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ImanishiMass SpectrometryShotgun proteomicsNot availableHelaSimulated spectral libraryPhosphopeptideLc-ms/msSynthetic peptidehttp://www.ebi.ac.uk/pride/archive/projects/PXD000474Hela CellHuman HeLa cell proteins were digested with trypsin and enriched for phosphopeptides using TiO2. Half of the enriched sample was dephosphorylated by alkaline phosphatase treatment. Both samples were analyzed by LC-MS/MS in duplicate runs using an LTQ Orbitrap Velos instrument. MS and MS/MS spectra were acquired in profile and centroid modes of the Orbitrap, respectively. Four fragmentation techniques were used: ETD, ion trap CID, MSA, and HCD (beam-type CID). Also, 20 synthetic phosphopeptides were mixed into 2 groups (10 peptides each), where phosphopeptide isoforms were separated from each other. Both the mixtures were analyzed by LC-MS/MS in triplicates, using the LTQ Orbitrap Velos with CID fragmentation (MS in profile mode of Orbitrap and MS/MS in centroid mode of LTQ) and also using a Q Exactive instrument with HCD fragmentation (both MS and MS/MS in profile mode).PrideNot availablephosphorylated residueiodoacetamide derivatized residuemonohydroxylated residueOrbitrap and Q Exactive data files were converted to mzXML files using Trans Proteomic Pipeline (TPP) version 4.4 VUVUZELA revision 1, and then to mgf files using MzXML2Search. The HeLa data were searched with Mascot v2.4.1 against a concatenated forward-reverse SwissProt database (2010_03, Homo sapiens, total 40530 sequences). Mascot v2.2.6 was used only for creating a spectral library of simulated HeLa phosphopeptides (see below). Sequest searches were done using the SORCERER platform. FDR was estimated using the target-decoy strategy by PeptideProphet. For phosphorylation site localization, Ascore v2008.04.16f BETA was used on the Sequest results. Another localizaton tool phosphoRS v3.0 (considering neutral loss ions disabled) was used on the Mascot search results, by submitting the mgf files to Proteome Discoverer v1.4.0.288 after adjusting file formats. For the synthetic phosphopeptide data obtained using the Q Exactive, Ascore v2011.01.12a BETA was used on Sequest. PhosphoRS (the initial version implemented in the Proteome Discoverer v1.3.0.339) and phosphoRS v3.0 (the neutral loss option enabled or disabled) were used on Mascot v2.4.0, by submitting the Q Exactive raw data to Proteome Discoverer without the file conversion. Also, to evaluate PTM score, MaxQuant v1.3.0.5 was used by submitting the raw data. From the HCD spectra of dephosphorylated peptides identified by Mascot v2,2,6 with PeptideProphet minimum probability of 0.90 (0.35% FDR), spectra of singly phosphorylated peptides were computationally simulated using a newly developed program, SimPhospho. These spectra and decoy entries were used to compile a consensus spectral library (SimHeLa, total 23126 spectra), by using SpectraST (version 4.0, released beta). The HeLa phosphopeptide HCD data (mzXML) were searched against SimHeLa library by SpectraST (SimSpectraST searching). The results were validated by PeptideProphet. DeltaDot score was recalculated to discriminate alternative phosphorylation sites. The SpectraST search was repeated via Proteome Discoverer only for viewing annotated spectral matches (which covered the same matches at 1% FDR and 1% FLR, there was some difference in the scores though). Also SimHela library was comparatively evaluated before and after adding large sets of spectra. CID spectral libraries of mouse and yeast phosphopeptides (51,420 and 18,412 spectra, respectively) were downloaded from PeptideAtlas, supplemented with decoy entries, and then joined to SimHeLa library. An HCD spectral library of the 20 synthetic phosphopeptides was created. The Q Exactive raw data were converted to mzXML and then mgf files, followed by searching with Mascot v2.4.1 against the SwissProt database. All spectra correctly assigned by Mascot were used for generating consensus spectra (31 spectra, no decoy). Also, CID spectral libraries of experimentally obtained and computationally simulated human phosphopeptides (18,066 