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• W2136145101 abstract "Respiratory complex I (NADH:quinone oxidoreductase) is an entry point to the electron transport chain in the mitochondria of many eukaryotes. It is a large, multisubunit enzyme with a hydrophilic domain in the matrix and a hydrophobic domain in the mitochondrial inner membrane. Here we present a comprehensive analysis of the protein composition and post-translational modifications of complex I from Pichia pastoris, using a combination of proteomic and bioinformatic approaches. Forty-one subunits were identified in P. pastoris complex I, comprising the 14 core (conserved) subunits and 27 supernumerary subunits; seven of the core subunits are mitochondrial encoded. Three of the supernumerary subunits (named NUSM, NUTM, and NUUM) have not been observed previously in any species of complex I. However, homologues to all three of them are present in either Yarrowia lipolytica or Pichia angusta complex I. P. pastoris complex I has 39 subunits in common with Y. lipolytica complex I, 37 in common with N. crassa complex I, and 35 in common with the bovine enzyme. The mitochondrial encoded subunits (translated by the mold mitochondrial genetic code) retain their N-α-formyl methionine residues. At least eight subunits are N-α-acetylated, but the N-terminal modifications of the nuclear encoded subunits are not well-conserved. A combination of two methods of protein separation (SDS-PAGE and HPLC) and three different mass spectrometry techniques (peptide mass fingerprinting, tandem MS and molecular mass measurements) were required to define the protein complement of P. pastoris complex I. This requirement highlights the need for inclusive and comprehensive strategies for the characterization of challenging membrane-bound protein complexes containing both hydrophilic and hydrophobic components. Respiratory complex I (NADH:quinone oxidoreductase) is an entry point to the electron transport chain in the mitochondria of many eukaryotes. It is a large, multisubunit enzyme with a hydrophilic domain in the matrix and a hydrophobic domain in the mitochondrial inner membrane. Here we present a comprehensive analysis of the protein composition and post-translational modifications of complex I from Pichia pastoris, using a combination of proteomic and bioinformatic approaches. Forty-one subunits were identified in P. pastoris complex I, comprising the 14 core (conserved) subunits and 27 supernumerary subunits; seven of the core subunits are mitochondrial encoded. Three of the supernumerary subunits (named NUSM, NUTM, and NUUM) have not been observed previously in any species of complex I. However, homologues to all three of them are present in either Yarrowia lipolytica or Pichia angusta complex I. P. pastoris complex I has 39 subunits in common with Y. lipolytica complex I, 37 in common with N. crassa complex I, and 35 in common with the bovine enzyme. The mitochondrial encoded subunits (translated by the mold mitochondrial genetic code) retain their N-α-formyl methionine residues. At least eight subunits are N-α-acetylated, but the N-terminal modifications of the nuclear encoded subunits are not well-conserved. A combination of two methods of protein separation (SDS-PAGE and HPLC) and three different mass spectrometry techniques (peptide mass fingerprinting, tandem MS and molecular mass measurements) were required to define the protein complement of P. pastoris complex I. This requirement highlights the need for inclusive and comprehensive strategies for the characterization of challenging membrane-bound protein complexes containing both hydrophilic and hydrophobic components. Respiratory complex I (NADH:quinone oxidoreductase) is an entry point to the electron transport chain in mitochondria and many aerobic bacteria. It couples NADH oxidation and quinone reduction to proton translocation across the inner mitochondrial (or plasma) membrane, so it is central to energy transduction. Consequently, complex I dysfunctions are linked to an increasing number of neuromuscular and neurodegenerative diseases, including Parkinson's disease, as well as to oxidative stress and aging (1DiMauro S. Schon E.A. Mitochondrial respiratory-chain diseases.N. Engl. J. Med. 2003; 