Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics

Nat Commun. 2015 Mar 27;6:6622. doi: 10.1038/ncomms7622.


Our ability to model the dynamics of signal transduction networks will depend on accurate methods to quantify levels of protein phosphorylation on a global scale. Here we describe a motif-targeting quantitation method for phosphorylation stoichiometry typing. Proteome-wide phosphorylation stoichiometry can be obtained by a simple phosphoproteomic workflow integrating dephosphorylation and isotope tagging with enzymatic kinase reaction. Proof-of-concept experiments using CK2-, MAPK- and EGFR-targeting assays in lung cancer cells demonstrate the advantage of kinase-targeted complexity reduction, resulting in deeper phosphoproteome quantification. We measure the phosphorylation stoichiometry of >1,000 phosphorylation sites including 366 low-abundance tyrosine phosphorylation sites, with high reproducibility and using small sample sizes. Comparing drug-resistant and sensitive lung cancer cells, we reveal that post-translational phosphorylation changes are significantly more dramatic than those at the protein and messenger RNA levels, and suggest potential drug targets within the kinase-substrate network associated with acquired drug resistance.

Publication types

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

MeSH terms

  • Algorithms
  • Amino Acid Motifs
  • Blotting, Western
  • Cell Line, Tumor
  • Chromatography, Liquid
  • Drug Resistance, Neoplasm*
  • Humans
  • Lung Neoplasms / metabolism*
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Protein Processing, Post-Translational*
  • Protein Structure, Tertiary
  • Proteomics*
  • RNA, Messenger / metabolism*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction
  • Tandem Mass Spectrometry


  • Phosphoproteins
  • RNA, Messenger