Protein Complexes in the Archaeon Methanothermobacter thermautotrophicus Analyzed by Blue Native/SDS-PAGE and Mass Spectrometry
2005; Elsevier BV; Volume: 4; Issue: 11 Linguagem: Inglês
10.1074/mcp.m500171-mcp200
ISSN1535-9484
AutoresMurtada H. Farhoud, Hans J. C. T. Wessels, Peter J.M. Steenbakkers, Sandy Mattijssen, Ron A. Wevers, Baziel G.M. van Engelen, Mike S. M. Jetten, Jan Smeitink, Lambert P. van den Heuvel, Jan T. Keltjens,
Tópico(s)Protein Structure and Dynamics
ResumoMethanothermobacter thermautotrophicus is a thermophilic archaeon that produces methane as the end product of its primary metabolism. The biochemistry of methane formation has been extensively studied and is catalyzed by individual enzymes and proteins that are organized in protein complexes. Although much is known of the protein complexes involved in methanogenesis, only limited information is available on the associations of proteins involved in other cell processes of M. thermautotrophicus. To visualize and identify interacting and individual proteins of M. thermautotrophicus on a proteome-wide scale, protein preparations were separated using blue native electrophoresis followed by SDS-PAGE. A total of 361 proteins, corresponding to almost 20% of the predicted proteome, was identified using peptide mass fingerprinting after MALDI-TOF MS. All previously characterized complexes involved in energy generation could be visualized. Furthermore the expression and association of the heterodisulfide reductase and methylviologen-reducing hydrogenase complexes depended on culture conditions. Also homomeric supercomplexes of the ATP synthase stalk subcomplex and the N5-methyl-5,6,7,8-tetrahydromethanopterin:coenzyme M methyltransferase complex were separated. Chemical cross-linking experiments confirmed that the multimerization of both complexes was not experimentally induced. A considerable number of previously uncharacterized protein complexes were reproducibly visualized. These included an exosome-like complex consisting of four exosome core subunits, which associated with a tRNA-intron endonuclease, thereby expanding the constituency of archaeal exosomes. The results presented show the presence of novel complexes and demonstrate the added value of including blue native gel electrophoresis followed by SDS-PAGE in discovering protein complexes that are involved in catabolic, anabolic, and general cell processes. Methanothermobacter thermautotrophicus is a thermophilic archaeon that produces methane as the end product of its primary metabolism. The biochemistry of methane formation has been extensively studied and is catalyzed by individual enzymes and proteins that are organized in protein complexes. Although much is known of the protein complexes involved in methanogenesis, only limited information is available on the associations of proteins involved in other cell processes of M. thermautotrophicus. To visualize and identify interacting and individual proteins of M. thermautotrophicus on a proteome-wide scale, protein preparations were separated using blue native electrophoresis followed by SDS-PAGE. A total of 361 proteins, corresponding to almost 20% of the predicted proteome, was identified using peptide mass fingerprinting after MALDI-TOF MS. All previously characterized complexes involved in energy generation could be visualized. Furthermore the expression and association of the heterodisulfide reductase and methylviologen-reducing hydrogenase complexes depended on culture conditions. Also homomeric supercomplexes of the ATP synthase stalk subcomplex and the N5-methyl-5,6,7,8-tetrahydromethanopterin:coenzyme M methyltransferase complex were separated. Chemical cross-linking experiments confirmed that the multimerization of both complexes was not experimentally induced. A considerable number of previously uncharacterized protein complexes were reproducibly visualized. These included an exosome-like complex consisting of four exosome core subunits, which associated with a tRNA-intron endonuclease, thereby expanding the constituency of archaeal exosomes. The results presented show the presence of novel complexes and demonstrate the added value of including blue native gel electrophoresis followed by SDS-PAGE in discovering protein complexes that are involved in