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- Title
Sensitive multiplexed analysis of kinase activities and activity-based kinase identification.
- Authors
Kubota, Kazuishi; Anjum, Rana; Yonghao Yu; Kunz, Ryan C.; Andersen, Jannik N.; Kraus, Manfred; Keilhack, Heike; Nagashima, Kumiko; Krauss, Stefan; Paweletz, Cloud; Hendrickson, Ronald C.; Feldman, Adam S.; Chin-Lee Wu; Rush, John; Villén, Judit; Gygi, Steven P.
- Abstract
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.
- Subjects
CANCER; MASS spectrometry; PEPTIDES; CELL lines; PHARMACOLOGY; PHOSPHORYLATION
- Publication
Nature Biotechnology, 2009, Vol 27, Issue 10, p933
- ISSN
1087-0156
- Publication type
Article
- DOI
10.1038/nbt.1566