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

TitleStable isotope labeling of phosphoproteins for large-scale phosphorylation rate determination.
Publication TypeJournal Articles
Year of Publication2014
AuthorsMolden RC, Goya J, Khan Z, Garcia BA
JournalMol Cell Proteomics
Volume13
Issue4
Pagination1106-18
Date Published2014 Apr
ISSN1535-9484
Keywordscell cycle, HEK293 Cells, HeLa Cells, HUMANS, Isotope Labeling, Kinetics, Peptide Mapping, Phosphoproteins, Phosphorylation, proteomics, Tandem Mass Spectrometry
Abstract

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 [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.

DOI10.1074/mcp.O113.036145
Alternate JournalMol. Cell Proteomics
PubMed ID24532841
PubMed Central IDPMC3977188
Grant ListP50 GM071508 / GM / NIGMS NIH HHS / United States