1. A high-throughput, multiplexed kinase assay using a benchtop orbitrap mass spectrometer to investigate the effect of kinase inhibitors on kinase signaling pathways.
- Author
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Kunz RC, McAllister FE, Rush J, and Gygi SP
- Subjects
- Amino Acid Sequence, Cell Line, Tumor, Humans, Inhibitory Concentration 50, Peptides chemistry, Peptides metabolism, Software, Enzyme Assays methods, Mass Spectrometry methods, Protein Kinase Inhibitors pharmacology, Protein Kinases metabolism, Signal Transduction drug effects
- Abstract
Protein phosphorylation is an important and ubiquitous post-translational modification in eukaryotic biological systems. The KAYAK (Kinase ActivitY Assay for Kinome profiling) assay measures the phosphorylation rates of dozens of peptide substrates simultaneously, directly from cell lysates. Here, we simplified the assay by removing the phosphopeptide enrichment step, increasing throughput while maintaining similar data quality. We term this new method, direct-KAYAK, because kinase activities were measured directly from reaction mixtures after desalting. In addition, new peptides were included to profile additional kinase pathways and redundant substrate peptides were removed. Finally, the method is now performed in 96-well plate format using a benchtop orbitrap mass spectrometer and the Pinpoint software package for improved data analysis. We applied the new high-throughput method to measure IC(50) values for kinases involved in monocyte-to-macrophage differentiation, a process important for inflammation and the immune response.
- Published
- 2012
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