Kinases have been the target of extensive research, and there has been significant interest in developing assays that measure the endogenous activities of kinases and their perturbation by small molecules. Many small molecule kinase inhibitors have extensive polypharmacology, so it is important to understand the pathways and off-targets that a potential new kinase inhibitor affects in vivo.
Kinase activity assay for kinome profiling (KAYAK) is a method that allows the phosphorylation of a diverse set of kinase peptide substrates to be simultaneously prosecuted. This new article* describes a modification to the technique, called direct-KAYAK, wherein the substrate set (light substrate peptides) was modified, the phosphopeptide enrichment step was removed, and the data analysis was streamlined (see Figure 1). The method can be run in a relatively high-throughput 96-plate format with a benchtop orbitrap mass spectrometer.
The heavy-phosphopeptides that are added following the acid quench step are complementary in sequence to the light peptides and have similar physicochemical properties, allowing them to co-elute with the light peptides but to be distinguished by their different m/z ratios in the mass spectrometer. This enables quantification and site-specific localization.