Profiling Kinase Inhibition In Cancer Cell Extracts Using Active-Site Probes And Targeted High Resolution, Accurate Mass Spectrometry

Abstract Protein kinases are enzymes that play key roles in signal transduction pathways which regulate cellular processes including growth, division, differentiation and metabolism. Kinase inhibitors are essential tools used by researchers to understand kinase function and are also therapeutic compounds used in treating cancer and other diseases. Active-site probes are one technology that can be used to determine the specificity and potency of kinase inhibitors. Desthiobiotin-ATP and -ADP are two nucleotide derivatives that have been shown to selectively label kinase active sites. Using these probes to enrich kinase active-site peptides, we identified over 150 kinases from K562 chronic myelogenous leukemia and A549 lung carcinoma cell extracts using high resolution, accurate mass (HR/AM) spectrometry. We also assessed specificity of kinase inhibitors including staurosporine and wortmannin by determining the relative quantitation of kinase active-site peptides after drug treatment. In addition, we validated kinase inhibitor targets using a parallel Western blot workflow. Overall, kinase inhibition measured using high resolution, accurate mass spectrometry had strong correlation with Western blot data and enabled global profiling of kinase inhibitor target and off-targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-218. doi:10.1158/1538-7445.AM2011-LB-218