Profiling Proteins Involved in Peroxynitrite Homeostasis Using ROS/RNS Conditional Proteomics
Peroxynitrite (ONOO<sup>–</sup>), the product of the diffusion-controlled reaction of superoxide (O<sub>2</sub><sup>•–</sup>) with nitric oxide (NO<sup>•</sup>), plays a crucial role in oxidative and nitrative stress and modulates key physiological pro...
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2025
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| Summary: | Peroxynitrite (ONOO<sup>–</sup>), the product of the diffusion-controlled reaction of superoxide (O<sub>2</sub><sup>•–</sup>) with nitric oxide (NO<sup>•</sup>), plays a crucial role in oxidative and nitrative stress and modulates key physiological processes such as redox signaling. While biological ONOO<sup>–</sup> is conventionally analyzed using 3-nitrotyrosine antibodies and fluorescent sensors, such probes lack specificity and sensitivity, making high-throughput and comprehensive profiling of ONOO<sup>–</sup>-associated proteins challenging. In this study, we used a conditional proteomics approach to investigate ONOO<sup>–</sup> homeostasis by identifying its protein neighbors in cells. We developed Peroxynitrite-responsive protein Labeling reagents (<b>Porp-L</b>) and, for the first time, discovered 2,6-dichlorophenol as an ideal moiety that can be selectively and rapidly activated by ONOO<sup>–</sup> for labeling of proximal proteins. The reaction of <b>Porp-L</b> with ONOO<sup>–</sup> generated several short-lived reactive intermediates that can modify Tyr, His, and Lys residues on the protein surface. We have demonstrated the <b>Porp-L</b>-based conditional proteomics in immune-stimulated macrophages, which indeed identified proteins known to be involved in the generation and modification of ONOO<sup>–</sup> and revealed the endoplasmic reticulum (ER) as a ONOO<sup>–</sup> hot spot. Moreover, we discovered a previously unknown role for Ero1a, an ER-resident protein, in the formation of ONOO<sup>–</sup>. Overall, <b>Porp-L</b> represent a promising research tool for advancing our understanding of the biological roles of ONOO<sup>–</sup>. |
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