Leveraging Chemical Proteomics and Mass Spectrometry to Investigate Kinases

Chemical proteomics is a field that employs chemical probes to detect enzyme activity, identify drug targets, and uncover novel small-molecule inhibitors. Chemical proteomics is frequently used in combination with liquid chromatography-mass spectrometry (LC-MS), a powerful tool to separate complex mixtures and identify probe-modified peptides. Throughout this dissertation, various chemoproteomic techniques are applied in attempt to study kinases. In chapter two, we first used an ATP-acyl phosphate probe to explain off-target activity of diacylglycerol kinase alpha inhibitors. Deconstruction of these inhibitors revealed that a thiazolopyrimidinone fragment was non-selective with regards to kinases and ATPases. In chapter three, we discovered that azetidines, 4-membered nitrogen heterocycles, are susceptible to nucleophilic attack from biological nucleophiles. We studied how to leverage this reaction and identify new small-molecule binding sites on diacylglycerol kinase alpha. Chapter four addresses challenges with LC-MS analysis for chemical proteomics. As probes become larger and increasingly hydrophobic, peptide separation and identification by LC-MS becomes more difficult. We found that traditional analytical methods needed to improve by altering chromatographic and fragmentation techniques of probe-modified peptides. By updating our methodology, we identified targets of a sulfur-triazole exchange probe capable of labeling kinases in situ. As chemical proteomics expands into numerous research sectors, the methods described in this study pave the way for improved LC-MS identification of probe targets.