348: 2656-2668Crossref PubMed Scopus (1321) Google Scholar). Complex I is an l-shaped assembly, with a hydrophobic “arm” in the inner mitochondrial (or plasma) membrane, and a hydrophilic arm extending into the mitochondrial matrix (or cytoplasm) (2Guénebaut V. Schlitt A. Weiss H. Leonard K. Friedrich T. Consistent structure between bacterial and mitochondrial NADH:ubiquinone oxidoreductase (complex I).J. Mol. Biol. 1998; 276: 105-112Crossref PubMed Scopus (204) Google Scholar). The 14 core subunits of complex I are sufficient for catalysis and conserved in all complex I–encoding species; they comprise two sets of seven subunits that correspond to the two domains (3Walker J.E. The NADH-ubiquinone oxidoreductase (complex I) of respiratory chains.Q. Rev. Biophys. 1992; 25: 253-324Crossref PubMed Scopus (681) Google Scholar, 4Hirst J. Towards the molecular mechanism of respiratory complex I. Biochem.J. 2010; 425: 327-339Google Scholar). The seven hydrophobic subunits are encoded by the mitochondrial genome in eukaryotes, and the seven nuclear-encoded hydrophilic subunits ligate the flavin mononucleotide (which catalyzes NADH oxidation), and the eight (or nine) iron-sulfur clusters (which transfer electrons from the flavin to quinone). The structure of the hydrophilic domain of complex I from Thermus thermophilus, comprising the seven hydrophilic core subunits and a frataxin-like supernumerary subunit, shows how the subunits and cofactors are arranged (5Sazanov L.A. Hinchliffe P. Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus.Science. 2006; 311: 1430-1436Crossref PubMed Scopus (666) Google Scholar). In addition to the 14 core subunits, eukaryotic complexes I contain a variable number of supernumerary subunits also. The roles of most of the supernumerary subunits are not well defined, although specific functions for some of them have been proposed (6Hirst J. Carroll J. Fearnley I.M. Shannon R.J. Walker J.E. The nuclear encoded subunits of complex I from bovine heart mitochondria.Biochim. Biophys. Acta. 2003; 1604: 135-150Crossref PubMed Scopus (333) Google Scholar). Twenty-one supernumerary subunits are common to all eukaryotic complexes I, but additional subunits vary between fungi, plants, and animals, as well as between individual species (7Gabaldón T. Rainey D. Huynen M.A. Tracing the evolution of a large protein complex in the eukaryotes, NADH:ubiquinone oxidoreductase (complex I).J. Mol. Biol. 2005; 348: 857-870Crossref PubMed Scopus (191) Google Scholar). The most intensively characterized eukaryotic complex I is the 1 MDa complex from Bos taurus that contains 45 dissimilar subunits, all with homologues in the human genome (6Hirst J. Carroll J. Fearnley I.M. Shannon R.J. Walker J.E. The nuclear encoded subunits of complex I from bovine heart mitochondria.Biochim. Biophys. Acta. 2003; 1604: 135-150Crossref PubMed Scopus (333) Google Scholar, 8Carroll J. Fearnley I.M. Skehel J.M. Shannon R.J. Hirst J. Walker J.E. Bovine complex I is a complex of 45 different subunits.J. Biol. Chem. 2006; 281: 32724-32727Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 9Carroll J. Fearnley I.M. Shannon R.J. Hirst J. Walker J.E. Analysis of the subunit composition of complex I from bovine heart mitochondria.Mol. Cell. Proteomics. 2003; 2: 117-126Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 10Murray J. Zhang B. Taylor S.W. Oglesbee D. Fahy E. Marusich M.F. Ghosh S.S. Capaldi R.A. The subunit composition of the human NADH dehydrogenase obtained by rapid one-step immunoprecipitation.J. Biol. Chem. 2003; 278: 13619-13622Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The subunit composition of the complexes I from two fungi, Neurospora crassa and Yarrowia lipolytica, have been analyzed previously. Thirty-nine subunits have been identified in N. crassa complex I (11Marques I. Duarte M. Assunção J. Ushakova A.V. Videira A. Composition of complex I from Neurospora crassa and disruption of two “accessory” subunits.Biochim. Biophys. Acta. 2005; 1707: 211-220Crossref PubMed Scopus (52) Google Scholar), and four of them do not have mammalian homologues. The mitochondrial genome sequence of Y. lipolytica was published in 2001 (12Kerscher S. Durstewitz G. Casaregola S. Gaillardin C. Brandt U. The complete mitochondrial genome of Yarrowia lipolytica.Comp. Funct. Genom. 