halophiles laboratory manual and Oesterhelt (21Oesterhelt D. Stoeckenius W. Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane. Methods Enzymol. 1974; 31: 667-678Google Scholar, 22DasSarma S. Robb F.T. Place A.R. Sowers K.R. Schreier H.J. Fleischmann E.M. Archaea: A Laboratory Manual: Halophiles, Cold Spring Harbor Laboratory Press, Plainview, NY. 1995; Google Scholar). One liter of Halobacterium sp. NRC-1 culture was grown to OD600 = ∼2.0 and pelleted by centrifugation at 7,500 rpm at 4 °C for 10 min. Pellets were resuspended in 20 ml basal salt solution containing 0.5 mg each of DNaseI and RNaseA and 1 mm of proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by osmotic shock against a 40× excess of deionized water within a dialysis tubing bag (Spectra/Por® membrane MWCO: 3,500; Spectrum, Rancho Dominguez, CA). Cell debris was removed by centrifugation at 10,000 × g for 30 min. The remaining cell lysates were then separated into the soluble and membrane fractions by ultracentrifugation at 53,000 × g for 2 h. The membrane fraction, a pellet at the bottom of the tube, and the soluble fraction, the aqueous supernatant portion, were then collected. The membrane was loaded on top of 30% sucrose cushion and ultracentrifuged at 53,000 × g at 10 °C overnight. The membrane fraction was collected and washed three times in 10 ml basal salt solution using a hand-held electrical homogenizer (Tissue-Tearor; Fisher, Pittsburgh, PA). Membrane proteins were then collected by centrifugation at 53,000 × g for 2 h at 10 °C. The pellet was resuspended in residual basal salt solution and then transferred to a microcentrifuge tube. The residual aqueous basal salt solution was removed by a brief spin at 14,000 rpm. The soluble protein fraction was dialyzed against five changes of 100× volume of deionized water at 4 °C to reduce the salt concentration that in excess might inhibit the protease reaction and mass spectrometry analysis. One hundred micrograms of proteins were digested with 2 μg of trypsin (Promega, Madison, WI) in 50 mm sodium bicarbonate (pH 8.3) at 37 °C overnight. Soluble proteins were lyophilized after digestion. Membrane proteins were digested in the presence of 0.5% SDS to aid solubilization. After the protease reaction, SDS was removed by precipitating proteins with 70% acetone or by chromatography using a cation exchange cartridge (OASIS MCX; Waters, Milford, MA) according to manufacturer's procedure. The proteins were lyophilized and stored at −80 °C and resuspended in 100 μl of 0.4% acetic solution prior to mass spectrometer analysis. Trypsin-digested peptides were analyzed by μLC-ESI-MS/MS using an LCQ-DECA mass spectrometer (Thermo Finnigan, San Jose, CA) equipped with a C18 trap ESI-emitter/micro-LC column. Trypsin-digested peptides (2 μg) were loaded to a Hewlett Packard/Agilent 1100 Series high-pressure LC system using a Famos Autosampler (Dionex, San Francisco, CA). The peptides bound to the C18 matrix were eluted by acetonitrile gradient (5% to 35%) by mixing acetonitrile with 0.4% acetic acid in water. The eluted peptides were injected into the mass spectrometer by nano-ESI (19Figeys D. Ducret A. Yates 3rd, J.R. Aebersold R. Protein identification by solid phase microextraction-capillary zone electrophoresis-microelectrospray-tandem mass spectrometry. Nat. Biotechnol. 1996; 14: 1579-1583Google Scholar, 23Ducret A. Van Oostveen I. Eng J.K. Yates 3rd, J.R. Aebersold R. High throughput protein characterization by automated reverse-phase chromatography/electrospray tandem mass spectrometry. Protein Sci. 1998; 7: 706-719Google Scholar). Mass spectra were acquired by data-dependent ion selection from a full range as well as discrete and narrow survey scan m/z ranges to increase the number of identifications. Proteins were identified from tandem mass spectra using the SEQUEST (24Eng J.K. McCormack A.L. Yates 3rd, J.R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Google Scholar) database