Maly, Dustin JBrush, Daniel2026-04-202026-04-202026Brush_washington_0250E_29317.pdfhttps://hdl.handle.net/1773/55461Thesis (Ph.D.)--University of Washington, 2026Protein kinases are regulated by local structural and microenvironmental features that are not fully captured by existing chemoproteomic methods, and comprehensive maps of lysine reactivity across the human kinome remain limited. Here, we developed a kinobead-coupled DIA-MS workflow to quantify kinase lysine reactivity in electrophile-treated samples by monitoring the intensity depletion of unmodified lysine-containing peptides. Using immobilized DB65 kinobeads to enrich folded kinases, we consistently quantified >1,000 lysines across >100 protein kinases and achieved ~4-fold deeper lysine coverage per kinase than previously reported proteome-wide lysine-reactivity datasets. Applying this framework to multiple lysine-reactive electrophile classes generated quantitative kinase engagement landscapes and showed that electrophile choice probes distinct subsets of kinase lysines. Formaldehyde- and activated ester-based labeling produced different patterns of kinase lysine engagement, indicating that kinase lysine behavior is not captured by a single global reactivity axis. Extending the same readout to lysine-reactive fragment electrophiles further showed that fragments do not broadly engage kinase lysines, but instead concentrate high-magnitude liganding responses at a smaller subset of sites, consistent with increased dependence on molecular recognition. Within this panel, a squarate ester fragment, ASQ, displayed a scout fragment-like profile, combining near-baseline median behavior with a distinct high-magnitude responder tail. Together, these studies establish a scalable platform for profiling chemically distinct kinase lysine environments and prioritizing candidate lysines for future mechanistic and covalent ligand discovery studies.application/pdfen-USnoneChemistryChemistryThesis