and 35,099 spectra, respectively) were downloaded from PeptideAtlas, joined together, and then supplemented with decoy entries. For spectral searching of the 20 synthetic phosphopeptides, the above-mentioned two libraries, as well as SimHeLa library with and without adding the yeast/mouse spectra, were used. For analysis of the synthetic phosphopeptide dataset generated by Marx et al. (PXD000138), a simulated spectral library was created as described above, with some modifications. The 96 Orbitrap Velos HCD raw data, which contain both the phosphopeptides and the non-phosphorylated counterparts, were converted to mzXML and then mgf files. The data were searched with Mascot v2.4.1 against the provided sequence database. PeptideProphet validated non-phosphorylated peptides with minimum probability of 0.95 were used for the phosphorylation simulation, after applying Mascot delta score of 10 as an additional cutoff. A consensus spectral library of the simulated phosphopeptides was created with decoy entries (SimMarx library, total 285,252 spectra) and used for SpectraST searching as described above. Mascot searching was repeated for phosphoRS 3 with and without the neutral loss considering, via Proteome Discoverer 1.4 after adjusting mgf file formats. To comparatively evaluate Mascot delta score, Mascot searching of the HeLa dephosphorylated data was repeated via Proteome Discoverer 1.4.ProteomicsGarry L. CorthalsLTQ Orbitrap VelosQ ExactivePARTIALTurku Centre for Biotechnology, University of Turku and Åbo Akademi University, FinlandHomo Sapiens (human)25774671 Suni V, Imanishi SY, Maiolica A, Aebersold R, Corthals GL. Confident site localization using a simulated phosphopeptide spectral library. J Proteome Res. 2015 Mar 16susima@meijo-u.ac.jpTechnicalBiomedicalMeijo UniversityWe have investigated if phosphopeptide identification and simultaneous site localization can be achieved by spectral library searching. This allows taking advantage of comparison of specific spectral features, which would lead to improved discrimination of differential localizations. For building a library, we propose a spectral simulation strategy where all possible single phosphorylations can be simply and accurately (re)constructed on enzymatically dephosphorylated peptides, by predicting the diagnostic fragmentation events produced in beam-type CID. To demonstrate the performance of our approach, enriched HeLa phosphopeptides were dephosphorylated with alkaline phosphatase and analyzed with higher energy collisional dissociation (HCD), which were then used for creating a spectral library of simulated phosphopeptides. Spectral library searching using SpectraST was performed on data sets of synthetic phosphopeptides and the HeLa phosphopeptides, and subsequently compared to Mascot and Sequest database searching followed by phosphoRS and Ascore afforded localization, respectively. Our approach successfully led to accurate localization, and it outperformed other methods, when phosphopeptides were covered by the library. These results suggest that the searching with simulated spectral libraries serves as a crucial approach for both supplementing and validating the phosphorylation sites obtained by database searching and localization tools. For future development, simulation of multiply phosphorylated peptides remains to be implemented.Confident site localization using a simulated phosphopeptide spectral library.Suni Veronika V, Imanishi Susumu Y SY, Maiolica Alessio A, Aebersold Ruedi R, Corthals Garry L GL407l(4)17, l(4)13, Gli, artificial sequence, Procedures, single-organism developmental process, region or site annotation, AW549739, Cid[Mel], Ci[D], A4, combined T cell and B cell immunodeficiency, combined T and B cell immunodeficiency, TYPE, CENP-A, phosphorylation, binding site, CENP-C, DAGA4, peptide, CenpA, Orthophosphoric-monoester phosphohydrolase (alkaline optimum), Polypeptides, l(4)102ABc, Techniques, peptido, Method, establishment and maintenance of position, Studies, establishment and maintenance of substrate location, synthetic genetic interaction (sensu