catabolic, anabolic, and general cell processes. Methanothermobacter thermautotrophicus is a thermophilic archaeon that generates energy from the reduction of carbon dioxide to methane. In contrast to mostly mesophilic methanogens, M. thermautotrophicus can use hydrogen and carbon dioxide only as substrates and is therefore regarded as a metabolic specialist. It has a relatively simple primary metabolism comprising eight consecutive steps (1Deppenmeier U. The unique biochemistry of methanogenesis.Prog. Nucleic Acids Res. Mol. Biol. 2002; 71: 223-283Google Scholar). Methane production, or methanogenesis, starts with the reduction of carbon dioxide to a formyl moiety that is bound to the C1 carrier methanofuran. The endergonic reaction is catalyzed by a membrane-bound formylmethanofuran dehydrogenase complex and is driven by a gradient of sodium (2de Poorter L.M.I. Geerts W.G. Theuvenet A.P.R. Keltjens J.T. Bioenergetics of the formyl-methanofuran dehydrogenase and heterodisulfide reductase reactions in Methanothermobacter thermautotrophicus..Eur. J. Biochem. 2003; 270: 66-75Google Scholar). Next the formyl moiety is transferred to a second C1 carrier and reduced by individual, cytosolic enzymes to a methyl moiety. Hereafter the methyl group is transferred to coenzyme M by the membrane-bound N5-methyl-5,6,7,8-tetrahydromethanopterin:coenzyme M methyltransferase complex that simultaneously generates a sodium gradient (3Gartner P. Ecker A. Fischer R. Linder D. Fuchs G. Thauer R.K. Purification and properties of N5-methyltetrahydromethanopterin coenzyme-M methyltransferase from Methanobacterium thermoautotrophicum..Eur. J. Biochem. 1993; 213: 537-545Google Scholar). Finally the methyl group is then reduced to methane by the cytosolic methyl coenzyme M reductase complex (4Ellermann J. Hedderich R. Bocher R. Thauer R.K. The final step in methane formation—investigations with highly purified methyl-CoM reductase (component C) from Methanobacterium thermoautotrophicum (strain Marburg).Eur. J. Biochem. 1988; 172: 669-677Google Scholar). Next to methane, the mixed disulfide of coenzyme M and coenzyme B is formed. The heterodisulfide is reduced by the heterodisulfide reductase complex that is supplied with reducing equivalents by the methylviologen-reducing hydrogenase (5Setzke E. Hedderich R. Heiden S. Thauer R.K. H2-Heterodisulfide oxidoreductase complex from Methanobacterium thermoautotrophicum—composition and properties.Eur. J. Biochem. 1994; 220: 139-148Google Scholar). The proton motive force that is generated in this step is finally used by a membrane-bound ATP synthase complex for the production of ATP (6Coskun U. Chaban Y.L. Lingl A. Muller V. Keegstra W. Boekema E.J. Gruber G. Structure and subunit arrangement of the A-type ATP synthase complex from the archaeon Methanococcus jannaschii visualized by electron microscopy.J. Biol. Chem. 2004; 279: 38644-38648Google Scholar). Next to the generation of ATP, methanogenesis also provides carbon for cell growth through the activity of the acetyl-CoA decarbonylase/synthase complex (7Lange S. Fuchs G. Autotrophic synthesis of activated acetic acid from CO2 in Methanobacterium thermoautotrophicum—synthesis from tetrahydromethanopterin-bound C-1 units and carbon monoxide.Eur. J. Biochem. 1987; 163: 147-154Google Scholar). In natural ecosystems the amount of hydrogen is the limiting substrate for growth and can vary 4 orders of magnitude in availability. As an adaptation, M. thermautotrophicus expresses isoenzymes with a high or low affinity for hydrogen, e.g. methyl coenzyme M reductases I and II are expressed under culturing conditions with low and high hydrogen gassing regimes ("MCR 1The abbreviations used are: MCR, methyl coenzyme M reductase; methyltransferase, N5-methyl-5,6,7,8-tetrahydromethanopterin:coenzyme M methyltransferase; BN-PAGE, blue native gel electrophoresis followed by SDS-PAGE; TES, N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; NHSS, N-hydroxysulfosuccinimide; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; 2D, two-dimensional. 