MassIVEapplication/xmlftp://massive-ftp.ucsd.edu/x01/MSV000080223/primaryOK200ProteomicsDr Albert J. R. Heck,Orbitrap FusionLTQ Orbitrap EliteHomo Sapiens (ncbitaxon:9606)https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=8b443ed1a848487c84cf04bb18a22651MassIVE628UNIMOD:21 - "Phosphorylation."UNIMOD:4 - "Iodoacetamide derivative."UNIMOD:1 - "Acetylation."UNIMOD:35 - "Oxidation or Hydroxylation."Although mass-spectrometry-based screens enable thousands of protein phosphorylation sites to be monitored simultaneously, they often do not cover important regulatory sites. Here, we hypothesized that this is due to the fact that nearly all large-scale phosphoproteome studies are initiated by trypsin digestion. We tested this hypothesis using multiple proteases for protein digestion prior to Ti(4+)-IMAC-based enrichment. This approach increases the size of the detectable phosphoproteome substantially and confirms the considerable tryptic bias in public repositories. We define and make available a less biased human phosphopeptide atlas of 37,771 unique phosphopeptides, correlating to 18,430 unique phosphosites, of which fewer than 1/3 were identified in more than one protease data set. We demonstrate that each protein phosphorylation site can be linked to a preferred protease, enhancing its detection by mass spectrometry (MS). For specific sites, this approach increases their detectability by more than 1,000-fold.An Augmented Multiple-Protease-Based Human Phosphopeptide Atlas.Giansanti Piero P, Aye Thin Thin TT, van den Toorn Henk H, Peng Mao M, van Breukelen Bas B, Heck Albert J R AJl(2)k04204, big, FBgn0003149, scale tissue, OG12X, human being, Ass-1, peltate hair, Degradation, number, Mbp1, 6330543G17Rik, Spectrum Analyses, froggy, Gyltl1a, Human, l(2)k05821, large, Para, Homo sapiens, l(2)k15606, mPM, AA408052, Mass, fold, Analysis, PM/mPM., CG5939, myd, Man, Mass Spectroscopy, Shot, Mass Spectrum Analysis, study, l(2)k10821, Man (Taxonomy), MRM, Analyses, MDDGB6, plant peltate hair, C1, Su(Mir)1, Mbp-1, LARGE, Estimated, Protein Degradations, Protein Degradation, ASS, BPFD#36, great, site, l(2)CA4, Prx3, single organism signaling, close to, Degradations, PLATEST, gyltl1b-b, kop, DmelCG5939, Modern, CG18076, CG18637, antibodies, Protein Digestion, Spectrum Analysis, Digestions, Cell, results, shortstop/kakapo, Multiple Reaction Monitoring, Spectroscopy, near to, MS, Phosphopeptide, Proteolyses, prm, Digestion, MDDGA6, mKIAA0609, DmelCG18076, Protein, l(3)S010605, scales, KIAA0609, region, Mass Spectrum Analyses, fg, Mass Spectrum, l(2)k05434, gyltl1b, scale, Grv, 0106/05, l(2)k03010, Protein Digestions, mdc1d, Spectrometry, expanded, kak, SHOT, human, OG12, Atlases, MDC1D, signalling process, enr, l(3)10631, enlarged, grv, approaches, Modern Man, cardinality, vicinity of, Og12x, 42/4, Platelets, pm, PMProteinase, human being, Man (Taxonomy), Proteolytic Enzyme, C1, Modern, Protease, Proteolytic, Peptide, Proteinases, human, Human, Esteroproteases, Atlases., Peptidase, Hydrolase, Enzyme, Phosphopeptide, Homo sapiens, Modern Man, Peptidases, Proteases, Peptide Hydrolase, Man, Proteolytic Enzymessodium salt, big, Scientific Bias, scale tissue, DmelCG8566, Dunc104, human being, Proteolytic Enzyme, Ass-1, Ecological Fallacy, peltate hair, Truncation, Degradation, Mbp1, FBN, Systematic Bias, Protease, DmKlp53D, Spectrum Analyses, froggy, Gyltl1a, Human, Esteroproteases, protein amino acid phosphorylation, large, Hydrolase, 1H-imidazoleacetic acid, Homo sapiens, ECTOL1, Epidemiologic Biase, Mass, fold, Ecological, Proteases, Analysis, Peptide Hydrolase, myd, Man, WMS, Mass Spectroscopy, Fallacies, Mass Spectrum Analysis, FACT80, Ecological Biases, Man (Taxonomy), Analyses, FACT, MDDGB6, plant peltate hair, C1, Spectrum, Aggregation, beta-Trypsin, imidazolyl-4-acetic acid, Mbp-1, LARGE, Proteolytic, Protein Degradations, Protein Degradation, Proteinases, OCTD, ASS, BPFD#36, IMAC, Enzyme, MIP-4a, Statistical Biases, great, Ecological Bias, site, Systematic, imac, GPHYSD2, Epidemiologic Biases, AA408052., SGS, Biase, Fallacy, Proteinase, Degradations, Scientific, Data Set, gyltl1b-b, TSC-1, Epidemiologic, Modern, imidazole-4-acetic acid, Truncation Biases, Ecological Fallacies, Protein Digestion, KIF1B, Digestions, Spectrum Analysis, Peptide, ACMICD, Spectroscopy, Outcome Measurement Errors, Experimental, MS, Phosphopeptide, Proteolyses, Digestion, MDDGA6, Unc104, mKIAA0609, Protein, Peptidases, Errors, MFS1, CG8566, scales, Truncation Bias, KIAA0609, region, Mass Spectrum Analyses, WMS2, fg, imidazole-4-acetic acid hydrochloride, Mass Spectrum, Bias, gyltl1b, PRSS, scale, Protein Digestions, mdc1d, Spectrometry, expanded, Tripcellim, Biases, MIP-4alpha, Klp53D, MASS, Outcome Measurement, beta Trypsin, Unc-104, human, Atlases, Experimental Bias, Trypure, MDC1D, Peptidase, Outcome Measurement Error, DmCG8566, SCYA26, enr, enlarged, Error, Aggregation Bias, Modern Man, SSKS, Statistical Bias, T160, Statistical, hypothesis, Proteolytic EnzymesHuman, Atlases., human being, Phosphopeptide, Man (Taxonomy), Homo sapiens, Man, human, Modern Man, C1, ModernPXD001428trueMultiple protease based human phosphopeptide atlasSite-specific phospho-antibodies are commonly used to study these changes, however only a limited number of good antibodies is available, covering just a few signaling nodes in important pathways. It is highly unlikely that specific antibodies, covering the more than hundred thousands of estimated phosphosites in human cells, will become available soon. Mass spectrometry (MS)-based approaches are becoming of interest, as these are able to monitor at least thousands of sites simultaneously. However, it has become apparent that important signaling nodes, detectable by phosphosite specific-antibodies are quite often not observed in these large-scale in-depth MS-based studies. Here we addressed this issue by using multiple proteases for protein digestion, thereby increasing the size of the accessible and detectable phosphoproteome. We demonstrate that nearly each phosphosite has a preferred protease which favors detection by MS. For specific sites the use of alternative proteases increases the intensity by more than 1,000 fold. Based on the results we define and make publicly available a human phosphopeptide atlas of more than 37,771 unique phosphopeptides, correlating to over 18,000 unique phosphosites, that will be useful for both shot-gun as well as targeted MRM/PRM/SWATH based phosphoproteomics studies.Tue Oct 04 14:01:00 BST 2016MSV00008022326074081