MPA-KCro: Antibody-Free Crotonylome Profiling via 2‑Mercaptophosphonic Acid Michael Addition and Titanium(IV)-Immobilized Metal Affinity Chromatography Enrichment

Protein lysine crotonylation (KCro), a metabolically linked epigenetic modification, lacks comprehensive profiling methods due to expensive, cross-reactive antibodies. Herein, we developed an antibody-free strategy, MPA-KCro, exploiting 2-mercaptophosphonic acid (2-MPA) as a bifunctional probe. The thiol group undergoes selective Michael addition with the α,β-unsaturated crotonyl moiety, while the phosphonate handle enables efficient Ti4+-IMAC enrichment. Critically, the bio-orthogonal C–P bond resists enzymatic hydrolysis, preventing interference from endogenous phosphoproteins, and a characteristic immonium ion (m/z 294.09) enables unambiguous site-specific determination of Kcro. Applied to HeLa cell histones, the MPA-KCro method identified 22 crotonylation sites, including well-characterized residues (H3.1 K23, H2B K5) and 12 novel sites, enabling proteome-wide crotonylation mapping and functional investigation of the emerging epigenetic mark.