Protein-Peptide Turnover Profiling reveals wiring of phosphorylation during protein maturation
Post-translational modifications (PTMs) regulate various aspects of protein function, including degradation. Mass spectrometric methods that rely on pulsed metabolic labeling are very popular to quantify turnover rates on a proteome-wide scale. Such data have often been interpreted in the context of protein proteolytic stability. Here, we combine theoretical kinetic modeling with experimental pulsed stable isotope labeling of amino acids in cell culture (pSILAC) for the study of protein phosphorylation. We demonstrate that metabolic labeling combined with PTM-specific enrichment does not measure effects of PTMs on protein stability. Rather, it reveals the relative order of PTM addition and removal along a proteins lifetime--a fundamentally different metric. We use this framework to identify temporal phosphorylation sites on cell cycle-specific factors and protein complex assembly intermediates. Our results open up an entirely new aspect in the study of PTMs, by tying them into the context of a proteins lifetime.