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–.