High-Throughput Assessment of Kinome-Wide Activation States
23 Pages Posted: 31 Oct 2018 Publication Status: PublishedMore...
Protein kinases play a pivotal role in cellular communication and aberrant kinase activity has been linked to a variety of disorders. Methods to directly probe kinase activation states in cells have been lacking and until now kinase activity has mainly been extracted from either kinase protein expression levels or data on substrate phosphorylation. Here, we describe a novel 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 (MS), we provide highly specific assays for 248 peptides, covering 221 phosphorylation sites 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 TNFα induced RIPK1 activation, which has thus far only been reported using a protein expression system in combination with an in vitro kinase assay. Moreover, our methodology enables the detection of 46 t-loop phosphorylation sites directly from a breast tumor 14G needle biopsy.
Suggested Citation: Suggested Citation