search engine to search against the Halobacterium NRC-1 predicted protein database (3Ng W.V. Kennedy S.P. Mahairas G.G. Berquist B. Pan M. Shukla H.D. Lasky S.R. Baliga N.S. Thorsson V. Sbrogna J. Swartzell S. Weir D. Hall J. Dahl T.A. Welti R. Goo Y.A. Leithauser B. Keller K. Cruz R. Danson M.J. Hough D.W. Maddocks D.G. Jablonski P.E. Krebs M.P. Angevine C.M. Dale H. Isenbarger T.A. Peck R.F. Pohlschroder M. Spudich J.L. Jung K.W. Alam M. Freitas T. Hou S. Daniels C.J. Dennis P.P. Omer A.D. Ebhardt H. Lowe T.M. Liang P. Riley M. Hood L. DasSarma S. Genome sequence of Halobacterium species NRC-1. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12176-12181Google Scholar). Halobacterium putative proteins were analyzed for the presence of transmembrane domains using the TMpred (25Hofmann K. Stoffel W. Tmbase—A database of membrane-spanning protein segments. Biol. Chem. Hoppe-Seyler. 1993; 374: 166Google Scholar) and TMHMM programs (26Sonnhammer E.L. von Heijne G. Krogh A. Pfam: Multiple sequence alignments and HMM-profiles of protein domains. Nucleic Acids Res. 1998; 26: 320-322Google Scholar, 27Krogh A. Larsson B. von Heijne G. Sonnhammer E.L. Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. J. Mol. Biol. 2001; 305: 567-580Google Scholar). The TMpred program predicts membrane-spanning regions (MSRs) and their orientation based on the statistical analysis of TMbase, a database of transmembrane proteins and their helical membrane-spanning domains. The prediction is based on an algorithm using a combination of several weight-matrices for investigating the local properties of amino acid sequences. The program TMHMM, on the other hand, takes a global approach to determine the topology of an entire protein based on Hidden Markov models. The stand-alone TMpred program was installed on a SUN Microsystem Enterprise 420R server. TMHMM (v. 2.0) was run through the web interface (www.cbs.dtu.dk/services/TMHMM). The Halobacterium NRC-1 genome encodes 2682 putative protein-coding genes (3Ng W.V. Kennedy S.P. Mahairas G.G. Berquist B. Pan M. Shukla H.D. Lasky S.R. Baliga N.S. Thorsson V. Sbrogna J. Swartzell S. Weir D. Hall J. Dahl T.A. Welti R. Goo Y.A. Leithauser B. Keller K. Cruz R. Danson M.J. Hough D.W. Maddocks D.G. Jablonski P.E. Krebs M.P. Angevine C.M. Dale H. Isenbarger T.A. Peck R.F. Pohlschroder M. Spudich J.L. Jung K.W. Alam M. Freitas T. Hou S. Daniels C.J. Dennis P.P. Omer A.D. Ebhardt H. Lowe T.M. Liang P. Riley M. Hood L. DasSarma S. Genome sequence of Halobacterium species NRC-1. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12176-12181Google Scholar). Among these, 2413 genes are unique. The TMHMM program predicted 544 membrane proteins containing 1 to 24 MSR(s), among which 163 were annotated proteins, 122 were conserved hypothetical proteins (CHP), and 259 were hypothetical proteins (HP). On the other hand, TMpred detected 929 membrane proteins containing 1 to 22 MSR(s) with total score more than 1000, among which 377 were annotated proteins, 202 were CHP, and 350 were HP. A score >500 is considered to be statistically significant in TMpred prediction (25Hofmann K. Stoffel W. Tmbase—A database of membrane-spanning protein segments. Biol. Chem. Hoppe-Seyler. 1993; 374: 166Google Scholar). TMpred also detected all 544 membrane proteins predicted by the TMHMM program with a minimal TMpred total score of 1194. Two micrograms of trypsin-digested peptide mixtures from the membrane and soluble proteins were analyzed by μLC-ESI-MS/MS with the following different m/z ranges from which ions were selected for collision-induced dissociation (CID): 1 (400–2000 m/z), 4 (400∼800, 800∼1200, 1200∼1600, and 1600∼2000 m/z), or 16 (400∼500, 500∼600, 600∼700, …, and 1900∼2000 m/z). Using a different m/z range has been shown to increase the number of novel peptides selected for CID (28Spahr C.S. Davis M.T. McGinley M.D. Robinson J.H. Bures E.J. Beierle J. Mort J. Courchesne P.L. Chen K. Wahl R.C. Yu W. Luethy R. Patterson S.D. Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry. I. Profiling an unfractionated tryptic digest. Proteomics. 2001; 1: 93-107Google Scholar, 29Yi E.C. Marelli M. Lee H. Purvine S.O. Aebersold R. Aitchison J.D. Goodlett D.R. Approaching complete peroxisome characterization by gas-phase fractionation. Electrophoresis. 2002; 23: 3205-3216Google Scholar). The tandem mass spectra were analyzed using the SEQUEST database search program (24Eng J.K. McCormack A.L. Yates 3rd, J.R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Google Scholar) with the Halobacterium NRC-1 protein database. Search results were processed using the INTERACT web interface, a software tool that allows internet-based data display, data filtering, and data sorting (30Han D.k. Eng J. Zhou H. Aebersold R. Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry. Nat. Biotechnol. 2001; 19: 946-951Google Scholar). Recently developed statistical modeling algorithms to compute probabilities associated with peptide (PeptideProphet™) (31Nesvizhskii A.L. Keller A. Kolker E. Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry. Anal. Chem. in press. 2003; Google Scholar) and protein (ProteinProphet™) (32Keller A. Nesvizhskii A.I. Kolker E Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem. 2002; 74: 5383-5392Google Scholar) sequence assignments that distinguish correct from incorrect database search results were used to validate the search results. These tools allowed assigning probabilities to all identifications and offering standardized interpretation of results by reducing the need for manual verification. In particular, these tools enabled rapid and objective evaluation of large proteomic datasets. A detailed application of these tools has been recently published (33von Haller P.D. Yi E.C. Donohoe S. Vaughn K. Keller A. Nesvizhskii A.I. Eng J. Li X. Goodlett D.R. Aebersold R. Watts J.D. The application of new software tools to quantitative protein profiling via ICAT and tandem mass spectrometry: II. Evaluation of tandem mass spectrometry methodologies for large-scale protein analysis and the application of statistical tools for data analysis and interpretation. Mol. Cell. Proteomics. 2003; 2: 428-442Google Scholar). More information on these applications can be found on the Proteomics pages at http://www.systemsbiology.org/ and they are open source. In this study, we report proteins with probability at least 0.5. Probability 0.5 means that according to the statistical model, the sequence match given is 50% likely to be correct. These resulted in the identification of 426 proteins with false-positive rate of 3.7% (Fig. 1). In the MS/MS analysis, 165 proteins were identified only in the membrane fraction but not in the soluble protein fraction (Table I). There were 100 (60.6%) annotated proteins, 29 (17.6%) CHP, and 36 (21.8%) HP. The transmembrane domain prediction program TMHMM predicted 90 (54.5%) proteins containing 1–22 membrane domains, and TMpred detected 123 (74.5%) proteins with 1–22 membrane domains with scores greater than 1000. No membrane domain was detected by either program in 31 (18.8%) proteins.Table IProteins identified in the membrane fractionFunctional CategoryGene IDProbabilityProteinPutative FunctionTMHMMTMpredAmino acid metabolismVNG0104G1SerA3Phosphoglycerate dehydrogenase02VNG1814G1CarBCarbamoyl-phosphate synthase large subunit00VNG2120G1YusMProline dehydrogenase00VNG2418G1AspC1Aspartate aminotransferase01*TMpred total score < 1000.VNG2421G1HalO-acetyl homoserine02Cell envelope componentVNG0180G0.98HopHalorhodopsin77VNG0321G1IdsBifunctional short chain isoprenyl diphosphate synthase11VNG1187G1Pan1Membrane protein01VNG1467G1BopBacteriorhodopsin67VNG1660G1Sop1Sensory rhodopsin I77VNG1764G0.98Sop2Sensory rhodopsin II77VNG6265G1YcdHAdhesion protein01Cellular processVNG0129G1Hsp4Heat shock protease protein44VNG0355G1Htr14Htr14 transducer12VNG0375G1SecEProtein translocase11VNG0614G0.79Htr16Htr16 