Profiling proteins involved in peroxynitrite homeostasis using ROS/RNS conditional proteomics
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Peroxynitrite (ONOO − ), the product of the diffusion-controlled reaction of superoxide (O 2 •− ) 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 P er o xynitrite- r esponsive p rotein L abeling 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 − .