MassIVEapplication/xmlftp://massive-ftp.ucsd.edu/v01/MSV000082409/primary200OK0000Maurizio Roncihttps://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=d3b4ae451f8c49468675513ebff2624dmaurizio.ronci@unich.itMassIVE192UNIMOD:21 - "Phosphorylation."UNIMOD:4 - "Iodoacetamide derivative."UNIMOD:7 - "Deamidation."UNIMOD:1 - "Acetylation."UNIMOD:35 - "Oxidation or Hydroxylation."ProteomicsOrbitrap FusionHomo Sapiens (ncbitaxon:9606)University G. d'AnnunzioMitochondria are undeniably the cell powerhouse, directly affecting cell survival and fate. Growing evidence suggest that mitochondrial protein repertoire affects metabolic activity and plays an important role in determining cell proliferation/differentiation or quiescence shift. Consequently, the bioenergetic status of a cell is associated with the quality and abundance of the mitochondrial populations and proteomes. Mitochondrial morphology changes in the development of different cellular functions associated with metabolic switches. It is therefore reasonable to speculate that different cell lines do contain different mitochondrial-associated proteins, and the investigation of these pools may well represent a source for mining missing proteins (MPs). A very effective approach to increase the number of IDs through mass spectrometry consists of reducing the complexity of the biological samples by fractionation. The present study aims at investigating the mitochondrial proteome of five phenotypically different cell lines, possibly expressing some of the MPs, through an enrichment-fractionation approach at the organelle and protein level. We demonstrate a substantial increase in the proteome coverage, which, in turn, increases the likelihood of detecting low abundant proteins, often falling in the category of MPs, and resulting, for the present study, in the identification of METTL12, FAM163A, and RGS13. All MS data have been deposited to the MassIVE data repository ( https://massive.ucsd.edu ) with the data set identifier MSV000082409 and PXD010446.Sequential Fractionation Strategy Identifies Three Missing Proteins in the Mitochondrial Proteome of Commonly Used Cell Lines.Ronci Maurizio M, Pieroni Luisa L, Greco Viviana V, Scotti Luca L, Marini Federica F, Carregari Victor C VC, Cunsolo Vincenzo V, Foti Salvatore S, Aceto Antonio A, Urbani Andrea A0Activity, Product, Metabolisms, postnatal development, Bioenergetics, histology, growth and development, protein, Organelle, Viabilities, Mitochondrial Contractions, Cell Growth in Number, dmTAF[[II]]230, School-Age, protein polypeptide chains, Roles, Biological, Cell Number Growth, Line, Falls, Concepts, Analysis, Expenditure, Biological Product, protein aggregate, anatomy and histology, Viability, Mass Spectrum Analysis, Growth, me75, Biologic Drugs, TFIID TAF250, Analyses, cel, FATE, Natural, Moods, Accidental Fall, proteins, Biological Drugs, D17Mit170, T1, Biological Medicine, Role Concepts, Medicine, Medicines, Biologic Drug, Data Set., dTAF[[II]]230, Biologic Products, TAF200, Mitochondrial Protein, TAFII-250, Ids, TAF250/230, Tl3, Tl2, Spectrum Analysis, Accidental, Spectroscopy, TAFII250, Role Concept, ATP synthase D chain, Playthings and Play, School Age, Biopharmaceuticals, CT43, Role, Plaything, Cellular, Biologic Product, Slip and Fall, NDSP, Lines, Populations, Mitochondrial, Biological Medicines, Biological Drug, Toys, Spectrometry, Biologics, CG17603, TAF[[II]], RP11-12M5.2, Energy Metabolisms, Taf250, SR3-5, Proliferation, A230106N23Rik, Cell Number, G0 phase, School-Age Population, Falling, Proteomes, TAF230, d230, Plays, MPS2, Biologic Pharmaceuticals, MPS, Gene, dTAFII250, Spectrum Analyses, Fall and Slip, protein-containing complex, EfW1, mitochondrial, morphology, polypeptide