Sensitive multiplexed analysis of kinase activities and activity-based kinase identification

Nat Biotechnol. 2009 Oct;27(10):933-40. doi: 10.1038/nbt.1566. Epub 2009 Oct 4.


Constitutive activation of one or more kinase signaling pathways is a hallmark of many cancers. Here we extend the previously described mass spectrometry-based KAYAK approach by monitoring kinase activities from multiple signaling pathways simultaneously. This improved single-reaction strategy, which quantifies the phosphorylation of 90 synthetic peptides in a single mass spectrometry run, is compatible with nanogram to microgram amounts of cell lysate. Furthermore, the approach enhances kinase monospecificity through substrate competition effects, faithfully reporting the signatures of many signaling pathways after mitogen stimulation or of basal pathway activation differences across a panel of well-studied cancer cell lines. Hierarchical clustering of activities from related experiments groups peptides phosphorylated by similar kinases together and, when combined with pathway alteration using pharmacological inhibitors, distinguishes underlying differences in potency, off-target effects and genetic backgrounds. Finally, we introduce a strategy to identify the kinase, and even associated protein complex members, responsible for phosphorylation events of interest.

Publication types

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

MeSH terms

  • CDC2 Protein Kinase
  • Cell Cycle / drug effects
  • Cells, Cultured
  • Cluster Analysis
  • Computational Biology / methods*
  • Cyclin B / metabolism
  • Cyclin-Dependent Kinases
  • Epidermal Growth Factor / pharmacology
  • HeLa Cells
  • Humans
  • Insulin / pharmacology
  • Mass Spectrometry / methods*
  • Phosphorylation
  • Protein Kinases / metabolism*
  • Proto-Oncogene Proteins c-akt / metabolism
  • Receptor Protein-Tyrosine Kinases / metabolism
  • Reproducibility of Results
  • Signal Transduction
  • Tetradecanoylphorbol Acetate / pharmacology


  • Cyclin B
  • Insulin
  • Epidermal Growth Factor
  • Protein Kinases
  • Receptor Protein-Tyrosine Kinases
  • Proto-Oncogene Proteins c-akt
  • CDC2 Protein Kinase
  • CDK1 protein, human
  • Cyclin-Dependent Kinases
  • Tetradecanoylphorbol Acetate