Journal
CELL SYSTEMS
Volume 9, Issue 4, Pages 366-+Publisher
CELL PRESS
DOI: 10.1016/j.cels.2019.08.005
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Funding
- Netherlands Organization for Scientific Research (NWO) through a VIDI grant [723.012.102]
- Proteins@Work, a program of the National Roadmap Large-Scale Research Facilities of the Netherlands [184.032.201]
- Landsteiner Foundation for Blood Transfusion Research [LSBR 1517]
- Dutch Cancer Society [NKI-2013-5799, 10304]
- European Research Council grant under the European Union's Seventh Framework Programme (FP7/2007-2013)/ERC synergy grant [319661 COMBATCANCER]
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Aberrant kinase activity has been linked to a variety of disorders; however, methods to probe kinase activation states in cells have been lacking. Until now, kinase activity has mainly been deduced from either protein expression or substrate phosphorylation levels. Here, we describe a strategy to directly infer kinase activation through targeted quantification of T-loop phosphorylation, which serves as a critical activation switch in a majority of protein kinases. Combining selective phosphopeptide enrichment with robust targeted mass spectrometry, we provide highly specific assays for 248 peptides, covering 221 phosphosites in the T-loop region of 178 human kinases. Using these assays, we monitored the activation of 63 kinases through 73 T-loop phosphosites across different cell types, primary cells, and patient-derived tissue material. The sensitivity of our assays is highlighted by the reproducible detection of TNF-alpha-induced RIPK1 activation and the detection of 46 T-loop phosphorylation sites from a breast tumor needle biopsy.
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