inequality), MAM, SCG3, Siah, Technique, C79691, CenH3[Cid], CG13329, CenH3[CID], positional, BcDNA:RE21270, peptides, HeLa, DmelCG13329, synthetic genetic interaction defined by inequality, procedures, Lccp, X-linked combined immunodeficiency, Study, mKIAA0989, CI, Methodological Studies, Cenp-A, site, CG2125, peptidos, RCB0007, CiD, combined immunodeficiency, establishment and maintenance of substance location, Ce, data, Ci, Phosphorylations, cenH3, artificial gene, synthetic DNA, Procedure, Peptide, collisionally activated dissociation, results, LGMD2C, CID, ciD, Cid, polypeptide, development, single organism localization, CAD, Phosphopeptide, HCD, ci[D], Ci155, synthetic, ci-D, establishment and maintenance of localization, techniques, Library, region, Data Base, cenpA, DmelCG2125, positional polypeptide feature, ci155, CenH3, DMDA1, Ci/Gli, Ci/GLI, CenH3/CID, CENP-A/Cid, CENP-A/CID, INSDC_feature:misc_binding, CENPA, MASCOT, single-organism localization, Methodological, synthetic constructs, Methodological Study, congenital combined immunodeficiency, SYNTHETIC CONSTRUCT sequences, DMDA, localisation, binding_or_interaction_site, SCARMD2, artificial, Peptid, Polypeptide, CenpA/CID, Ci-155, Peptide., establishment and maintenance of cellular component location, methodology, CID/CENP-Al(4)17, Bru, l(4)13, Gli, posttranslational modification, artificial sequence, delta, ion, Raw, determination, Cid[Mel], Ci[D], Somatomedin-C, Mbp1, DELTA AND GAMMA, Aip1, AIP1, combined T cell and B cell immunodeficiency, betaTub1, contrasted, phosphorylation, Green heme-binding protein, CENP-A, binding site, CENP-C, UCRBP, peptide, Polypeptides, YIN-YANG-1, B1t, peptido, Sev-1, NADPH-dependent diaphorase, 0926/11, 9630007J19Rik, establishment and maintenance of substrate location, myd, C79691, CG13329, Svc, HEL-S-10, D, posttranslational amino acid modification, BcDNA:RE21270, DELTA, kus, beta1tub, peptides, Biliverdin reductase B, CG9277, FLR, C1, somatomedin-C, SF, T, beta-particle, Mbp-1, Kitl, Myo5, Y, Estimated, Mast cell growth factor, Lccp, DELTA1, 1t, d, CG10724, e, CI, e-, beta1Tub, IGF1, data., flr, Mechano growth factor, AI661011, Cenp-A, delta D1, RCB0007, Consensus Development, Soluble KIT ligand, Delta, house mouse, Sl, CG3619, MyoVA, CiD, IGF-I, establishment and maintenance of substance location, Ce, steel factor, BETA 56D, Ci, DL, PLATEST, Data Set, 1053/14, gyltl1b-b, T5E21.12, mouse, somatomedin, IBP1, results, collisionally activated dissociation, sKITLG, CID, ciD, Cid, Programs, single organism localization, l(1)G0108, HCD, DL1, Ionen, NF-E1, MDDGA6, mechano growth factor, mKIAA0609, FLOWERING TIME CONTROL PROTEIN FCA ALPHA, ci-D, Igf-1, AI413174, region, T5E21_12, cenpA, fg, beta-tub, Yeast, SHEP7, gyltl1b, positional polypeptide feature, Beta <eudicots>, PTM, mast cell growth factor, Ci/Gli, Ci/GLI, CenH3/CID, CENP-A/Cid, CENP-A/CID, FR, CENPA, mice, DmelCG3619, GHBP, Stem cell factor, mdc1d, expanded, FCAALL.331, MASCOT, beta56D, STAT5, DmelCG9277, GBS, 1304/03, human, CT12133, beta1-tub, beta(-), DTB2, congenital combined immunodeficiency, Gbeta5, stem cell factor, Kitlg, beta-Tub, MDC1D, SYNTHETIC CONSTRUCT sequences, KL-1, enr, enlarged, post-translational modification, i6, DL4180C, d-120J, KITLG, artificial, NADPH-flavin reductase, Mouse, Polypeptide, l(3)92Ab, beta-Tub56D, Gb, Ci-155, Platelets, betaTub, Specbeta, establishment and maintenance of cellular component location, beta1-Tubulin, big, l(3)j8C3, Clo, human being, AW549739, region or site annotation, Slf, SLF, anon-WO0118547.269, e(-), FPH2, electron, Development, combined T and B cell immunodeficiency, froggy, Gyltl1a, CenpA, l(4)102ABc, large, B-spec, betaspec, Consensus, establishment and maintenance of position, Del(8)44H, synthetic genetic interaction (sensu inequality), 1.5.1.30, Siah, CenH3[Cid], CenH3[CID], positional, betatub(56D), 1.3.1.24, 1119/09, iones, MDDGB6, Elektron, HeLa, beta[[1]] tubulin, INO80S, beta1t, DmelCG13329, synthetic genetic interaction defined by inequality, TPP, posttranslational protein modification, ions, man, Probabilities, anon-EST:fe1B3, X-linked combined