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High-Throughput Assessment of Kinome-wide Activation States.

Authors
  • Schmidlin, Thierry1
  • Debets, Donna O1
  • van Gelder, Charlotte A G H1
  • Stecker, Kelly E1
  • Rontogianni, Stamatia1
  • van den Eshof, Bart L2
  • Kemper, Kristel3
  • Lips, Esther H4
  • van den Biggelaar, Maartje2
  • Peeper, Daniel S3
  • Heck, Albert J R1
  • Altelaar, Maarten5
  • 1 Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands; Netherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, the Netherlands. , (Netherlands)
  • 2 Sanquin Research, Department of Molecular and Cellular Hemostasis, Amsterdam, the Netherlands. , (Netherlands)
  • 3 Division of Molecular Oncology, Oncode Institute, The Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands. , (Netherlands)
  • 4 Department of Molecular Pathology, The Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands. , (Netherlands)
  • 5 Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands; Netherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, the Netherlands; Mass Spectrometry and Proteomics Facility, The Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands. Electronic address: [email protected] , (Netherlands)
Type
Published Article
Journal
Cell systems
Publication Date
Oct 23, 2019
Volume
9
Issue
4
Identifiers
DOI: 10.1016/j.cels.2019.08.005
PMID: 31521607
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

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. Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.

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