Kinase-centric chemoproteomics utilizing targeted affinity enrichment and state-of-the-art quantitative mass spectrometry analyses

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Protein phosphorylation by kinases serves as an important molecular switch in most cellular processes. Kinases activate and shut down signaling cascades, mediate protein-protein interactions and their dysregulation commonly leads to diseases like cancer. Because they act as integrators and effectors of cellular stimuli, the abundance and activity of kinases are often used as reporters for the cellular state. Kinase inhibitors have recently been used as affinity agents together with quantitative mass spectrometry (MS) analyses to profile kinase inhibitor selectivity. While demonstrating the ability to enrich kinases for global proteomic analyses, these studies did not functionally interrogate specific signaling pathways or the activation states of kinases. Here we suggest a strategy utilizing targeted affinity enrichment and quantitative mass spectrometry analyses enabling the in depth characterization of abundance of kinases and the functional state of the corresponding signaling modules. To achieve kinase-centric protein enrichment, two series of small molecule (SM) kinase inhibitor-based affinity agents will be prepared. The first series comprises type I kinase inhibitor analogs allowing selective or global, activation-state independent, enrichment of kinases and their interaction partners. The second series consists of type II kinase inhibitors facilitating activation state-dependant enrichment of kinases and associated proteins. In a proof of concept study, affinity matrices prepared from these inhibitor analogs will be used for the pathway-specific and activation state-dependent enrichment of kinases and associated cellular components involved in epidermal growth factor (EGFR) - mitogen activated protein kinase (MAPK) signaling. Seven different NCI60 cell lines resembling important human cancer pathologies will serve as the model proteomes. The enriched sub-proteomes will be analyzed by quantitative MS in regard to (i) enrichment efficiency of EGFR - MAPK signaling components, (ii) accuracy and reproducibility of the identification and quantitation of these components and (iii) the ability to characterize the enriched kinases and associated proteins, including post-translational modifications (PTMs). This should facilitate the functional characterization of the EGFR - MAPK signaling module. On a later stage, we wish to apply our approach to the superordinate ErbB signaling network and proteomes derived from patient samples. Our long term goal is to provide novel analytical tools for functional cancer proteomics enabling rapid and in depth characterization of aberrant kinase signaling. The proteome-level information obtained should complement genome-level information obtained from sequencing cancer genomes and thus extend the analytical armory for the fight against cancer.