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

High-Throughput Kinase Activity Mapping (HT-KAM) system: biochemical assay

Jean-Philippe Coppé, Miki Mori, Bo Pan

Abstract

Mapping the phospho-catalytic profile of kinase enzymes in cells or tissues remains a challenge. Here, we introduce a practical biochemical assay to measure the enzymatic activity of kinases using peptides as surrogate sensors to identify kinases in tumor biopsies and cell lines. The platform relies on collections of peptide probes that are derived from biological target sites of kinases, and that operate as distinct combinatorial peptide sets to simultaneously distinguish and measure the phospho-catalytic activity of many kinase enzymes. The assay is modular by design: users can adapt probe libraries and assay conditions to their needs. We named this functional proteomic platform 'High-Throughput Kinase Activity Mapping (HT-KAM) system'. The procedure described in this Protocol Exchange chapter focuses on detailing the biochemical assay, and 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

Introduction

Reagents

Equipment

Procedure

Timing

Troubleshooting

Anticipated Results

References

Acknowledgements

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

High-Throughput Kinase Activity Mapping (HT-KAM) system: biochemical assay

Jean-Philippe Coppé, Miki Mori, Bo Pan

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Abstract

Mapping the phospho-catalytic profile of kinase enzymes in cells or tissues remains a challenge. Here, we introduce a practical biochemical assay to measure the enzymatic activity of kinases using peptides as surrogate sensors to identify kinases in tumor biopsies and cell lines. The platform relies on collections of peptide probes that are derived from biological target sites of kinases, and that operate as distinct combinatorial peptide sets to simultaneously distinguish and measure the phospho-catalytic activity of many kinase enzymes. The assay is modular by design: users can adapt probe libraries and assay conditions to their needs. We named this functional proteomic platform 'High-Throughput Kinase Activity Mapping (HT-KAM) system'. The procedure described in this Protocol Exchange chapter focuses on detailing the biochemical assay, and 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

Anticipated Results

References

Acknowledgements

Learn more about our company.