To date, the effects of specific modification types and sites on protein lifetime have not been systematically illustrated. Here, we describe a proteomic method, DeltaSILAC, to quantitatively assess the impact of site-specific phosphorylation on the turnover of thousands of proteins in live cells. Based on the accurate and reproducible mass spectrometry-based method, a pulse labeling approach using stable isotope-labeled amino acids in cells (pSILAC), phosphoproteomics, and a unique 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 method represents a generalizable approach and provides a rich resource for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology.

• Keywords
• DeltaSILAC, data-independent acquisition, mass spectrometry, phosphomodiform, phosphorylation, protein lifetime, protein turnover, proteomics, pulse SILAC,
• MeSH
• Cell Cycle physiology   MeSH
• Cyclin-Dependent Kinases genetics   metabolism   MeSH
• Phosphoproteins chemistry   metabolism   MeSH
• Phosphorylation MeSH
• Glutamates metabolism   MeSH
• Mass Spectrometry methods   MeSH
• Isotope Labeling methods   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Peptides metabolism   MeSH
• Peroxiredoxin VI chemistry   metabolism   MeSH
• Proteolysis * MeSH
• Proteome genetics   metabolism   MeSH
• Proteomics methods   MeSH
• Amino Acid Sequence MeSH
• RNA Splicing Factors chemistry   metabolism   MeSH
• Signal Transduction genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Cyclin-Dependent Kinases MeSH
• Phosphoproteins MeSH
• Glutamates MeSH
• Peptides MeSH
• Peroxiredoxin VI MeSH
• PRDX6 protein, human MeSH Browser
• Proteome MeSH
• RNA Splicing Factors MeSH
• SF3B1 protein, human MeSH Browser