Imaging and Profiling of Proteins under Oxidative Conditions in Cells and Tissues by Hydrogen-Peroxide-Responsive Labeling

Reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂) can inflict damage to biomolecules under oxidative stress and also act as signaling molecules at physiological levels. Here we developed a unique chemical tool to elucidate the biological roles of ROS using both fluorescence imaging and conditional proteomics. H₂O₂-responsive protein labeling reagents (Hyp-L) were designed to selectively tag proteins under the oxidative conditions in living cells and tissues. The Hyp-L signal remained even after sample fixation, which was compatible with conventional immunostaining. Moreover, Hyp-L allowed proteomic profiling of the labeled proteins using a conditional proteomics workflow. The integrative analysis enabled the identification of ROS generation and/or accumulation sites with a subcellular resolution. For the first time, we characterized that autophagosomes were enriched with H₂O₂ in activated macrophages. Hyp-L was further applied to mouse brain tissues and clearly revealed oxidative stress within mitochondria by the conditional proteomics.