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

Kazuishi Kubota, Rana Anjum, Yonghao Yu, Ryan C. Kunz, Jannik N. Andersen, Manfred Kraus, Heike Keilhack, Kumiko Nagashima, Stefan Krauss, Cloud Paweletz, Ronald C. Hendrickson, Adam S. Feldman, Chin Lee Wu, John Rush, Judit Villén, Steven P. Gygi

Research output: Contribution to journalArticle

75 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)933-940
Number of pages8
JournalNature Biotechnology
Issue number10
Publication statusPublished - Oct 2009


ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Biotechnology
  • Molecular Medicine
  • Bioengineering
  • Biomedical Engineering

Cite this

Kubota, K., Anjum, R., Yu, Y., Kunz, R. C., Andersen, J. N., Kraus, M., ... Gygi, S. P. (2009). Sensitive multiplexed analysis of kinase activities and activity-based kinase identification. Nature Biotechnology, 27(10), 933-940.