Methods, compositions and kits for high throughput kinase activity screening using mass spectrometry and stable isotopes
US10407712B2 · kind B2 · utility
Assignee
Inventors
Key dates
| Filing date | Oct 16, 2017 |
| Grant date | Sep 10, 2019 |
| Priority date | — |
| Expiry date | Oct 16, 2037 |
Classification
- Technology area (CPC G)Physics
- CPC primaryG01N33/6848
- WIPO fieldMeasurement
- WIPO sectorInstruments
Abstract
A mass-spectrometry-based method and substrates are provided herein for large scale kinome activity profiling directly from crude lysates using 90 chemically synthesized peptide substrates with amino acid sequences derived from known phosphoproteins. Quantification of peptide phosphorylation rates was achieved via the use of stable isotope labeled synthetic peptides. A method and substrates for obtaining 90 simultaneous activity measurements in a single-reaction format were developed and validated. The kinome activity profiling strategy was successfully applied with lysates of: cells manipulated by combination of mitogen stimulation, pharmacological perturbation and siRNA-directed kinase knockdown; seven different breast cancer cell lines treated with gefitinib; and each of normal and cancerous tissue samples from renal cell carcinoma patients. This method concurrently measures multiple peptide phosphorylation rates to provide a diagnostic fingerprint pattern for activated kinases, protein phosphatases, modulators of these enzymes, and pathways (kinome) from as little starting material as a few cells.
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