on 12-03-201204:02 AM - edited on 10-15-202106:11 AM by Closed Account
Peterman S, Bomgarden R, Viner R Application Note 574 Protein kinases are key enzymes involved in a wide array of complex cellular functions and pathways. Misregulation or mutation of protein kinases underlies numerous disease states, including tumorigenesis, making them ideal candidates for drug development. However, identifying specific kinase inhibitors is challenging due to the high degree of homology among subfamily members of the 500+ human kinases. In addition, overlapping kinase substrate specificity and crosstalk between cellular signaling pathways can confound attempts to identify kinase inhibitor targets in vivo.
An emerging technology for identifying kinase inhibitor targets is based on chemical proteomic profiling of kinase inhibitor specificity and binding affinity. This technology combines mass spectrometry (MS)-based identification and quantitation with small molecule probe binding and enrichment to determine kinase active site occupancy during inhibition. One of these methods uses novel biotinylated ATP and/or ADP probes that irreversibly react with conserved lysine residues of kinase ATP binding sites. Selective enrichment of active-site peptides from labeled kinase digests dramatically reduces background matrix and increases signal for MS analysis of low abundance kinase peptides. Using this method, more than 400 different protein and lipid kinases from various mammalian tissues and cell lines have been identified and functionally assayed using targeted acquisition on an ion trap mass spectrometer. The assays are available commercially from ActivX Biosciences as their KiNativ™ kinase profiling services.
Our approach incorporates desthiobiotin-ATP and -ADP probes with a hybrid quadrupole-Orbitrap™ mass spectrometer into an integrated workflow for global kinase identification and inhibition analysis (Figure 1). This workflow leverages the unique capabilities of the mass spectrometer for acquisition of MS and MS/MS spectra in unbiased or targeted modes with Orbitrap ion detection for high resolution and mass accuracy. A multiplexed data acquisition method was also employed to maximize instrument duty cycle and quantification of low-abundance kinase peptides through selected ion monitoring (SIM) on a nano-LC timescale. Overall, this approach resulted in significant improvements to kinase active-site peptide detection and relative quantitation for kinase inhibitor profiling.