Covalent capture and genetic code expansion enables chemoproteomic profiling and functional characterization of lysine acetoacetylation.

Lysine acetoacetylation (Kacac) driven by metabolite acetoacetic acid represents a molecular mechanism by which ketone bodies regulate cellular functions beyond energy provision. However, comprehensive characterization of Kacac has been hindered by technical limitations in detection and functional validation. Here, we report an integrated platform for systematic Kacac investigation. Exploiting the unique reactive ketone carbonyl moiety, we developed Aca-Bio, a hydroxylamine-based probe enabling specific enrichment of Kacac peptides through ketone-targeted covalent labeling and pH-controlled reversible enrichment. Application to mouse liver identified 260 Kacac sites across 125 proteins, revealing notable enrichment in metabolic pathways. Concurrently, we established a genetic code expansion system enabling site-specific Kacac incorporation. Using this approach, we demonstrated that K310acac in HMGCS2 substantially attenuates catalytic activity through impaired substrate binding. This dual-platform approach establishes a comprehensive framework for global profiling and site-specific functional characterization of Kacac, thereby facilitating systematic exploration of its physiological roles and pathological implications.