Water oxidation-driven histidine dioxidation enables probe-free proximity labeling

Photocatalytic proximity labeling (photo-PL) has emerged as a powerful tool for spatial proteomics in subcellular compartments. However, many photo-PL toolboxes rely on singlet oxygen (1O2) to generate highly unstable endoperoxide intermediates that must be trapped immediately by high concentrations of exogenous probes. This constraint can bias spatial proteome coverage, particularly in dynamic and heterogeneous compartments such as endosomes and exosomes, where probe accessibility is intrinsically nonuniform. Here, we develop IDM, an organic photocatalyst that generates hydroxyl (*OH) and superoxide (O *-) radicals via water oxidation instead of 1O , enabling a synergistic dual-radical mechanism for probe-free proximal protein mapping in live cells (PF-Map). The resulting radicals dioxidize proximal histidine residues into a persistent dioxidized state (His-2O) that is thermodynamically stabilized as lactam tautomers, which remain electrophilic and chemically addressable after cell lysis. By decoupling histidine oxidation from live{square}cell probe capture, PF{square}Map can minimize spatial bias arising from heterogeneous probe distribution. Applying PF{square}Map to intracellular vesicle trafficking, we find that both PF{square}Map and a probe{square}dependent workflow (PD{square}Map) robustly identify exosome markers, whereas PF{square}Map additionally reveals a hidden vesicle trafficking-related subproteome that PD-Map underestimated. Together, we establish a minimally biased photocatalytic strategy for spatial protein mapping in complex biological systems. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/731337v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@13da43dorg.highwire.dtl.DTLVardef@1e9cb7corg.highwire.dtl.DTLVardef@2e7557org.highwire.dtl.DTLVardef@19e16c_HPS_FORMAT_FIGEXP M_FIG C_FIG