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High-Throughput Assessment of Kinome-Wide Activation States

23 Pages Posted: 31 Oct 2018 Sneak Peek Status: Published

See all articles by Thierry Schmidlin

Thierry Schmidlin

Utrecht University - Biomolecular Mass Spectrometry and Proteomics Group

Stamatia Rontogianni

Utrecht University - Biomolecular Mass Spectrometry and Proteomics Group

Charlotte A. G. H. van Gelder

Utrecht University - Biomolecular Mass Spectrometry and Proteomics Group

Kelly E. Stecker

Utrecht University - Biomolecular Mass Spectrometry and Proteomics Group

Bart L. van den Eshof

Sanquin Research - Department of Molecular and Cellular Hemostasis

Kristel Kemper

The Netherlands Cancer Institute - Division of Molecular Oncology

Esther H. Lips

The Netherlands Cancer Institute - Department of Molecular Pathology

Maartje van den Biggelaar

Sanquin Research - Department of Molecular and Cellular Hemostasis

Daniel S. Peeper

The Netherlands Cancer Institute - Division of Molecular Oncology

Albert J. R. Heck

Utrecht University - Biomolecular Mass Spectrometry and Proteomics Group

A.F. Maarten Altelaar

Utrecht University - Biomolecular Mass Spectrometry and Proteomics Group

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Abstract

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 TNF╬▒ 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

Schmidlin, Thierry and Rontogianni, Stamatia and van Gelder, Charlotte A. G. H. and Stecker, Kelly E. and Eshof, Bart L. van den and Kemper, Kristel and Lips, Esther H. and Biggelaar, Maartje van den and Peeper, Daniel S. and Heck, Albert J. R. and Altelaar, A.F. Maarten, High-Throughput Assessment of Kinome-Wide Activation States (October 30, 2018). Available at SSRN: https://ssrn.com/abstract=3275288 or http://dx.doi.org/10.2139/ssrn.3275288
This is a paper under consideration at Cell Press and has not been peer-reviewed.

Thierry Schmidlin

Utrecht University - Biomolecular Mass Spectrometry and Proteomics Group

Padualaan 8,
Utrecht, 3584 CH
Netherlands

Stamatia Rontogianni

Utrecht University - Biomolecular Mass Spectrometry and Proteomics Group

Padualaan 8,
Utrecht, 3584 CH
Netherlands

Charlotte A. G. H. Van Gelder

Utrecht University - Biomolecular Mass Spectrometry and Proteomics Group

Padualaan 8,
Utrecht, 3584 CH
Netherlands

Kelly E. Stecker

Utrecht University - Biomolecular Mass Spectrometry and Proteomics Group

Padualaan 8,
Utrecht, 3584 CH
Netherlands

Bart L. van den Eshof

Sanquin Research - Department of Molecular and Cellular Hemostasis

Amsterdam
Netherlands

Kristel Kemper

The Netherlands Cancer Institute - Division of Molecular Oncology

Plesmanlaan 121
Amsterdam, 1066 CX
Netherlands

Esther H. Lips

The Netherlands Cancer Institute - Department of Molecular Pathology

Plesmanlaan 121
Amsterdam, 1066 CX
Netherlands

Maartje van den Biggelaar

Sanquin Research - Department of Molecular and Cellular Hemostasis

Amsterdam
Netherlands

Daniel S. Peeper

The Netherlands Cancer Institute - Division of Molecular Oncology

Plesmanlaan 121
Amsterdam, 1066 CX
Netherlands

Albert J. R. Heck

Utrecht University - Biomolecular Mass Spectrometry and Proteomics Group

Padualaan 8,
Utrecht, 3584 CH
Netherlands

A.F. Maarten Altelaar (Contact Author)

Utrecht University - Biomolecular Mass Spectrometry and Proteomics Group ( email )

Padualaan 8,
Utrecht, 3584 CH
Netherlands

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