RESUMO
The enormous diversity of kinases and their pivotal role in cell signaling have set kinases in the focus of biomedical research. Profiling the kinome of tissues of different origin is essential for biomarker discovery. In drug research, it is necessary to comprehend the specificity profile of a given kinase inhibitor. Capture Compound Mass Spectrometry (CCMS) (Koster et al., Assay Drug. Dev. Technol. 5:381-390, 2007) addresses the need for a tool to physically isolate and reliably profile the binders of kinase inhibitors directly in biological samples. Capture Compounds™ are trifunctional probes: a selectivity function consisting of the kinase inhibitor interacts reversibly with the native target proteins in equilibrium, a photoactivatable reactivity function forms an irreversible covalent bond to the target protein upon irradiation, and a sorting function allows the captured protein(s) to be isolated and identified by mass spectrometric analysis in an affinity-driven manner. Capture Compounds™ with any kinase inhibitor as selectivity function can be synthesized. We here used staurosporine as the selectivity function because it targets and, therefore, allows profiling a broad range of kinases (Romano and Giordano, Cell Cycle 7:3364-3668, 2008). Furthermore, we give an example of the application of the staurosporine Capture Compound to isolate kinases from human liver-derived HepG2 cells.