transducer11VNG0793G1Htr6Htr6 transducer22VNG0806G1Htr4Htr4 transducer23VNG0971G0.99CheAChemotaxis protein02VNG0976G1CheW1Chemotaxis protein00VNG1523G1Htr8Htr8 transducer55VNG1659G1Htr1Htr1 transducer12VNG1760G1Htr5Htr5 transducer22VNG1765G1Htr2Htr2 transducer13VNG1801G1Hsp1Small heat shock protein00VNG1856G1Htr3Htr3 transducer11VNG1987G1SecDProtein-export membrane protein65VNG1988G1SecFProtein-export membrane protein65VNG5025G, VNG6024G1GvpH1GvpH protein, cluster A00VNG5030G, VNG6029G1GvpA1GvpA protein, cluster A00VNG5033G, VNG6032G1GvpN1GvpN protein, cluster A00Cofactor metabolismVNG1635G0.98CbiMCobalamin biosynthesis protein66VNG1776G1NirHHeme biosynthesis protein00DNA replication, repair, and recombinationVNG0884G1Top6ADNA topoisomerase VI subunit A00VNG2372G1Rad24cDNA repair protein00VNG2473G1RadA1DNA repair protein01Energy metabolismVNG0412G1FolPDihydropteroate synthase00VNG0637G1NdhG5NADH dehydrogenase/oxidoreductase01VNG0639G1NdhG4NADH dehydrogenase/oxidoreductase67VNG0646G1NuoLF420H2:quinone oxidoreductase chain L1715VNG0648G1NdhG3NADH dehydrogenase/oxidoreductase1110VNG0657G1CoxA2Cytochrome c oxidase subunit I1314VNG0665G1CoxB1Cytochrome c oxidase subunit II33VNG0891G1YjlDNADH dehydrogenase01VNG1308G1SdhBSuccinate dehydrogenase subunit B01VNG1498G1CelMEndoglucanase00VNG2141G1AtpCH+-transporting ATP synthase subunit C01*TMpred total score < 1000.VNG2143G1AtpKH+-transporting ATP synthase subunit K22VNG2193G1CoxA1Cytochrome c oxidase subunit I1011VNG2195G1CoxB2Cytochrome c oxidase subunit II00VNG5055G, VNG5242G, VNG6053G, VNG6473G1CydACytochrome d oxidase chain I911MiscellaneousVNG0249G1FbrCytochrome-like protein02VNG0303G1LonATP-dependent proteinase homolog12VNG0459G1NodPModulation protein00VNG0540G1ImpImmunogenic protein01*TMpred total score < 1000.VNG0620G1EdpProteinase IV homolog13VNG0635G1NolBNADH dehydrogenase/oxidoreductase-like protein33VNG0640G0.98NolDNADH dehydrogenase/oxidoreductase-like protein00VNG0795G1HcpCHalocyanin precursor-like02VNG1428G1HtlAHtr-like protein77VNG1932G1NolANADH dehydrogenase/oxidoreductase-like protein02VNG2086G1HpbPossible phosphate binding protein01VNG2196G1HcpBHalocyanin precursor-like33VNG2308G1HlpHemolysin protein23VNG2320G1HdrDHeterodisulfide reductase56VNG2358G1AppAOligopeptide binding protein01VNG6301G1AphAlkaline phosphatase01Nucleotide metabolismVNG0632G1PurKPhosphoribosylaminoimidazole carboxylase ATP binding subunit00VNG1408G1UshUDP-sugar hydrolase22VNG2507G0.99PyrDDihydroorotate dehydrogenase00TranslationVNG0177G1Rpl15e50S ribosomal protein L15E00VNG0551G1Rpl44e50S ribosomal protein L44E00VNG1433G1Rps17e30S ribosomal protein S17E00VNG1866G0.98MapMethionyl aminopeptidase00VNG2469G1Rpl39e50S ribosomal proteins L39E00TransportVNG0002G1YvrOAmino acid ABC transporter, ATP-binding protein01*TMpred total score < 1000.VNG0174G1Cat1Cationic amino acid transporter1110VNG0453G1PstA2Phosphate ABC transporter permease1414VNG0455G1PstC2Phosphate ABC transporter permease78VNG0457G1PhoXPhosphate ABC transporter periplasmic phosphate-binding01VNG0794G1YufNABC transporter (lipoprotein)01*TMpred total score < 1000.VNG0924G1IbpIron-binding protein02VNG1634G0.97CbiNCobalt transport protein22VNG1762G0.99ProXPutative ABC transporter01VNG2343G1YkfDOligopeptide ABC transporter ATP-binding00VNG2346G1DppC2Dipeptide ABC transporter permease89VNG2347G1DppB1Dipeptide ABC transporter permease67VNG2359G0.99AppBOligopeptide ABC permease76VNG2378G1NosF1Copper transport ATP-binding protein00VNG2483G1PstA1Phosphate ABC transporter permease1214VNG2486G1YqgGPhosphate ABC transporter binding01VNG2527G1DppDDipeptide ABC transporter ATP-binding01*TMpred total score < 1000.VNG2529G1DppB2Dipeptide ABC transporter permease77VNG6277G1UgpBGlycerol-3-phosphate-binding protein01VNG6313G1NhaC3Na+/H+ antiporter1111UncharacterizedVNG0593G0.99DmdDiphosphomevalonate decarboxylase01VNG0748G1PrkAKinase anchor protein00VNG1068G1TotTransmembrane
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