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Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics.

ABSTRACT: Our ability to model the dynamics of signal transduction networks will depend on accurate methods to quantify levels of protein phosphorylation on a global scale. Here we describe a motif-targeting quantitation method for phosphorylation stoichiometry typing. Proteome-wide phosphorylation stoichiometry can be obtained by a simple phosphoproteomic workflow integrating dephosphorylation and isotope tagging with enzymatic kinase reaction. Proof-of-concept experiments using CK2-, MAPK- and EGFR-targeting assays in lung cancer cells demonstrate the advantage of kinase-targeted complexity reduction, resulting in deeper phosphoproteome quantification. We measure the phosphorylation stoichiometry of >1,000 phosphorylation sites including 366 low-abundance tyrosine phosphorylation sites, with high reproducibility and using small sample sizes. Comparing drug-resistant and sensitive lung cancer cells, we reveal that post-translational phosphorylation changes are significantly more dramatic than those at the protein and messenger RNA levels, and suggest potential drug targets within the kinase-substrate network associated with acquired drug resistance.

SUBMITTER: Tsai CF

PROVIDER: S-EPMC4389224 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics.

Tsai Chia-Feng CF Wang Yi-Ting YT Yen Hsin-Yung HY Tsou Chih-Chiang CC Ku Wei-Chi WC Lin Pei-Yi PY Chen Hsuan-Yu HY Nesvizhskii Alexey I AI Ishihama Yasushi Y Chen Yu-Ju YJ

Nature communications 20150327

Our ability to model the dynamics of signal transduction networks will depend on accurate methods to quantify levels of protein phosphorylation on a global scale. Here we describe a motif-targeting quantitation method for phosphorylation stoichiometry typing. Proteome-wide phosphorylation stoichiometry can be obtained by a simple phosphoproteomic workflow integrating dephosphorylation and isotope tagging with enzymatic kinase reaction. Proof-of-concept experiments using CK2-, MAPK- and EGFR-targ ...[more]

PMID: 25814448

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Project description:Signals that control responses to stimuli and cellular function are transmitted through the dynamic phosphorylation of thousands of proteins by protein kinases. Many techniques have been developed to study phosphorylation dynamics, including several mass spectrometry (MS)-based methods. Over the past few decades, substantial developments have been made in MS techniques for the large-scale identification of proteins and their post-translational modifications. Nevertheless, all of the current MS-based techniques for quantifying protein phosphorylation dynamics rely on the measurement of changes in peptide abundance levels, and many methods suffer from low confidence in phosphopeptide identification due to poor fragmentation. Here we have optimized an approach for the stable isotope labeling of amino acids by phosphate using [?-¹?O?]ATP in nucleo to determine global site-specific phosphorylation rates. The advantages of this metabolic labeling technique are increased confidence in phosphorylated peptide identification, direct labeling of phosphorylation sites, measurement phosphorylation rates, and the identification of actively phosphorylated sites in a cell-like environment. In this study we calculated approximate rate constants for over 1,000 phosphorylation sites based on labeling progress curves. We measured a wide range of phosphorylation rate constants from 0.34 min?¹ to 0.001 min?¹. Finally, we applied stable isotope labeling of amino acids by phosphate to identify sites that have different phosphorylation kinetics during G1/S and M phase. We found that most sites had very similar phosphorylation rates under both conditions; however, a small subset of sites on proteins involved in the mitotic spindle were more actively phosphorylated during M phase, whereas proteins involved in DNA replication and transcription were more actively phosphorylated during G1/S phase. The data have been deposited to the ProteomeXchange with the identifier PXD000680.

Project description:BACKGROUND: Phosphorylation is the most prevalent post-translational modification on eukaryotic proteins. Multisite phosphorylation enables a specific combination of phosphosites to determine the speed, specificity and duration of biological response. Until recent years, the lack of high quality data limited the possibility for analyzing the properties of phosphorylation at the proteome scale and in the context of a wide range of conditions. Thanks to advances of mass spectrometry technologies, thousands of phosphosites from in-vivo experiments were identified and archived in the public domain. Such resource is appropriate to derive an unbiased view on the phosphosites properties in eukaryotes and on their functional relevance. RESULTS: We present statistically rigorous tests on the spatial and functional properties of a collection of approximately 70,000 reported phosphosites. We show that the distribution of phosphosites positioning along the protein tends to occur as dense clusters of Serine/Threonines (pS/pT) and between Serine/Threonines and Tyrosines, but generally not as much between Tyrosines (pY) only. This phenomenon is more ubiquitous than anticipated and is pertinent for most eukaryotic proteins: for proteins with > or = 2 phosphosites, 54% of all pS/pT sites are within 4 amino acids of another site. We found a strong tendency for clustered pS/pT to be activated by the same kinase. Large-scale analyses of phosphopeptides are thus consistent with a cooperative function within the cluster. CONCLUSIONS: We present evidence supporting the notion that clusters of pS/pT but generally not pY should be considered as the elementary building blocks in phosphorylation regulation. Indeed, closely positioned sites tend to be activated by the same kinase, a signal that overrides the tendency of a protein to be activated by a single or only few kinases. Within these clusters, coordination and positional dependency is evident. We postulate that cellular regulation takes advantage of such design. Specifically, phosphosite clusters may increase the robustness of the effectiveness of phosphorylation-dependent response. REVIEWERS: Reviewed by Joel Bader, Frank Eisenhaber, Emmanuel Levy (nominated by Sarah Teichmann). For the full reviews, please go to the Reviewers' comments section.

Dataset Information

Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics.

Publications

Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics.

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