immunodeficiency, mKIAA0989, BPFD#36, Con, LMPY, SPEC8, great, DmelCG10724, blz, flail, site, CG2125, dmDelta, peptidos, CG5870, BVR-B, combined immunodeficiency, beta-tubulin56D, Spec-beta, data, Dmbeta1, l(3)05151, hematopoietic growth factor KL, 1440/11, Phosphorylations, Tubulin, artificial gene, cenH3, SDR43U1, synthetic DNA, Dbv, i168, BVRB, Peptide, polypeptide, Phosphopeptide, CAD, ci[D], Mus, Ci155, chemical analysis, 0495/20, synthetic, sequence, betaSpec, establishment and maintenance of localization, b-Spec, Library, Data Base, Biliverdin-IX beta-reductase, DmelCG5870, l(1)G0074, aip1, l(1)G0198, Col4a-1, DmelCG2125, ci155, CenH3, negatron, beta[[1]]-tubulin, INSDC_feature:misc_binding, 1423/11, b spectrin, beta1, single-organism localization, E(ls)2, MVa, synthetic constructs, primary structure of sequence macromolecule, beta, post-translational amino acid modification, Tub, localisation, binding_or_interaction_site, 1485/04, Ion, beta-spec, MGF, Mgf, c-Kit ligand, BETA, Peptid, assay, SCF, CenpA/CID, Spec, CID/CENP-Aestablishment and maintenance of substance location, positional, single organism localization, localisation, binding_or_interaction_site, positional polypeptide feature, establishment and maintenance of cellular component location., region or site annotation, INSDC_feature:misc_binding, establishment and maintenance of position, site, establishment and maintenance of localization, establishment and maintenance of substrate location, single-organism localization, Library, region, binding siteestablishment and maintenance of substance location, positional, single organism localization, Library., localisation, binding_or_interaction_site, positional polypeptide feature, region or site annotation, INSDC_feature:misc_binding, establishment and maintenance of position, site, establishment and maintenance of localization, establishment and maintenance of substrate location, single-organism localization, region, establishment and maintenance of cellular component location, binding siteliquid chromatography tandem mass spectroscopy, l(4)17, l(4)13, Gli, human being, artificial sequence, Cid[Mel], Ci[D], instrument configuration, Preptin, A4, Gene, splitted from, combined T cell and B cell immunodeficiency, Insulin-like growth factor II, combined T and B cell immunodeficiency, CENP-A, TYPE, LC-MS-MS, CENP-C, myopathy with lactic acidosis and sideroblastic anemia, MYH9.11, DAGA4, Orthophosphoric-monoester phosphohydrolase (alkaline optimum), CenpA, peptide, l(4)102ABc, Polypeptides, peptido, Multiple system atrophy (disorder), IT, LC-MSMS, Shy-dragger syndrome (formerly), Gene Products, MLASA, Shy-Drager Syndrome, MYH9_11, synthetic genetic interaction (sensu inequality), MAM, Siah, SCG3, Shy-Drager syndrome, Pure, CenH3[Cid], CG13329, CenH3[CID], BcDNA:RE21270, LCMSMS, and siderblastic anemia, autonomic failure, peptides, HeLa, beta-Trypsin, DmelCG13329, mitochondrial myopathy and sideroblastic anaemia, myopathy with lactic acidosis and sideroblastic anaemia, synthetic genetic interaction defined by inequality, proteins, sideroblastic anaemia and mitochondrial myopathy, Msa, MSA, man, X-linked combined immunodeficiency, CI, discontiguous, sample, Cenp-A, and siderblastic anaemia, CG2125, peptidos, ETD, CiD, combined immunodeficiency, LC-MS2, Ce, HCD., Ci, Proteins, LC-MS/MS, cenH3, artificial gene, synthetic DNA, not genetically inherited, collisionally activated dissociation, Peptide, CID, ciD, Cid, MARIPOSA, LGMD2C, hypotension, mitochondrial myopathy and sideroblastic anemia, 5730442G03Rik, polypeptide, LC/MS/MS, Phosphopeptide, CAD, REGULATORY PARTICLE NON-ATPASE SUBUNIT 5A, HCD, divided_from, ci[D], Ci155, multiple system atrophy, lactic acidosis and sideroblastic anemia, Protein, lactic acidosis and sideroblastic anaemia, Shy-McGee-Drager syndrome, synthetic, ci-D, HELA cell, multisystem atrophy, susceptibility to multiple system atrophy 1, sideroblastic anemia and mitochondrial myopathy, cenpA, IGF-II, Multiplication-stimulating polypeptide, DmelCG2125, ci155, CenH3, Ci/Gli, Ci/GLI, CenH3/CID, DMDA1, instrument, CENP-A/Cid, CENP-A/CID, CENPA, orthostatic hypotension syndrome, Tripcellim, RPN5A, beta Trypsin, synthetic constructs, human, sample