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      The functional landscape of the human phosphoproteome

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          Abstract

          Protein phosphorylation is a key post-translational modification regulating protein function in almost all cellular processes. Although tens of thousands of phosphorylation sites have been identified in human cells, approaches to determine the functional importance of each phosphosite are lacking. Here, we manually curated 112 datasets of phospho-enriched proteins generated from 104 different human cell types or tissues. We reanalyzed the 6,801 proteomics experiments that passed our quality control criteria, creating a reference phosphoproteome containing 119,809 human phosphosites. To prioritize functional sites, we used machine learning to identify 59 features indicative of proteomic, structural, regulatory or evolutionary relevance and integrate them into a single functional score. Our approach identifies regulatory phosphosites across different molecular mechanisms, processes and diseases, and reveals genetic susceptibilities at a genomic scale. Several novel regulatory phosphosites were experimentally validated, including a role in neuronal differentiation for phosphosites in SMARCC2, a member of the SWI/SNF chromatin remodeling complex.

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          Most cited references38

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          Functional Characterization of the S. cerevisiae Genome by Gene Deletion and Parallel Analysis

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            Ultradeep human phosphoproteome reveals a distinct regulatory nature of Tyr and Ser/Thr-based signaling.

            Regulatory protein phosphorylation controls normal and pathophysiological signaling in eukaryotic cells. Despite great advances in mass-spectrometry-based proteomics, the extent, localization, and site-specific stoichiometry of this posttranslational modification (PTM) are unknown. Here, we develop a stringent experimental and computational workflow, capable of mapping more than 50,000 distinct phosphorylated peptides in a single human cancer cell line. We detected more than three-quarters of cellular proteins as phosphoproteins and determined very high stoichiometries in mitosis or growth factor signaling by label-free quantitation. The proportion of phospho-Tyr drastically decreases as coverage of the phosphoproteome increases, whereas Ser/Thr sites saturate only for technical reasons. Tyrosine phosphorylation is maintained at especially low stoichiometric levels in the absence of specific signaling events. Unexpectedly, it is enriched on higher-abundance proteins, and this correlates with the substrate KM values of tyrosine kinases. Our data suggest that P-Tyr should be considered a functionally separate PTM of eukaryotic proteomes. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
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              Tracking cancer drugs in living cells by thermal profiling of the proteome.

              The thermal stability of proteins can be used to assess ligand binding in living cells. We have generalized this concept by determining the thermal profiles of more than 7000 proteins in human cells by means of mass spectrometry. Monitoring the effects of small-molecule ligands on the profiles delineated more than 50 targets for the kinase inhibitor staurosporine. We identified the heme biosynthesis enzyme ferrochelatase as a target of kinase inhibitors and suggest that its inhibition causes the phototoxicity observed with vemurafenib and alectinib. Thermal shifts were also observed for downstream effectors of drug treatment. In live cells, dasatinib induced shifts in BCR-ABL pathway proteins, including CRK/CRKL. Thermal proteome profiling provides an unbiased measure of drug-target engagement and facilitates identification of markers for drug efficacy and toxicity. Copyright © 2014, American Association for the Advancement of Science.
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                Author and article information

                Journal
                9604648
                Nat Biotechnol
                Nat. Biotechnol.
                Nature biotechnology
                1087-0156
                1546-1696
                06 November 2019
                09 December 2019
                March 2020
                09 June 2020
                : 38
                : 3
                : 365-373
                Affiliations
                [1 ]European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, CB10 1SD, Cambridge, UK
                [2 ]European Molecular Biology Laboratory, Genome Biology Unit, 69117 Heidelberg, Germany
                [3 ]Gladstone Institute of Data Science and Biotechnology, J. David Gladstone Institutes, San Francisco, CA 94158, USA
                [4 ]Department of Cellular and Molecular Pharmacology and the Quantitative Biosciences Institute (QBI), University of California, San Francisco, CA 94158, USA
                Author notes
                [# ]Correspondence to: David Ochoa ( ochoa@ 123456ebi.ac.uk ) and Pedro Beltrao ( pbeltrao@ 123456ebi.ac.uk )
                Article
                EMS84831
                10.1038/s41587-019-0344-3
                7100915
                31819260
                231283bc-b303-4f5c-b86f-3b1aa1f28bb4

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                Biotechnology
                Biotechnology

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