1The abbreviations used are: MCR, methyl coenzyme M reductase; methyltransferase, N5-methyl-5,6,7,8-tetrahydromethanopterin:coenzyme M methyltransferase; BN-PAGE, blue native gel electrophoresis followed by SDS-PAGE; TES, N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; NHSS, N-hydroxysulfosuccinimide; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; 2D, two-dimensional. I and MCR II culture conditions"), respectively (8Bonacker L.G. Baudner S. Thauer R.K. Differential expression of the 2 methyl-coenzyme M-reductases in Methanobacterium thermoautotrophicum as determined immunochemically via isoenzyme-specific antisera.Eur. J. Biochem. 1992; 206: 87-92Google Scholar). In a closely related species, Methanothermobacter marburgensis, it was shown that complexes of the primary metabolism that are coupled functionally can also interact physically. The purification of heterodisulfide reductase activity resulted in the purification of the heterodisulfide reductase protein complex that also contained the methylviologen-reducing hydrogenase complex (5Setzke E. Hedderich R. Heiden S. Thauer R.K. H2-Heterodisulfide oxidoreductase complex from Methanobacterium thermoautotrophicum—composition and properties.Eur. J. Biochem. 1994; 220: 139-148Google Scholar). In M. thermautotrophicus, similarly it was speculated that the association into high molecular weight complexes in concentrated solutions of purified methyl coenzyme M reductase and F420-dependent hydrogenase proteins could also play a role in vivo (9Wackett L.P. Hartwieg E.A. King J.A. Ormejohnson W.H. Walsh C.T. Electron-microscopy of nickel-containing methanogenic enzymes—methyl reductase and F420-reducing hydrogenase.J. Bacteriol. 1987; 169: 718-727Google Scholar). Electron microscopy studies of enzymes and complexes involved in methanogenesis of Methanococcus voltae and Methanosarcina mazei Go¨1 showed the associations of methanogenesis enzymes into supercomplexes, named methanoreductosomes (10Mayer F. Rohde M. Salzmann M. Jussofie A. Gottschalk G. The methanoreductosome—a high-molecular-weight enzyme complex in the methanogenic bacterium strain Go1 that contains components of the methylreductase system.J. Bacteriol. 1988; 170: 1438-1444Google Scholar). These results corroborate the generally accepted idea that the protein content of a cell is not merely a pool of individual and inert proteins but that the proteome of a living cell is an intricate network of proteins that interact and communicate. So far, research on M. thermautotrophicus has focused mainly on the enzymes and enzyme complexes that are involved in methanogenesis, and therefore only limited data are available on interacting proteins involved in other cellular processes. To explore interacting (and non-interacting) proteins of an organism on a proteome-wide scale, only a limited number of techniques are available. One established method is the use of blue native electrophoresis followed by SDS-PAGE (or BN-PAGE) (11Camacho-Carvajal M.M. Wollscheid B. Aebersold R. Steimle V. Schamel W.W.A. Two-dimensional blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates. A proteomics approach.Mol. Cell. Proteomics. 2004; 3: 176-182Google Scholar). In this method, proteins and protein complexes are solubilized by a mild detergent, charged with Coomassie dye, and separated natively in a first dimension depending on charge, size, and shape. In a second dimension, proteins and protein complexes are reduced and denatured with SDS and subunits, and individual proteins are separated by molecular weight. So far BN-PAGE has been applied mainly to membrane-associated complexes of organelles (12Brookes P.S. Pinner A. Ramachandran A. Coward L. Barnes S. Kim H. Darley-Usmar V.M. High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of mitochondria and signaling complexes.Proteomics. 2002; 2: 969-977Google Scholar, 13Nijtmans L.G.J. Henderson N.S. Holt I.J. Blue native electrophoresis to study mitochondrial and other protein complexes.Methods. 2002; 26: 327-334Google Scholar) sometimes after dialysis of protein preparations (11Camacho-Carvajal M.M. Wollscheid B. Aebersold R. Steimle V. Schamel W.W.A. Two-dimensional blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates. A proteomics approach.Mol. Cell. Proteomics. 