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

Signals that control response to stimuli and cellular function are transmitted through 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 last 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 (SILAP) using [gamma-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 500 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 SILAP to determine sites that have different phosphorylation kinetics during G1/S and M phase. We found that most sites have 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, while proteins involved in DNA replication and transcription were more actively phosphorylated during G1/S phase.