Our ultimate aim was to develop and apply this technology to obtain insight into the molecular adaptations of drug-resistant cells with the goal of using this information to rationally target kinases contributing to imatinib resistance. Using multiple, structurally distinct kinase inhibitors, this MALDI-TOF/TOF MS based technology provides a high throughput, quantitative approach to interrogate the kinome as described earlier. Importantly, these studies demonstrated that kinase binding to MIBs was a function of both activity and expression, hence MIBs can be used to profile the ����activation state���� of the kinome. Our studies confirm this and show the utility of the MIB/MS approach to study kinome adaptations in drug-resistant cells and have identified significant quantitative differences in the kinomes of MYL and MYL-R cells. Multiple peptides with 95% confidence were obtained from these samples, allowing the quantification of multiple kinases simultaneously. Lyn is a SFK with an established role in promoting the survival of imatinib-resistant CML cells from patients and cell lines such as MYL-R independently of BCR-ABL mutations. MIB/MS confirmed the increased expression and activation of Lyn in MYL-R cells as reported initially by Ito and others. Using MIB/MS we also detected a substantial number of kinases not previously reported to be increased or decreased in imatinib-resistant cells. In three independent experiments our MIB/MS approach identified and quantified a total of 153 kinases, nearly 50% of the estimated expressed kinome. For the purpose of establishing a MYL-R kinome profile, the significance of these quantifications was established through statistical analysis and only kinase abundance ratios with Benjamini-Hochberg q-values,0.2 were considered to be significantly different. The MYL-R kinome profile revealed upregulation of multiple kinases involved in cell growth, anti-apoptosis and stress signaling. This included kinases such as MEK2 and ERK2, IKKa and others NEK9, PRPK, AAKG1, RIPK2 and PRKDC. The increased binding of MEK2 and IKKa to MIBs was confirmed to be activity dependent by two independent criteria. First, a greater amount of the phosphorylated kinases was captured on MIBs as determined by immunoblotting and second, this binding was reversed by phosphatase treatment of the samples. These studies Evofosfamide illustrate that kinase capture measured by MIB/MS is both a function of changes in kinase expression and kinase activation as reported earlier. In support of a pivotal role for Lyn in MYL-R cells, treatment with dasatinib, a Lyn and SFK inhibitor, prevented the binding of a large number of these kinases to MIBs. Further evidence for Lyn as a regulator of the MEK/ERK pathway was supported by our shRNA data and is consistent with earlier Torin 1 mTOR inhibitor observations demonstrating Lyn as an activator of MEK. By contrast, the mechanism by which Lyn regulates IKKa or other kinases in MYL-R cells remains to be elucidated.