2004; 3: 176-182Google Scholar) and often in combination with the use of antibodies. In this study we present the results of a proteome-wide examination of interacting and individual proteins of M. thermautotrophicus using blue native/SDS-PAGE combined with mass spectrometry. This resulted in the identification of 361 proteins, corresponding to almost 20% of the predicted proteome, and visualization of a significant number of proteins that are part of enzyme complexes. These allowed, among others, the identification of an exosome-like complex of M. thermautotrophicus. Next to the homologs of four exosome core subunits identified previously (14Evguenieva-Hackenberg E. Walter P. Hochleitner E. Lottspeich F. Klug G. An exosome-like complex in Sulfolobus solfataricus.EMBO Rep. 2003; 4: 889-893Google Scholar), the M. thermautotrophicus exosome additionally contains a tRNA-intron endonuclease. In summary, the results presented here give, for the first time, an overview of interacting and individual proteins of the archaeon M. thermautotrophicus on a proteome-wide scale using BN-PAGE. The genome coverage and the protein complexes identified by this technique clearly indicate the added value of including BN-PAGE in proteomic research and allow the study of protein complexes involved in primary metabolism, anabolism, and general cell processes. M. thermautotrophicus (DSM1053) was cultured in a 12-liter fed-batch fermentor containing 10 liters of medium. Cells were cultured under two different conditions, one favoring the expression of MCR I and the other favoring MCR II expression (8Bonacker L.G. Baudner S. Thauer R.K. Differential expression of the 2 methyl-coenzyme M-reductases in Methanobacterium thermoautotrophicum as determined immunochemically via isoenzyme-specific antisera.Eur. J. Biochem. 1992; 206: 87-92Google Scholar). "MCR I" cells were cultured in N medium (15Pennings J.L.A. Vermeij P. de Poorter L.M.I. Keltjens J.T. Vogels G.D. Adaptation of methane formation and enzyme contents during growth of Methanobacterium thermoautotrophicum (strain Delta H) in a fed-batch fermentor.Antonie Leeuwenhoek. 2000; 77: 281-291Google Scholar) at 65 °C with a gas flow of 2.0 liters/min of 80% H2, 20% CO2 (v/v) and harvested at an optical density at 600 nm of 5. "MCR II" cells were cultured in MII medium (15Pennings J.L.A. Vermeij P. de Poorter L.M.I. Keltjens J.T. Vogels G.D. Adaptation of methane formation and enzyme contents during growth of Methanobacterium thermoautotrophicum (strain Delta H) in a fed-batch fermentor.Antonie Leeuwenhoek. 2000; 77: 281-291Google Scholar) at 55 °C with a gas flow of 5.0 liters/min of 80% H2, 20% CO2 (v/v) and harvested at an optical density at 600 nm of 1.8. After centrifugation, cell-free extracts were prepared by suspending the pellet in 1 volume of 50 mm TES, pH 7.0, 1 mm DTT, including both DNase and RNase at a final concentration of 10 μg/ml. Cell suspensions were passed through a French pressure cell operated at 138 megapascals. After centrifugation for 15 min at 10,000 × g at 4 °C, the cytosolic fraction was obtained as the clarified supernatant, and the resulting pellet is referred to as the membrane fraction. Both fractions were stored at −20 °C. Before protein solubilization, the membrane fraction was washed three times with a solution containing 400 mm sorbitol, 25 mm NaCl, and 7.5 mm imidazole, pH 7.0, to reduce contaminating cytosolic proteins. Cytosolic and membrane protein preparations were diluted with 2–3 volumes of solubilization buffer (50 mm NaCl, 5 mm 6-aminocaproic acid, 1 mm EDTA, and 50 mm imidazole, pH 7.0) containing either 2% (v/v) laurylmaltoside, 1% (v/v) digitonin, or 1% (v/v) Triton X-100. After a 15-min incubation on ice with occasional vortexing samples were centrifuged for 30 min at 12,000 × g at 4 °C. Supernatants were transferred to clean tubes, and protein concentrations were determined with the 2D-Quant kit (Amersham Biosciences). Finally 4 μl of 750 mm 6-aminohexanoic acid with 5% (w/v) Serva Blue G was added per 100 μl of supernatant. To investigate the association of membrane-bound complexes, the proteins present in the membrane fraction were treated with the 8-Å linker dimethyl 3,3′-dithiopropionimidate dihydrochloride (Sigma) and the "zero-length" carbodiimide cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC, Molecular Probes) in the presence of N-hydroxysulfosuccinimide (NHSS, Molecular Probes) (16Staros J.V. Wright R.W. Swingle D.M. Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions.Anal. Biochem. 