Global and site-specific effect of phosphorylation on protein turnover

The effects of specific modification types and sites on protein lifetime have not been illustrated in large scale. We describe a proteomic method, DeltaSILAC, to quantify the impact of site-specific phosphorylation on the turnover of thousands of proteins in live cells. Being configured on the accurate and reproducible mass spectrometry, the pulse labeling approach using stable isotope-labeled amino acids in cells (pSILAC), phosphoproteomics, as well as a novel peptide-level matching strategy, our DeltaSILAC profiling revealed a global, unexpected delaying effect of many phosphosites on protein turnover. We further found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness, enriched in Cyclin-dependent kinase substrates, and evolutionarily conserved, whereas the Glutamic acids surrounding phosphosites significantly delay protein turnover. Our investigation provides a generalizable approach and a rich resource for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology.