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–.

过氧亚硝基阴离子（Peroxynitrite, ONOO–）是超氧阴离子（superoxide, O2•–）与一氧化氮（nitric oxide, NO•）经扩散控制反应生成的产物，在氧化应激与硝化应激中发挥关键作用，并调控氧化还原信号传导等核心生理过程。传统上，生物体系中的ONOO–通常通过3-硝基酪氨酸抗体（3-nitrotyrosine antibodies）与荧光传感器（fluorescent sensors）进行检测，但这类探针存在特异性与灵敏度不足的缺陷，使得高通量、系统性分析ONOO–关联蛋白质组的工作极具挑战性。本研究采用条件蛋白质组学（conditional proteomics）策略，通过鉴定细胞内ONOO–的蛋白质邻近互作分子，探究其稳态调控机制。我们开发了过氧亚硝基响应性蛋白质标记试剂（Peroxynitrite-responsive protein Labeling reagents, Porp-L），并首次发现2,6-二氯苯酚可被ONOO–选择性且快速活化，用于邻近蛋白质的特异性标记。Porp-L与ONOO–反应可生成多种短寿命活性中间体，能够对蛋白质表面的酪氨酸（Tyr）、组氨酸（His）与赖氨酸（Lys）残基进行化学修饰。我们在免疫刺激巨噬细胞中验证了基于Porp-L的条件蛋白质组学方法，不仅成功鉴定出已知参与ONOO–生成与修饰的蛋白质，还揭示内质网（ER）为ONOO–的富集热点区域。此外，我们还发现了内质网驻留蛋白Ero1a此前未被报道的ONOO–生成相关功能。综上，Porp-L是一款极具应用前景的研究工具，可助力我们深入解析ONOO–的生物学功能。