population, orthostatic, Protein Gene Products, congenital combined immunodeficiency, Gene Proteins, Trypure, EMBRYO DEFECTIVE 2107, SYNTHETIC CONSTRUCT sequences, myopathy, DMDA, lactic acidosis, liquid chromatography-tandem mass spectroscopy, SCARMD2, liquid chromatography tandem mass spectrometry, artificial, Peptid, Polypeptide, Multiplication-stimulating activity, CenpA/CID, Ci-155, CID/CENP-A, MyopathyForms, establishment and maintenance of substance location, HCD., ion, region or site annotation, Proteins, Phosphorylations, FBN, Gene, phosphorylation, Peptide, predicted, binding site, Programs, ACMICD, polypeptide, peptide, Polypeptides, single organism localization, Workflows, ClvPrd, peptido, Ionen, sensitive, ECTOL1, Protein, Gene Products, establishment and maintenance of position, MFS1, establishment and maintenance of localization, establishment and maintenance of substrate location, Work Flow, Library, sensitivity, region, WMS, WMS2, hypersensitivity, positional, hypersensitive, positional polypeptide feature, peptides, INSDC_feature:misc_binding, iones, proteins, single-organism localization, MASS, ions, Protein Gene Products, OCTD, allergic reaction, Gene Proteins, localisation, binding_or_interaction_site, Ion, hypersensitivity reaction disease, SSKS, site, Peptid, peptidos, hypersensitivity reaction, Polypeptide, GPHYSD2, Work Flows, establishment and maintenance of cellular component location, SGS0trueSimulated phosphopeptide spectral library for confident site localizationWe have developed a new workflow to unambiguously localize phosphorylation sites on proteins. We demonstrate that spectral matching of phosphopeptide datasets against a library of the well-simulated spectra provided higher sensitivity for confident site localization than other tested programs. To computationally simulate tandem mass spectra representing all possible singly phosphorylated forms of a peptide, characteristic fragment ions are predicted from ions of their dephosphorylated form generated by beam-type collision-induced dissociation.2015-03-172014-08-26PXD00047444544641524991755931173880433304323598390632716016032935811275838511879471351225117750585557146835364921589103062915936413885803327159100226388869151355881464796928280383654583339858011825906329572725639286745134081694530135547714245073264435551274432714694559729157546478422422089643174471270139011431932307411148489693529372512646907275802402165993665812165952941281283296543128033952321818431832920435315257554787575930797214321382426191096976226155153111795745586265281736309431796989361604885664678253175133555432046NCBITaxon:1313456586068860769938942795545351201488779594100264203676310761854312157457794144242324113NCBITaxon:1773102391011787099913170187367451011656915693983346633039925883910005899940343053115655442193287263178723666987246426872754761151043055180066125522856213616299767982510216998231174673116761080772446896776744685960039376511822635735784588086363266689NCBITaxon:26970491031298382413689975961231975228319662776689605960615729513722117110125673712334351052311009040549986387833578840193733496151982157574878272151908985076480418985623919026454637463601134506253145943NCBITaxon:6157977840812633803945264114595361235178616722724323074691123869NCBITaxon:2954424327762791292497036125180284812426428150475102993886599034694173475154155403632997966287732396289954865755846282382352286287173623139946360106884204399479685835584795444042607101678078NCBITaxon:61918402393128118361003618557919080270381692259106592260704618326070533454226070788531870299103209285124966842528619212638541094343197192875NCBITaxon:103592829126806395602959870448300641802223544322430857541298396427111699632810412169792726241238993643680109779135622334747269704958331803029760381858334240906114778712744141639130767494022432640817212816120425468761383708127442399011912744201274426519534911370267506028532189517731193501846451450511492412153239564818822970915800257125614801549031384736031423166385962859631015104932275969373332648373153NCBITaxon:961529722109767711211144937608333419110792762833322375615592924847974920036009434073352472515151438992374725774671