2001; 2: 80-90Crossref PubMed Scopus (52) Google Scholar) and the first assessment of the subunit composition of complex I from Y. lipolytica in 2002 described 37 subunits, identified by a combination of SDS-PAGE, MALDI-TOF mass spectrometry and Edman degradation (13Abdrakhmanova A. Zickermann V. Bostina M. Radermacher M. Schägger H. Kerscher S. Brandt U. Subunit composition of mitochondrial complex I from the yeast Yarrowia lipolytica.Biochim. Biophys. Acta. 2004; 1658: 148-156Crossref PubMed Scopus (77) Google Scholar). Subsequently, three additional subunits were described, and, in 2008, the masses of a set of subunits were measured using laser-induced liquid bead ion desorption MS. The accuracy of the molecular mass data was variable, but observed masses were paired with predicted masses within 130 Da (14Morgner N. Zickermann V. Kerscher S. Wittig I. Abdrakhmanova A. Barth H.-D. Brutschy B. Brandt U. Subunit mass fingerprinting of mitochondrial complex I.Biochim. Biophys. Acta. 2008; 1777: 1384-1391Crossref PubMed Scopus (69) Google Scholar). Thus, 40 subunits have been identified in Y. lipolytica complex I; 37 of them are found in N. crassa, and 35 in B. taurus. Pichia pastoris is a methylotrophic ascomycete, which is a commonly used over-expression host for recombinant proteins. The purification and characterization of complex I from P. pastoris, and the related species Pichia angusta, have been described previously (15Bridges H.R. Grgic L. Harbour M.E. Hirst J. The respiratory complexes I from the mitochondria of two Pichia species.Biochem. J. 2009; 422: 151-159Crossref PubMed Scopus (21) Google Scholar). Here we present a comprehensive analysis of the protein composition and post-translational modifications of complex I from P. pastoris, using a combination of proteomic and bioinformatic approaches. Proteomic techniques provide a rapid and efficient means of defining the composition of protein complexes. Here the subunits of P. pastoris complex I were resolved either by reverse-phase HPLC or by SDS-PAGE, and then analyzed by peptide mass fingerprinting and tandem mass spectrometry of tryptic peptides, by MALDI-TOF mass spectrometry. The analysis was complicated by the presence of hydrophobic membrane proteins, and by the presence of small subunits which produce few or no proteolytic fragments. After reverse-phase separation, the subunits were also analyzed by ESI-MS, and their molecular masses provided additional information on the subunit composition and amino acid sequences, and on stable post-translational modifications. Forty-one different proteins have been identified in complex I from P. pastoris, including three proteins (named NUSM, NUTM, and NUUM) that have not been detected previously in any species of complex I. Complex I was isolated from P. pastoris strain X-33, and from Y. lipolytica strain GB10, as described previously (15Bridges H.R. Grgic L. Harbour M.E. Hirst J. The respiratory complexes I from the mitochondria of two Pichia species.Biochem. J. 2009; 422: 151-159Crossref PubMed Scopus (21) Google Scholar, 16Kashani-Poor N. Kerscher S. Zickermann V. Brandt U. Efficient large scale purification of his-tagged proton translocating NADH:ubiquinone oxidoreductase (complex I) from the strictly aerobic yeast Yarrowia lipolytica.Biochim. Biophys. Acta. 2001; 1504: 363-370Crossref PubMed Scopus (74) Google Scholar, 17Dröse S. Galkin A. Brandt U. Proton pumping by complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica reconstituted into proteoliposomes.Biochim. Biophys. Acta. 2005; 1710: 87-95Crossref PubMed Scopus (40) Google Scholar). SDS-PAGE of 10-20 µg of protein was performed on 18-22% acrylamide gradient Laemmli-style gels (18Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar) for 4 h at 300 mV, and stained with 0.2% Coomassie Blue R250. For reverse-phase HPLC, complex I was precipitated by 20 volumes of cold ethanol, centrifuged at 16,000 × g for 3 min, and the supernatant, containing detergents and lipids, was discarded. The pellet was dissolved in 60% (v/v) formic acid, 15% (v/v) trifluoroethanol, and 1% (v/v) hexafluoroisopropanol, and injected onto a PLRP-S reverse phase HPLC column (1 mm i.d. × 75 mm, Varian Inc., Palo Alto, CA). Proteins were eluted at 50 µl min−1 in a linear gradient of 0-70% propan-2-ol and 15-20% trifluoroethanol, in 1% hexafluoroisopropanol and either 50 mm ammonium formate (pH 3.1) or 0.1% trifluoroacetic acid (pH 1.8) (19Carroll J. Fearnley I.M. Wang Q. Walker J.E. Measurement of the molecular masses of hydrophilic and hydrophobic subunits of ATP synthase and complex I in a single experiment.Anal. Biochem. 2009; 395: 249-255Crossref PubMed Scopus (30) Google Scholar). Protein bands were excised from the SDS-PAGE gels, diced into small cubes (approximately 1 mm3), and digested by trypsin as described previously (20Shevchenko A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of proteins from silver stained polyacrylamide gels.Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7831) Google Scholar). The tryptic peptides were analyzed by peptide mass fingerprinting and tandem MS using an Applied Biosystems/MDX SCIEX 4800 Plus MALDI TOF/TOF mass spectrometer, with α-cyano-4-hydroxy cinnamic acid as the matrix. Spectra were calibrated using the trypsin autolysis peptides at 2163.057 and 2273.160 Da, and a calcium-associated matrix ion at 1060.048 Da. Peptide sequences were obtained using the same spectrometer, by collision-induced dissociation, at a collision energy of 1 kV. Typically, tandem MS spectra contained data accumulated from 2,500 laser pulses. Fractions from HPLC were dried in a speed-vac centrifuge, then dissolved in 0.2% (w/v) ammonium bicarbonate, 0.5 mm CaCl2, and 12 ng/ml trypsin, and digested overnight at 37 °C. MALDI-TOF mass spectra were smoothed and monoisotopically labeled using the 4000 Data Explorer software (version 3.5.3, Applied Biosystems). Monoisotopic peak lists were generated using the peaks-to-mascot feature, with a minimum signal-to-noise ratio of 10, and a peak density of 10 (occasionally 15) peaks per 200 Da. Known peaks from human keratin, trypsin autolysis, and matrix associated ions were excluded (see Supplementary Information). Then, the peak lists were compared with the in-house P. pastoris protein sequence database (5,209 sequences, see below), or to the online National Center for Biotechnology Information (NCBInr) database (version 20080912 comprising 7,031,513 sequences) using the Mascot (Matrix Science Ltd., London, UK) search algorithm, version 2.1.0 (21Perkins D.N. Pappin D.J.C. Creasy D.M. Cottrell J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data.Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6814) Google Scholar). The significance threshold for peptide identification was p < 0.05, and a single missed cleavage was allowed. The mass tolerances were 70 ppm for peptide mass fingerprinting and 0.8 Da and 120 ppm for tandem MS. N-terminal acetylation and formylation, cysteine propionamidation, and methionine oxidation were selected as variable modifications in initial searches. Error tolerant searches were also performed to identify additional matches that rely on amino acid substitutions or further artifactual or unusual modifications. Fractions from reverse-phase HPLC were analyzed by ESI-MS in positive ion mode, either online with a Quattro Ultima triple quadrupole mass spectrometer (Waters-Micromass, Waters Corp., Milford, MA) scanning 700–2100 m/z every 5 s, or offline using a Q-TOF1 mass spectrometer (Waters-Micromass). Samples were loaded into the Q-TOF1 by flow injection into 50% isopropanol, and both spectrometers were calibrated with myoglobin and trypsinogen over the mass/charge range of 616 to 2181. Protein molecular masses were determined from series of multiply charged ions, using the component analysis function of MassLynx, version 3.4 (Waters-Micromass). The genome sequence of P. pastoris strain X-33 was obtained from Integrated Genomics Inc. (Chicago, IL) in flat file format, and comprised 13 contiguous segments (contigs): 12 nuclear contigs and the mitochondrial genome sequence. A set of approximately 5,000 ORFs and their translated protein sequences were obtained also. Consequently, a genomic DNA sequence database and a protein sequence database were assembled in FASTA format, and tblastn and blastp were used, respectively, to identify the sequences of known complex I subunits (NCBI BLAST v 2.2.16, statistical E-value threshold 1 × 10−10) (22Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar). Comparisons were performed with the amino acid sequences of the subunits of Y. lipolytica complex I plus two subunits (10.4 kDa and NURM) specific to N. crassa complex I (14Morgner N. Zickermann V. Kerscher S. Wittig I. Abdrakhmanova A. Barth H.