Systematic and Quantitative Assessment of Hydrogen Peroxide Reactivity With Cysteines Across Human Proteomes

Abstract

Protein cysteinyl residues are the mediators of hydrogen peroxide (H₂O₂)-dependent redox signaling. However, site-specific mapping of the selectivity and dynamics of these redox reactions in cells poses a major analytical challenge. Here we describe a chemoproteomic platform to systematically and quantitatively analyze the reactivity of thousands of cysteines toward H₂O₂ in human cells. We identified >900 H₂O₂-sensitive cysteines, which are defined as the H₂O₂-dependent redoxome. Although redox sites associated with antioxidative and metabolic functions are consistent, most of the H₂O₂-dependent redoxome varies dramatically between different cells. Structural analyses reveal that H₂O₂-sensitive cysteines are less conserved than their redox-insensitive counterparts and display distinct sequence motifs, structural features, and potential for crosstalk with lysine modifications. Notably, our chemoproteomic platform also provides an opportunity to predict oxidation-triggered protein conformational changes. The data are freely accessible as a resource at http://redox.ncpsb.org/OXID/.