4-ONE as a Chemoselective Biological Glue for Site-Resolved Protein Crosslinking and Proteomic Profiling

ABSTRACT: Protein crosslinking governs structure, stability, and interaction networks in biology, yet current chemical crosslinkers lack selectivity, produce heterogeneous products, and require pre-installed cleavable handles for analysis. Here, we report a conceptually distinct strategy that harnesses the intrinsic reactivity of the lipid peroxidation product 4-oxo-nonenal (4-ONE) to enable selective and programmable protein crosslinking. We show that 4-ONE operates as a biological glue that follows defined chemical logic: an initial Michael addition to cysteine activates a latent aldehyde function, which subsequently reacts with lysine to form a stable pyrrole crosslink. Building on this reactivity, we introduce COSMIC (Crosslink Oxidation to Sulfoxide for Mass-Cleavable Interactomics), a late-stage oxidation strategy that transforms stable crosslinks into mass-cleavable sulfoxides that generate distinct fragmentation signatures for lysine and cysteine, enabling direct site-resolution by LC-MS/MS. This platform enables peptide stapling and macrocyclization, protein conjugation, identification of hyperreactive Lys and Cys sites prone to 4-ONE modification, and live-cell crosslinking analysis. Together, these results establish a generalizable chemical strategy for decoding metabolite-driven protein crosslinking and its impact on toxicity.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Disease Free

SUBMITTER: Riley Hughes  

LAB HEAD: Monika Raj

PROVIDER: PXD070252 | Pride | 2026-05-26

REPOSITORIES: Pride