-D. Brutschy B. Brandt U. Subunit mass fingerprinting of mitochondrial complex I.Biochim. Biophys. Acta. 2008; 1777: 1384-1391Crossref PubMed Scopus (69) Google Scholar), and 10 additional subunits from B. taurus complex I (8Carroll J. Fearnley I.M. Skehel J.M. Shannon R.J. Hirst J. Walker J.E. Bovine complex I is a complex of 45 different subunits.J. Biol. Chem. 2006; 281: 32724-32727Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). Twenty-seven putative complex I subunits, homologous to known nuclear-encoded subunits from Y. lipolytica, were identified in the P. pastoris protein sequence database (although the ORF lengths for NUEM and NUKM required adjusting to produce proteins similar in size to their Y. lipolytica homologues, and a single base deletion was corrected in the C-terminal region of NUAM). A further five putative nuclear-encoded sequences were identified in the DNA sequence database, and were translated, along with their flanking regions, using the NCBInr ORF finder tool (ORFFINDER) (23Sayers E.W. Barrett T. Benson D.A. Bryant S.H. Canese K. Chetvernin V. Church D.M. DiCuccio M. Edgar R. Federhen S. Feolo M. Geer L.Y. Helmberg W. Kapustin Y. Landsman D. Lipman D.J. Madden T.L. Maglott D.R. Miller V. Mizrachi Il Ostell J. Pruitt K.D. Schuler G.D. Sequeira E. Sherry S.T. Shumway M. Sirotkin K. Souvorov A. Starchenko G. Tatusova T.A. Wagner L. Yaschenko E. Ye J. Database resources of the National Center for Biotechnology Information.Nucleic Acids Res. 2009; 37: D5-D15Crossref PubMed Scopus (738) Google Scholar). ORFs for proteins of similar size to the Y. lipolytica homologues were identified for two sequences, and single introns were predicted in the remaining three (NI9M, NB5M, and NIDM) using the prediction program AUGUSTUS (24Stanke M. Keller O. Gunduz I. Hayes A. Waack S. Morgenstern B. AUGUSTUS: ab initio prediction of alternative transcripts.Nucleic Acids Res. 2006; 34: W435-W439Crossref PubMed Scopus (1125) Google Scholar) with the organism set as Debaryomyces hansenii. The five additional sequences were added to the in-house P. pastoris protein sequence database. No homologues to the Y. lipolytica NUNM subunit, the N. crassa 10.4 kDa and NURM subunits, or the 10 subunits considered specific to mammalian complex I were identified. Finally, ORFFINDER was used to translate the P. pastoris mitochondrial genome, using both the “yeast” and the “mold, protozoan and coelenterate” codes (the yeast code is used by, for example, Saccharomyces cerevisiae (25Bonitz S.G. Berlani R. Coruzzi G. Li M. Macino G. Nobrega F.G. Nobrega M.P. Thalenfeld B.E. Tzagoloff A. Codon recognition rules in yeast mitochondria.Proc. Natl. Acad. Sci. U.S.A. 1980; 77: 3167-3170Crossref PubMed Scopus (220) Google Scholar) and Kluveromyces thermotolerans (26Talla E. Anthouard V. Bouchier C. Frangeul L. Dujon B. The complete mitochondrial genome of the yeast Kluyveromyces thermotolerans.FEBS Lett. 2005; 579: 30-40Crossref PubMed Scopus (30) Google Scholar) and the mold code by, for example, N. crassa (27Browning K.S. RajBhandary U.L. Cytochrome oxidase subunit III gene in Neurospora crassa mitochondria.J. Biol. Chem. 1982; 257: 5253-5256Abstract Full Text PDF PubMed Google Scholar) and Y. lipolytica (12Kerscher S. Durstewitz G. Casaregola S. Gaillardin C. Brandt U. The complete mitochondrial genome of Yarrowia lipolytica.Comp. Funct. Genom. 