chain, dmTAF1, Taf230, Metabolism, Survival, Mitochondrion, Gene Products, Mass, Mood, Low, Number Growth, RGD1565760, U99HG, Mass Spectroscopy, TAF250, Toy, Biologicals, Drugs, School-Age Populations, study, SIDS, Taf200, C1orf76, anatomy, dTAF[[II]]250, Playthings, cell, Contraction, Natural Product, Energy, Taf1p, Expenditures, Population, dTAF250, cell proliferation, Puppets, Play, School Age Population, TAF, Puppet, Biologic, Pharmaceuticals, Multiplication, Products, TAF[[II]]250, Cellular Proliferation, cou, Biologic Medicines, protein complex, Affects, Proteins, cell cycle quiescence, Cell Lines, total expressed protein, l(3)84Ab, BG:DS00004.13, Cell, dTAF230, Concept, Cell Viabilities, development, Contractions, MS, Lr, native protein, natural protein, p230, Protein, Biopharmaceutical, TAF[[II]]250/230, TFIID, Fall, Mass Spectrum Analyses, Energy Expenditure, Taf[[II]]250, Mass Spectrum, TAF[[II]]230, postnatal growth, Energy Expenditures, TAF[II]250, School Age Populations, Bioenergetic, Mitochondrial Contraction, Protein Gene Products, Drug, Gene Proteins, mitochondria, DmelCG17603, Cell Multiplication, Bra, Natural Products, Cell Viability, growth, General activity, TAF1Bru, IPP2A2, posttranslational modification, E 920, Meth, Raw, Hgfr, AI326280, OHO31, cleavage, 5730555F13Rik, Sialic acid-binding Ig-like lectin 1, protein, Organelle, phosphorylation, Kap-alpha2, Mutations, 5730420M11Rik, peptide, std, Cut, Polypeptides, protein polypeptide chains, srybeta, pen-2, peptido, l(1)7Ba, HGFR, l(1)7Bb, skc35, SPH3, Line, sry-beta, NEST:bs27c08, 2-amino-3-mercaptopropanoic acid, Cystein, CK1-gamma, Analysis, impalpha2, protein aggregate, ms(3)89B, WMS, Mass Spectrum Analysis, Svc, SER, sry alpha, SET, C, posttranslational amino acid modification, DmelCG6127, l(2)144/1, peptides, HPP, Analyses, TAF-I, MUB3_18, K, cisteina, M, 2-amino-3-(4-hydroxyphenyl)propanoic acid, 3-(p-Hydroxyphenyl)alanine, hypoplasia, epsilon-diaminocaproic acid, Dimp-alpha2, proteins, MUB3.18, Y, SN, CG11387, EG:196F3.2, imp-alpha2, THR, Thr, DmelCG4299, ser, Bd, IGAAD, set, c, childhood, LYS, Lys, DmelCG10574, Ice, EL2, ppm, Tyr, Lysin, Sa, CT, RG7MT1, GPHYSD2, Racemethionine, spider, SGS, Lzm, Diagnostic Findings, phapii, SIGNS SYMPTOMS, Lzp, oho-31, proto-oncogene c-Met, bs29g06.y1, HS3, PRO, phosphoethanol-aminuria, DESC, alphaKap2, acetylation, Ct, 2-Amino-4-(methylthio)butyric acid, 2-Amino-3-(p-hydroxyphenyl)propionic acid, StF-IT-1, Mitochondrial Protein, Search, 9130215G10Rik, CYS, Cys, Spectrum Analysis, IMPalpha2, ACMICD, Spectroscopy, CK1gamma, pyropoikilocytosis hereditary, CKIgamma, Methionin, Lzm-s1, Hmet, BcDNA:GH10590, CG14784, (2R)-2-amino-3-sulfanylpropanoic acid, fixed, and GLY protein 2, END, OCAIA, anon-WO0140519.258, Lines, Crystal, CG14783, HGF receptor, HGF/SF receptor, SHEP3, parent ion, Mitochondrial, and GLY protein 1, CK1, HLA-DR-associated protein II, PTM, DI-2, I-2Dm, Srybeta, Spectrometry, MASS, CG4299, alpha, hCMT1c, oho31, study protocol, c-Met, beta Trypsin, Sheep erythrocyte receptor, L-Cystein, I-2PP1, Speed, Lysm, CG17957, Hrr25, TAF-IBETA, precursor ion, post-translational modification, Biocatalyst, CG6127, TAF-Ibeta, Sry alpha, Polypeptide, Gish, DmelCG32810, Spider, i2pp2a, Clinical Finding, Rch1, Oho31, childhood hypophosphatasia, criteria, l(1)VE614, 2-amino-4-(methylsulfanyl)butanoic acid, Biocatalysts, Dalpha2, L-Zystein, CG4799, Goal, FBN, Gene, sryalpha, precursor, Spectrum Analyses, pen, hypophospatasia, guidelines, protein-containing complex, Chalk, PHAPII, Symptoms and Signs, importin alpha2/pendulin, method, HGF, reduced, polypeptide chain, ECTOL1, mel(3)8, DPend, method used in an experiment, Tina, Gene Products, Mass, smooth