Global and Site-Specific Effect of Phosphorylation on Protein Turnover

Dev Cell. 2021 Jan 11;56(1):111-124.e6. doi: 10.1016/j.devcel.2020.10.025. Epub 2020 Nov 24.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cell Cycle / physiology
  • Cell Line, Tumor
  • Cyclin-Dependent Kinases / genetics
  • Cyclin-Dependent Kinases / metabolism
  • Glutamates / metabolism
  • Humans
  • Isotope Labeling / methods*
  • Mass Spectrometry / methods*
  • Peptides / metabolism
  • Peroxiredoxin VI / chemistry
  • Peroxiredoxin VI / metabolism
  • Phosphoproteins / chemistry
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Proteolysis*
  • Proteome / genetics
  • Proteome / metabolism*
  • Proteomics / methods*
  • RNA Splicing Factors / chemistry
  • RNA Splicing Factors / metabolism
  • Signal Transduction / genetics


  • Glutamates
  • Peptides
  • Phosphoproteins
  • Proteome
  • RNA Splicing Factors
  • SF3B1 protein, human
  • PRDX6 protein, human
  • Peroxiredoxin VI
  • Cyclin-Dependent Kinases