Profiling Proteins Involved in Peroxynitrite Homeostasis Using ROS/RNS Conditional Proteomics

Peroxynitrite (ONOO^-), the product of the diffusion-controlled reaction of superoxide (O₂^•-) with nitric oxide (NO^•), plays a crucial role in oxidative and nitrative stress and modulates key physiological processes such as redox signaling. While biological ONOO^- is conventionally analyzed using 3-nitrotyrosine antibodies and fluorescent sensors, such probes lack specificity and sensitivity, making high-throughput and comprehensive profiling of ONOO^--associated proteins challenging. In this study, we used a conditional proteomics approach to investigate ONOO^- homeostasis by identifying its protein neighbors in cells. We developed Peroxynitrite-responsive protein Labeling reagents (Porp-L) and, for the first time, discovered 2,6-dichlorophenol as an ideal moiety that can be selectively and rapidly activated by ONOO^- for labeling of proximal proteins. The reaction of Porp-L with ONOO^- generated several short-lived reactive intermediates that can modify Tyr, His, and Lys residues on the protein surface. We have demonstrated the Porp-L-based conditional proteomics in immune-stimulated macrophages, which indeed identified proteins known to be involved in the generation and modification of ONOO^- and revealed the endoplasmic reticulum (ER) as a ONOO^- hot spot. Moreover, we discovered a previously unknown role for Ero1a, an ER-resident protein, in the formation of ONOO^-. Overall, Porp-L represent a promising research tool for advancing our understanding of the biological roles of ONOO^-.