A puromycin-dependent activity-based sensing probe for histochemical staining of hydrogen peroxide in cells and animal tissues

Hydrogen peroxide (H₂O₂) is a key member of the reactive oxygen species family of transient small molecules that has broad contributions to oxidative stress and redox signaling. The development of selective and sensitive chemical probes can enable the study of H₂O₂ biology in cell, tissue and animal models. Peroxymycin-1 is a histochemical activity–based sensing probe that responds to H₂O₂ via chemoselective boronate oxidation to release puromycin, which is then covalently incorporated into nascent proteins by the ribosome and can be detected by antibody staining. Here, we describe an optimized two-step, one-pot protocol for synthesizing Peroxymycin-1 with improved yields over our originally reported procedure. We also present detailed procedures for applying Peroxymycin-1 to a broad range of biological samples spanning cells to animal tissues for profiling H₂O₂ levels through histochemical detection by using commercially available anti-puromycin antibodies. The preparation of Peroxymycin-1 takes 9 h, the confocal imaging experiments of endogenous H₂O₂ levels across different cancer cell lines take 1 d, the dot blot analysis of mouse liver tissues takes 1 d and the confocal imaging of mouse liver tissues takes 3–4 d.