Protein kinases are key regulators of cell signaling. Importantly, perturbations in kinase-mediated signaling networks can often lead to human diseases such diabetes and cancer. Due to the low cellular abundance of most protein kinases, a kinase enrichment step is essential for mass spectrometry-based kinomic profiling. Chemical proteomics utilizing immobilized broad-spectrum kinase inhibitors as kinase-capturing tools represents one of the most efficient kinomic enrichment strategies. However, the existing kinase inhibitors are still inadequate in covering the entire kinome, hence there is a need for development of new broad-spectrum kinase inhibitors to fill this void.
In this study, we identified a novel bis-anilino pyrimidine exhibiting inhibitory activity against a broad range of kinases in vitro, and we further developed it into a kinase capture reagent CTx-0294885 (CTx). Kinomic enrichment using CTx-0294885 resin in MDA-MB-231 cells enabled the detection of over 235 kinases, including all members of the AKT family that had not been previously detected by other studies using existing broad-spectrum kinase inhibitors. Addition of CTx to a mixture of 3 commonly used reagents increased the number of kinase identifications to 261, representing the largest kinome coverage from a single cell line reported to date. Coupling phosphopeptide enrichment with affinity purification using the 4 inhibitors enabled the identification of over 800 high confidence kinase-derived phosphosites, approximately 10% of which localized to the activation loop, and including novel phosphosites on kinases BMP2K, MELK, HIPK2 and PRKDC.
We are currently applying the kinome enrichment strategies in combination with quantitative mass spectrometry in order to characterize the kinomes characteristic of specific breast cancer subsets and how they are regulated by particular oncogenic events.