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High-Throughput Kinase Activity Mapping (HT-KAM) system: analysis of phospho-catalytic profiles

Jean-Philippe Coppé, Christina Yau, Denise M. Wolf

Abstract

Phosphorylation networks intimately regulate mechanisms of response to therapies. Identifying the phospho-catalytic activities of kinases in biological samples remains a challenge. Here, we introduce a high-throughput system to detect kinases’ enzymatic activity using their biological peptide targets as phospho-sensors. Libraries of peptides operate as specific, distinct combinatorial peptide sets that simultaneously distinguish and measure the activity of a multiplicity of kinase enzymes. Our strategy provides access to a vast, untapped resource of meaningful measurements, whether readouts are interpreted irrespective of which enzymes phosphorylate which probes, or analyzed to convert global phospho-signatures into functional profiles of kinase activities. The procedure described in this Protocol Exchange chapter focuses on detailing the statistical and computational analysis steps that allow deconvoluting peptide phosphorylation profiles into kinase activity signatures. This is related to the Nature Cell Biology manuscript NCB-C36710, titled: "Mapping phospho-catalytic dependencies of therapy-resistant tumors reveals actionable vulnerabilities".

Keywords
kinase enzymes, phospho-catalytic activity screening, peptide sensors, combinatorial profiling, cell extracts, tissue biospecimen extracts, functional proteomics, signaling networks

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Preprint: Please note that this article has not completed peer review.

High-Throughput Kinase Activity Mapping (HT-KAM) system: analysis of phospho-catalytic profiles

Jean-Philippe Coppé, Christina Yau, Denise M. Wolf

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Abstract

Phosphorylation networks intimately regulate mechanisms of response to therapies. Identifying the phospho-catalytic activities of kinases in biological samples remains a challenge. Here, we introduce a high-throughput system to detect kinases’ enzymatic activity using their biological peptide targets as phospho-sensors. Libraries of peptides operate as specific, distinct combinatorial peptide sets that simultaneously distinguish and measure the activity of a multiplicity of kinase enzymes. Our strategy provides access to a vast, untapped resource of meaningful measurements, whether readouts are interpreted irrespective of which enzymes phosphorylate which probes, or analyzed to convert global phospho-signatures into functional profiles of kinase activities. The procedure described in this Protocol Exchange chapter focuses on detailing the statistical and computational analysis steps that allow deconvoluting peptide phosphorylation profiles into kinase activity signatures. This is related to the Nature Cell Biology manuscript NCB-C36710, titled: "Mapping phospho-catalytic dependencies of therapy-resistant tumors reveals actionable vulnerabilities".

Introduction

Reagents

Equipment

Procedure

Timing

Troubleshooting

References

Learn more about our company.