Ultradeep human phosphoproteome reveals a distinct regulatory nature of Tyr and Ser/Thr-based signaling

Cell Rep. 2014 Sep 11;8(5):1583-94. doi: 10.1016/j.celrep.2014.07.036. Epub 2014 Aug 21.


Regulatory protein phosphorylation controls normal and pathophysiological signaling in eukaryotic cells. Despite great advances in mass-spectrometry-based proteomics, the extent, localization, and site-specific stoichiometry of this posttranslational modification (PTM) are unknown. Here, we develop a stringent experimental and computational workflow, capable of mapping more than 50,000 distinct phosphorylated peptides in a single human cancer cell line. We detected more than three-quarters of cellular proteins as phosphoproteins and determined very high stoichiometries in mitosis or growth factor signaling by label-free quantitation. The proportion of phospho-Tyr drastically decreases as coverage of the phosphoproteome increases, whereas Ser/Thr sites saturate only for technical reasons. Tyrosine phosphorylation is maintained at especially low stoichiometric levels in the absence of specific signaling events. Unexpectedly, it is enriched on higher-abundance proteins, and this correlates with the substrate KM values of tyrosine kinases. Our data suggest that P-Tyr should be considered a functionally separate PTM of eukaryotic proteomes.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Algorithms
  • HeLa Cells
  • Humans
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Protein Processing, Post-Translational*
  • Proteome / metabolism*
  • Sequence Analysis, Protein / methods
  • Serine / metabolism
  • Signal Transduction*
  • Threonine / metabolism
  • Tyrosine / metabolism


  • Phosphoproteins
  • Proteome
  • Threonine
  • Tyrosine
  • Serine