Profiling Proteins Involved in Peroxynitrite Homeostasis Using ROS/RNS Conditional Proteomics
Hao Zhu, Hiroaki Uno, Kyoichi Matsuba, Itaru Hamachi
Abstract
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 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 – .