1986; 156: 220-222Google Scholar). For this, an aliquot of 200 μl of the membrane fraction of MCR I cells was diluted with 600 μl of a solution containing 75 mm imidazole, pH 7, 400 mm sorbitol, 25 mm NaCl, and 0.03% laurylmaltoside. The chemical cross-linking reaction was started by adding 40 μl of freshly prepared 10 mg/ml dimethyl 3,3′-dithiopropionimidate dihydrochloride or 12 μl each of an 80 mm EDAC and 80 mm NHSS solution and proceeded for 1 h on ice with occasional mixing. The reaction was stopped by the addition of ammonium acetate to a final concentration of 0.1 m. As a control, immediately after addition of the cross-linkers, a 200-μl aliquot of the reaction mixture was quenched by adding 50 μl of 0.5 m ammonium acetate. Protein complexes were isolated immediately with either laurylmaltoside or digitonin as described above. Blue native/SDS-PAGE two-dimensional gel electrophoresis was performed as described elsewhere (13Nijtmans L.G.J. Henderson N.S. Holt I.J. Blue native electrophoresis to study mitochondrial and other protein complexes.Methods. 2002; 26: 327-334Google Scholar, 17Scha¨gger H. de Coo R. Bauer M.F. Hofmann S. Godinot C. Brandt U. Significance of respirasomes for the assembly/stability of human respiratory chain complex I.J. Biol. Chem. 2004; 279: 36349-36353Google Scholar, 18Arnold I. Pfeiffer K. Neupert W. Stuart R.A. Scha¨gger H. Yeast mitochondrial F1F0-ATP synthase exists as a dimer: identification of three dimer-specific subunits.EMBO J. 1998; 17: 7170-7178Google Scholar) and performed on a Protean II xi system (Bio-Rad). For the blue native first dimension, protein preparations were separated on gels containing the following bis-/acrylamide gradients: 4–13, 5–15, 6–18, 8–20, and 10–22%. The ferritin monomer (440 kDa) and ferritin dimer (880 kDa) were included as markers. Samples were separated overnight at 15 °C at 100 V and typically finished after 16 h. The second dimension was performed as described elsewhere (19Scha¨gger H. von Jagow G. Tricine sodium dodecyl-sulfate polyacrylamide-gel electrophoresis for the separation of proteins in the range from 1-kDa to 100-kDa.Anal. Biochem. 1987; 166: 368-379Google Scholar) with some modifications. Individual lanes of the first dimension gel were excised with a razor blade and incubated in 1% (w/v) SDS and 1% (v/v) 2-mercaptoethanol for 1–2 h at room temperature. Next the first dimension lane, which was rinsed with milli-Q to remove excess 2-mercaptoethanol, was placed on top of a separation gel consisting of a 10% Tris-Tricine-SDS gel, which was cross-linked with piperazine diacrylamide. The flanking regions of the first dimension lane were filled with 10% native polyacrylamide gel. Using a notched inner glass plate, the Protean II xi system was converted into a four-gel system and started at a constant current of 5 mA per gel, which was raised to 10 mA after 45 min. Second dimension gels were run overnight at 15 °C and typically finished after 20 h. After the second dimension, gels were stained with colloidal Coomassie as described elsewhere (20Candiano G. Bruschi M. Musante L. Santucci L. Ghiggeri G.M. Carnemolla B. Orecchia P. Zardi L. Righetti P.G. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis.Electrophoresis. 2004; 25: 1327-1333Google Scholar) and scanned using an Amersham Biosciences Image Scanner. After spot picking, protein present in gel plugs was first reduced by incubating for 10 min in 50 μl 10 mm DTT at 60 °C followed by an alkylating incubation of 45 min in 50 μl of 50 mm iodoacetamide at room temperature in the dark. Hereafter in-gel trypsin digestion was performed as described elsewhere (21Shevchenko A. Jensen O.N. Podtelejnikov A.V. Sagliocco F. Wilm M. Vorm O. Mortensen P. Shevchenko A. Boucherie H. Mann M. Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels.Proc. Natl. Acad. Sci. U S A. 1996; 93: 14440-14445Google Scholar) except that overnight trypsin treatment was performed in 5 μl of 50 mm ammonium bicarbonate containing 5 mm n-octyl glucopyranoside per plug. Next protein fragments were extracted by adding 1 μl of 0.5% (v/v) trifluoroacetic acid, 5 mm n-octyl glucopyranoside and incubating for 2 h at room temperature and a final 1-min sonication step. After extraction, peptides were stored at −20 °C until further analysis. For MS analysis, 0.25 μl of extracted peptides was pipetted on a MALDI-TOF sample plate and directly mixed with an equal volume of sample buffer containing 20 mg/ml α-cyano-4-hydroxycinnamic acid in 0.05% (v/v) TFA, 50% (v/v) acetonitrile. MALDI-TOF MS measurements were performed in the mass range of 650–2,600 Da on a Bruker III mass spectrometer, set to reflectron mode, after calibration using a mixture of bradykinin fragment 1–7, angiotensin, synthetic peptide P14R, and adrenocorticotropic hormone fragment 18–39. Mass spectra were determined as the sum of 180 measurements. Monoisotopic peaks were manually selected, excluding background peaks. Peptide masses were exported to Biotools software and used to perform a MASCOT search in an in-house database of the M. thermautotrophicus predicted proteome. Search parameters allowed a mass deviation of ±0.3 Da, matching peptides containing one miscleavage, fixed modification of carbamidomethylated cysteines, and a variable modification of oxidized methionines. Proteins were regarded as identified proteins when the MASCOT search resulted in a Mowse score higher than 45 (corresponding to an expect score <10−4.5), which was calculated from the database size by the MASCOT software. A filter was included to recognize false-positives. A 2D gel electrophoresis study currently in progress has shown that proteins that are identified with matching peptides that mostly contain one miscleavage do not show a correlation between the predicted and experimental location on a 2D gel. 2M. H. Farhoud, J. C. T. Wessels, P. J. M. Steenbakkers, W. Pluk, E. Lasonder, I. Schmidt, R. A. Wevers, B. G. van Engelen, M. S. M. Jetten, J. A. Smeitink, L. P. van den Heuvel, and J. T. Keltjens, unpublished data. Therefore, proteins were only regarded as significantly identified when they showed a Mowse score higher than 45 and contained mostly matching peptides that did not contain a miscleavage. Similar criteria applied for the identification of multiple proteins in a single spot. Here proteins were regarded as identified when peptides unique to that protein matched the above mentioned criteria. All nanoflow LC-MS/MS experiments were performed on a 7-tesla Finnigan LTQ-FT mass spectrometer (Thermo Electron) equipped with a nanoelectrospray ion source (Proxeon Biosystems, Odense, Denmark). The LC part of the analytical system consisted of an Agilent Series 1100 nanoflow LC system (Waldbronn, Germany) comprising a solvent degasser, a nanoflow pump, and a thermostated microautosampler. Chromatographic separation of the peptides was performed in a 15-cm fused silica emitter (100-μm inner diameter; New Objective) packed in-house with methanol slurry of reverse-phase ReproSil-Pur C18-AQ 3-μm resin (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) at a constant pressure (20 bars) of helium. Then 5 μl of the tryptic peptide mixtures were autosampled onto the packed emitter with a flow of 600 nl/min for 20 min and then eluted with a 5-min gradient from 3 to 10% followed by a 25-min gradient from 10 to 30% acetonitrile in 0.5% acetic acid at a constant flow of 300 nl/min. The mass spectrometer was operated in the data-dependent mode to automatically switch between MS and MS/MS acquisition. Survey MS spectra (from m/z 350 to 2,000) were acquired in the FTICR with r = 50,000 at m/z 400 (after accumulation to a target value of 1,000,000). The three most intense ions were sequentially isolated for fragmentation in the linear ion trap using collisionally induced dissociation with normalized collision energy of 29% and a target value of 1,000. Former target ions selected for MS/MS were dynamically excluded for 30 s. Total cycle time was ∼3 s. Proteins were identified via automated database searching (Matrix Science, London, UK) of all tandem mass spectra against both NCBInr and an in-house curated M. thermautotrophicus database. Carbamidomethylcysteine was set as fixed modification, and oxidized methionine and protein N-acetylation were searched as variable modifications. Initial mass tolerances for protein identification on MS and MS/MS peaks were 10 ppm and 0.5 Da, respectively. The instrument setting for the MASCOT search was specified as "ESI-Trap." The proteins identified were annotated on basis of the presence