ER, l(2)k14401, F15E12.6, Del(8)44H, tiny, RCCP2, Finding, CK1[[gamma]], alpha2, Mass Spectroscopy, Search Engines, F15E12_6, Glass, CKI-related, Sryalpha, Siglec-1, scatter factor receptor, E-920, sry beta, ipp2a2, beta-Trypsin, imp alpha2, Acetylations, 2pp2a, albino, Hcys, posttranslational protein modification, kf, alpha2A-Kap, CG10574, AUTS9, Solution, OCTD, DmelCG5785, Enzyme, 2PP2A, 2-amino-4-(methylthio)butanoic acid, DmelCG11387, 10^[-6], Imp-alpha2, CYSTEINE, taf-ibeta, CMM8, Kpna2, dSET, dSet, CG6963, FREE CYSTEINE, peptidos, 2.1, AI838057, Tyrosin, (2R)-2-amino-3-mercaptopropanoic acid, metionina, CG7938, CD169, HHT1, anch, small, hypophosphatasia mild, Gene., Lyzs, Edg, DL-Methionine, protein complex, Proteins, igaad, srya, Cell Lines, Phosphorylations, Cell, Peptide, Engine, sryb, group, polypeptide, Signs and Symptoms, OCA1A, DmelCG4799, MS, native protein, natural protein, I-2PP2A, tirosina, SPTA, frag1, Protein, Dm I-2, I2PP2A, MFS1, Zystein, Mass Spectrum Analyses, WMS2, ATCMPG1, MET, Met, ATCMPG2, Mass Spectrum, sry-a, sry-b, PRSS, Col4a-1, DmelCG6963, underdeveloped, ensemble, 2-amino-3-sulfanylpropanoic acid, tyrosine-protein kinase Met, DmelCG7938, Tripcellim, hereditary pyropoikilocytosis, Kap alpha2, L-2-Amino-3-mercaptopropionic acid, CG5785, ana, (R)-2-amino-3-mercaptopropanoic acid, Rpw, 2-Amino-3-mercaptopropionic acid, post-translational amino acid modification, Protein Gene Products, plan specification, Gene Proteins, Trypure, sry-alpha, dSET/TAF-Ibeta, 2610030F17Rik, CT18858, anon-WO0118547.425, SSKS, alpha-amino-gamma-methylmercaptobutyric acid, E920, Peptid, Par4, variable, DmelCG17957, ORW1, AA407739, 5730455C01Rik, CG32810, sry, SF receptorProtein Gene Products, Gene Proteins, Mitochondrial, ATP synthase D chain, Protein, Gene Products, Proteins, Line, Cell Lines, total expressed protein, Cell., Gene, mitochondrial, Proteomes, Cell, LinesProtein Gene Products, Gene Proteins, Mitochondrial, ATP synthase D chain, Protein, Gene Products, Proteins, Line, Cell Lines, total expressed protein, Cell., Gene, mitochondrial, Proteomes, Cell, LinesPXD010446trueSequential Fractionation Strategy Identifies Nine Missing Proteins in The Mitochondrial Proteome of Commonly Used Cell LinesIdentification of mitochondrial proteins with a bottom up approach after organelle enrichment and 1D PAGE separation for 5 different cell lines. The bands were cut and the proteins reduced, alkylated and digested with trypsin according to a conventional protocol. Acquisition were performed on the Orbitrap Fusion Tribrid mass spectrometer in top speed mode with 3 seconds cycles. Raw data were processed using PEAKS Studio 7.5 (Bioinformatics Solutions Inc.) and searched using the PEAKS search engine and the SPIDER peptide mutation and homology search tool against neXtProt (July 2017; 42,151 total entries). Parent Mass Error Tolerance was set to 10.0 ppm and Fragment Mass Error Tolerance to 0.6 Da. Other search parameters were trypsin enzyme specificity, two missed cleavages per peptide, fixed Carbamidomethylation of Cys and variable Oxidation of Met, Deamidation of Gln and Asn (NQ), Phosphorylation of Ser, Thr and Tyr and Acetylation of Lys with two variable PTM per peptide. Non-specific cleavage was allowed to only one end of the peptide. FDR estimation was enabled, and precursor options corrected. To follow the HPP Mass Spectrometry Data Interpretation Guidelines Version 2.1 and in view of our goal of finding missing proteins, we set the FDR threshold on PSMs to 2%, typically resulting in FDR on peptides lower than 5% and we filtered out all the proteins identified with only one peptide, resulting in a FDR at the protein level of 0.0%.Mon May 28 01:33:00 BST 2018MSV00008240930284448