2001; 2: 80-90Crossref PubMed Scopus (52) Google Scholar)). Both codes produced viable sequences for the seven expected subunits of complex I, which were identified by comparison with the NCBInr database and added to the in-house P. pastoris database. In summary, 39 putative subunits of P. pastoris complex I (seven nuclear core subunits, seven mitochondrial core subunits, and 25 supernumerary subunits) were identified. All the DNA and protein sequences for the P. pastoris complex I subunits have been deposited in the European Molecular Biology Laboratory (EMBL) database and the respective accession numbers, along with the amino acid sequences, are presented in the Supplementary Information. Fig. 1 shows an analysis of complex I from P. pastoris by SDS-PAGE. Each band was excised from the gel, digested with trypsin, and the products were analyzed by peptide mass fingerprinting and tandem mass spectrometry. The unstained regions between the bands were analyzed also because the hydrophobic subunits of complex I do not stain intensively with Coomassie Blue. The data were compared against the in-house P. pastoris protein sequence database and the data are included in Table I and presented fully in Table S1. Thirty-four of the 39 predicted subunits were detected by peptide mass fingerprinting, and 35 were detected by tandem MS. All seven nuclear encoded core subunits were detected, and five of the seven mitochondrial encoded subunits; NU6M and NULM were not detected because, irrespective of the mitochondrial translation code used, their sequences do not contain any tryptic peptides with masses between 700 and 3,000 Da. Significantly more peptides were matched to sequences produced by translation with the mold mitochondrial genetic code (see Table I) than with the yeast mitochondrial code. In particular, subunit NU2M was only identified when the mold genetic code was used, and only the mold code explains a peptide mass and sequence data from subunit NU5M, corresponding to the peptide LIYYTFLNNPNSPK (ATA is translated as Ile by the mold code (underlined), and as Met by the yeast code). Twenty-three of the 25 predicted supernumerary subunits were detected; the ST1 and ACPM2 subunits were not. Two additional proteins, that have been named NUSM and NUUM and which are not similar to any known subunits of complex I, were detected also. Note that the preparation of complex I described here is highly pure: peptide mass fingerprinting and tandem MS analyses sporadically detected only four additional proteins: plasma membrane H+– adenosine triphosphatase, band 7 stomatin protein, 40s ribosomal subunit and a GatB/YeqY superfamily protein (genbank entries 254565045, 254569368, 254566987, and 254566543, respectively, from P. pastoris GS115). All four proteins already have known functions, are present in only low amounts (they were not visible in SDS-PAGE) and were not detected by molecular mass measurements, so they are considered low-level impurities.Table IA summary of peptide mass fingerprinting and tandem MS data for the 41 proteins identified in complex I from P. pastorisSubunitNumber of residuesPeptide mass fingerprintingTandem MSNumber of peptides1Sequence coverageScoreNumber of peptidesSequence coverageScore1. NUAM/75 kDa70254 (98)77%2982252%17952. NUBM/51 kDa46217 (11)47%1751437%7673. NUCM/49 kDa45120 (11)58%2501957%13284. NUGM/30 kDa25118 (15)80%2461875%12135. NUHM/24 kDa2159 (14)41%109942%4676. NUIM/TYKY222211 (23)41%1091253%7457. NUKM/PSST220412 (8)45%132632%4958. NU1M/ND13534 (28)7%1728%869. NU2M/ND25233 (24)6%1825%13210. NU3M/ND31412 (0)17%43217%8311. NU4M/ND434913 (24)7%1515%4812. NU5M/ND56426 (15)10%35610%26513. NU6M/ND64161––––––14. NULM/ND4L482––––––15. NUEM/39 kDa37722 (16)71%2911862%152616. NESM/ESSS2154 (15)27%23848%87017. NUJM/B14.721512 (45)55%114736%35818. NUXM19212 (13)65%1781063%56019. NUPM/PGIV1839 (8)57%107846%87820. NUZM18119 (12)84%2771269%91121. NUSM15412 (17)76%173761%52122. NIMM/MWFE15016 (14)62%1961052%59323. NB6M/B16.614610 (16)63%1411158%65024. N7BM/B17.21399 (16)53%147645%51325. NUYM/18 kDa1399 (20)67%108862%64426. NIAM/ASHI1327 (17)45%75643%30027. NUMM/13 kDa1288 (22)71%111966%51928. NUFM/B131266 (15)49%78658%61629. NB4M/B141217 (21)61%106656%43130. NI2M/B2211011 (19)78%165759%39331. NIPM/15 kDa10511 (17)75%145863%50632. ACPM1/SDAP15,692327%–327%33233. NIDM/PDSW915 (24)59%84448%27334. NI8M/B8896 (24)61%85556%39735. ACPM2/SDAP26,788220%–220%10036. NB8M/B18866 (18)65%67658%33437. NUTM585228%20327%5838. NB5M/B15808 (22)62%153536%26839. NI9M/B9785 (17)43%64326%33640. NUUM733 (18)54%40360%16541. NB2M/B12605 (17)31%68345%147The scores for peptide mass fingerprinting and tandem MS are Mascot scores, and the sum of the ions scores for matching peptides, respectively. The individual peptides and their scores for tandem MS, including error tolerant searches are presented in