High-Throughput Assessment of Kinome-wide Activation States

Cell Syst. 2019 Oct 23;9(4):366-374.e5. doi: 10.1016/j.cels.2019.08.005. Epub 2019 Sep 11.


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-α-induced RIPK1 activation and the detection of 46 T-loop phosphorylation sites from a breast tumor needle biopsy.

Keywords: SRM; T-loop phosphorylation; cancer; kinase; kinase activity; phosphoproteomics; proteomics; signaling; targeted mass spectrometry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biopsy, Needle
  • Breast Neoplasms / diagnosis*
  • Enzyme Activation
  • Female
  • High-Throughput Screening Assays / methods*
  • Humans
  • Jurkat Cells
  • Mass Spectrometry
  • Peptides / metabolism*
  • Phosphorylation
  • Proteomics / methods*
  • Receptor-Interacting Protein Serine-Threonine Kinases / metabolism*
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Tumor Necrosis Factor-alpha / metabolism


  • Peptides
  • Tumor Necrosis Factor-alpha
  • RIPK1 protein, human
  • Receptor-Interacting Protein Serine-Threonine Kinases