A high-throughput, multiplexed kinase assay using a benchtop orbitrap mass spectrometer to investigate the effect of kinase inhibitors on kinase signaling pathways.
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.
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, United States.
Ryan C
RC
Kunz
Fiona E
FE
McAllister
John
J
Rush
Steven P
SP
Gygi
D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D012984:Software
D010455:Peptides; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases