on 06-11-201206:01 AM - edited on 10-15-202111:00 AM by Closed Account
Bomgarden RD, Etienne CL, Viner R, Rosenblatt MM, Cichon E, Rogers JC. Application Note 515 Here, we examined the relative expression and inhibition of kinases in SH-SY5Y neuroblastoma cells stably expressing TrkA or TrkB. We employed a proteomic approach using desthiobiotin nucleotide probes to specifically capture and profile the kinome of each cell line using mass spectrometry to identify labeled kinase active-site peptides (Figure 1). In addition, we assessed staurosporine and lestaurtinib inhibition of protein kinases using kinase active-site probes in combination with Tandem Mass Tag (TMT) reagents and validated our results using a parallel Western blot workflow (Figure 2). Finally, because this approach allows for the specifi c enrichment of adenine nucleotide binding proteins of interest, an SRM-based workflow could be used to enable targeted quantification as an alternative means of validating the relative expression and inhibition of the kinases, and may be employed as a follow-up to this study.