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-.