Table S1. Scores of 50 for peptide mass fingerprinting and 23 for tandem MS were required for p < 0.05% (for comparison against the P. pastoris database). Number of residues and sequence coverage are based on the mature sequences; the supernumerary subunits are listed in order of the sequence length. The number of peptides is given alongside the number of unmatched masses (in brackets).Mature protein sequence is not known, number of residues and sequence coverage are based on the complete sequence.See Supplementary Information for peptide mass fingerprints and tandem MS spectra.No peptides were identified.Only observed following protein separation by reverse phase HPLC.Peptide mass fingerprinting data alone were insufficient for protein identification.Length of mature protein inferred from homology with other species. Open table in a new tab The scores for peptide mass fingerprinting and tandem MS are Mascot scores, and the sum of the ions scores for matching peptides, respectively. The individual peptides and their scores for tandem MS, including error tolerant searches are presented in Table S1. Scores of 50 for peptide mass fingerprinting and 23 for tandem MS were required for p < 0.05% (for comparison against the P. pastoris database). Number of residues and sequence coverage are based on the mature sequences; the supernumerary subunits are listed in order of the sequence length. The number of peptides is given alongside the number of unmatched masses (in brackets).Mature protein sequence is not known, number of residues and sequence coverage are based on the complete sequence.See Supplementary Information for peptide mass fingerprints and tandem MS spectra.No peptides were identified.Only observed following protein separation by reverse phase HPLC.Peptide mass fingerprinting data alone were insufficient for protein identification.Length of mature protein inferred from homology with other species. To evaluate the completeness of our analysis, complex I from Y. lipolytica was analyzed alongside that from P. pastoris, by comparison of mass spectrometry data with the online NCBInr database. Thirty-six of the 40 known subunits were detected. The second acyl carrier protein (ACPM2) and the mitochondrial encoded subunits NU2M, NU6M, and NULM were not detected; NU6M and NULM do not produce tryptic peptides with masses within the range analyzed. Fig. 2 shows a typical reverse-phase chromatogram for the fractionation of the subunits of P. pastoris complex I. The reverse-phase procedure is compatible with the recovery of intrinsic membrane proteins, and separates both hydrophilic and hydrophobic components in a form suitable for ESI MS (19Carroll J. Fearnley I.M. Wang Q. Walker J.E. Measurement of the molecular masses of hydrophilic and hydrophobic subunits of ATP synthase and complex I in a single experiment.Anal. Biochem. 2009; 395: 249-255Crossref PubMed Scopus (30) Google Scholar). The hydrophilic proteins elute first, and the highly hydrophobic proteins elute toward the end of the gradient. The eluting proteins were either collected manually and their intact molecular masses were analyzed by ESI MS, or the eluant from the column was analyzed online using ESI MS. In some experiments, portions of each fraction were digested with trypsin and analyzed using peptide mass fingerprinting and tandem MS to determine the order in which the proteins elute, and to correlate molecular masses with specific subunits. The combined data from all ESI-MS experiments are summarized in Table II. Note that, for some of the larger proteins, the peaks in the ESI spectra were broad; therefore, precise measurements were difficult.Table IICalculated and observed protein masses of complex I subunits from P. pastoris and their corresponding post-translational modificationsSubunitMas" @default.
• W2136145101 created "2016-06-24" @default.
• W2136145101 creator A5031090925 @default.
• W2136145101 creator A5031647363 @default.
• W2136145101 creator A5056046836 @default.
• W2136145101 date "2010-10-01" @default.
• W2136145101 modified "2023-10-18" @default.
• W2136145101 title "The Subunit Composition of Mitochondrial NADH:Ubiquinone Oxidoreductase (Complex I) From Pichia pastoris" @default.
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