<p>Intracellular reactive oxygen species (ROS) are elevated in the pathological environment. Studying protein conformations and protein-protein interactions (PPIs) at these ROS-rich locations is crucial to understanding protein functions under oxidative stress. Chemical cross-linking mass spectrometry (XL-MS) has made an increasing contribution to structural biology and interactome. Crosslinkers react globally with proteins throughout the entire environment, and the complex crosslinking information interferes with the objective analysis of proteins in specific microenvironments. Furthermore, the most commonly used crosslinkers target lysine residues, which limits the diversity and coverage of crosslinking information. Therefore, it is necessary to achieve multi-targeting crosslinking in ROS-rich specific locations. In this study, a multi-targeting crosslinker was developed for ROS-triggered protein conformation analysis, which combined the boronate oxidation and nucleophilic labeling to achieve protein crosslinking of multiple nucleophilic amino acids under ROS stimulation (RT-MN crosslinker). The crosslinking efficiency of RT-MN was evaluated in the simulated ROS environment with native protein, and the diverse distance restraint information was highly compatible with the crystal structure, indicating its ability to elucidate protein conformations. The study extends the ROS stimulus-response concept to crosslinkers, which has the potential to capture abundant protein conformation information by reacting with multiple types of amino acids in ROS-rich specific locations of intact cells.</p>