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<p>Lipid peroxidation products modify proteins during oxidative stress, but the residue-pair connectivity and structural consequences of these reactions remain difficult to define. Here, we redefine the lipid peroxidation product 4-oxo-2-nonenal (4-ONE) from a damaging electrophile into a chemoselective Cys-Lys covalent crosslinker. Through a chemoselective, two-step pathway, Michael addition to cysteine activates a latent aldehyde that cyclizes with lysine to form a stable pyrrole linkage under physiological conditions. We show that this chemistry supports late-stage peptide functionalization, macrocyclization and stapling, selective protein modification, and proteome-wide mapping of 4-ONE-reactive lysine and cysteine residues. Additionally, late-stage oxidation converts this pyrrole linkage into an MS-cleavable sulfoxide for site-resolved identification of linked residues through diagnostic link-site-containing fragments, a workflow we name COSMIc (Crosslink Oxidation to Sulfoxide for Mass-Cleavable Interactomics). COSMIc enables detection of structurally informative crosslinks in the human 26S proteasome, where peroxide-induced sulfoxide formation markedly improves fragment assignment and residue-pair confidence. Together, these findings repurpose 4-ONE from a toxic electrophile into a compact, metabolite-derived Cys-Lys crosslinker for covalent mapping and structural proteomics.</p>

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Reprogramming the lipid peroxidation product 4-ONE as a chemoselective cleavable crosslinker

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年6月25日

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  • Zachary E. Paikin,
  • John M. Talbott,
  • Anthony M. Ciancone,
  • Samrat Kundu,
  • Sukhendu Manna,
  • Bao Quang Gia Le,
  • Hitendra Negi,
  • Kylie J. Walters,
  • Francis J. O’Reilly,
  • Monika Raj

摘要

Lipid peroxidation products modify proteins during oxidative stress, but the residue-pair connectivity and structural consequences of these reactions remain difficult to define. Here, we redefine the lipid peroxidation product 4-oxo-2-nonenal (4-ONE) from a damaging electrophile into a chemoselective Cys-Lys covalent crosslinker. Through a chemoselective, two-step pathway, Michael addition to cysteine activates a latent aldehyde that cyclizes with lysine to form a stable pyrrole linkage under physiological conditions. We show that this chemistry supports late-stage peptide functionalization, macrocyclization and stapling, selective protein modification, and proteome-wide mapping of 4-ONE-reactive lysine and cysteine residues. Additionally, late-stage oxidation converts this pyrrole linkage into an MS-cleavable sulfoxide for site-resolved identification of linked residues through diagnostic link-site-containing fragments, a workflow we name COSMIc (Crosslink Oxidation to Sulfoxide for Mass-Cleavable Interactomics). COSMIc enables detection of structurally informative crosslinks in the human 26S proteasome, where peroxide-induced sulfoxide formation markedly improves fragment assignment and residue-pair confidence. Together, these findings repurpose 4-ONE from a toxic electrophile into a compact, metabolite-derived Cys-Lys crosslinker for covalent mapping and structural proteomics.