Paired-probe chemoproteomic strategy enables global site-resolved quantification of lysine redox states

Site-specific quantification of lysine (Lys) redox states remains unexplored, as current approaches identify only a limited number of modification sites without determining their stoichiometry. Allysine, an oxidized form of Lys, provides a model posttranslational modification (PTM) for addressing this challenge by requiring selective enrichment and site-specific stoichiometric quantification. Here, we developed a quantitative chemoproteomic platform for paired redox-state isotopic stoichiometry mapping of lysine, termed PRISM-Lys. We used an Am-alkyne probe for allysine enrichment by reductive amination and developed a complementary Ald-alkyne probe to label Lys, with identical reactions generating the same chemical product. Coupling this paired-probe strategy with a quantitative chemoproteomic workflow enables global site-resolved quantification and stoichiometric analysis of Lys redox states across cells and tissues. Applied to ferroptosis, PRISM-Lys revealed bidirectional, site-selective remodeling of Lys oxidation events associated with ferroptotic progression. Together, PRISM-Lys establishes a paired-chemistry framework for global, site-resolved quantification of Lys redox states, providing a new quantitative perspective for investigating protein oxidation in biological systems.