Stable isotope labeling of phosphoproteins for large-scale phosphorylation rate determination.

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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 [γ-¹⁸O₄]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.", "claims": [{"id": "claim-target-result", "statement": "Using [γ-18O4]ATP metabolic labeling and labeling progress curves, the study estimated phosphorylation rate constants for over 1,000 sites, ranging from 0.34 min−1 to 0.001 min−1, and deposited the source data in ProteomeXchange under PXD000680.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:24532841", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "In this study we calculated approximate rate constants for over 1,000 phosphorylation sites based on labeling progress curves."}, {"asset": "source:pmid:24532841", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "We measured a wide range of phosphorylation rate constants from 0.34 min−1 to 0.001 min−1."}, {"asset": "source:pmid:24532841", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "The data have been deposited to the ProteomeXchange with the identifier PXD000680."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "partial", "objective": "Using [γ-18O4]ATP metabolic labeling and labeling progress curves, the study estimated phosphorylation rate constants for over 1,000 sites, ranging from 0.34 min−1 to 0.001 min−1, and deposited the source data in ProteomeXchange under PXD000680.", "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:24532841"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "blocked", "blocked_on": "data:accession_absent", "description": "The GEO series accession resolves and its sample accessions are listed. 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