of domain signatures of the Pfam database (22Bateman A. Coin L. Durbin R. Finn R.D. Hollich V. Griffiths-Jones S. Khanna A. Marshall M. Moxon S. Sonnhammer E.L.L. Studholme D.J. Yeats C. Eddy S.R. The Pfam protein families database.Nucleic Acids Res. 2004; 32: D138-D141Google Scholar). Sequence similarities were determined using Blast 2 sequences (23Tatusova T.A. Madden T.L. BLAST 2 SEQUENCES, a new tool for comparing protein and nucleotide sequences.FEMS Microbiol. Lett. 1999; 174: 247-250Google Scholar) at default settings. The organization of genes into operons was determined from the gene organization at the PEDANT website (24Riley M.L. Schmidt T. Wagner C. Mewes H.W. Frishman D. The PEDANT genome database in 2005.Nucleic Acids Res. 2005; 33: D308-D310Google Scholar). Genes were considered to be part of an operon when the intergenic distance was smaller than 55 bp. The proteome of M. thermautotrophicus was analyzed using BN-PAGE to investigate the presence of enzyme complexes and individual enzymes. Biomass was collected after culturing under MCR I or MCR II conditions (see "Experimental Procedures"). For both culture conditions, lysed cells were separated into a cytosolic and a membrane fraction by centrifugation to reduce the complexity of the sample. The protein preparations were subjected to blue native gel electrophoresis directly without any additional cleaning procedures. Different first dimension gradients were applied to achieve maximum resolution of individual and complexed proteins. Typically individual proteins were optimally resolved with a first dimension gradient running from 10 to 20% bis-/acrylamide, whereas protein complexes showed maximum resolution with gradients starting at 4% bis-/acrylamide. Next to an optimization of the electrophoresis procedure and protein load, the identification of proteins also was optimized. The use of colloidal Coomassie staining in comparison to silver staining or traditional Coomassie staining enabled a high sensitivity while remaining completely compatible with mass spectrometry. Another improvement was the alkylation of cysteine residues after spot picking, which greatly reduced the number of background peaks after MALDI-TOF MS measurements and allowed the identification of multiple proteins in a single spot. An initial analysis of protein spots from duplicate gels and duplicate biological samples confirmed the reproducibility of BN-PAGE. The complete data set comprised the analysis of 1,550 protein spots by peptide mass fingerprinting. The data set included the optimization procedures, the appropriate reference protein spots from duplicate gels, and the identification of putative complex components originating from multiple blue native gels with different bis-/acrylamide gradients. The analysis showed that only a minor portion of the proteins identified appeared at multiple positions on gels and that no detectable protein degradation had taken place. The analysis of ∼875 protein spots of the proteins extracted from the membrane fraction allowed the identification of 300 different proteins, whereas the analysis of 675 protein spots from the cytosolic fraction identified 223 proteins. A total of 361 different proteins (for a reference map see Supplemental Fig. 1) were identified with an average Mowse score of 122 (which corresponds to an expect value less than 10−12) and an average protein sequence coverage of 37% (see Supplemental Table 1). Five percent of the predicted membrane proteins (34 of 407 proteins containing one or more predicted transmembrane-spanning regions) were retrieved. This was also reflected in the coverages of the different protein function categories that were recognized after initial analyses of the M. thermautotrophicus genome (25Smith D.R. DoucetteStamm L.A. Deloughery C. Lee H.M. Dubois J. Aldredge T. Bashirzadeh R. Blakely D. Cook R. Gilbert K. Harrison D. Hoang L. Keagle P. Lumm W. Pothier B. Qiu D.Y. Spadafora R. Vicaire R. Wang Y. Wierzbowski J. Gibson R. Jiwani N. Caruso A. Bush D. Safer H. Patwell D. Prabhakar S. McDougall S. Shimer G. Goyal A. Pietrokovski S. Church G.M. Daniels C.J. Mao J.I. Rice P. Nolling J. Reeve J.N. Complete genome sequence of Methanobacterium thermoautotrophicum Delta
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