Abstract
<title>Abstract</title> <p>Photocatalytic proximity labeling (PPL) has emerged as a powerful strategy for mapping protein interactomes with high spatial and temporal control. However, its labeling characteristics—distance, efficiency, and impact from oxidative side reactions—remain incompletely elucidated, and a systematic comparison across different photocatalysts and probes has not yet been established. Moreover, studying intracellular interactomes remains challenging for current photocatalysts. Here we introduce a semi-quantitative framework that integrates site-specific antibody–photocatalyst conjugation with mass spectrometry to elucidate PPL mechanism. By precisely positioning photocatalysts within the well-defined geometries of monoclonal antibodies and antibody–antigen/Protein A binary complexes, we directly quantified labeling efficiency, mapped labeling distances at nanometer resolution, and discriminated covalent tagging from photo-induced oxidation. Using this platform, we show that diazirine-derived carbenes label antibodies predominantly within ~ 4 nm of the catalytic center, whereas aryl-azide-derived nitrenes label more intensely on distal regions. This divergent pattern is explained by the distinct in-solution half-life of activated intermediates and their different susceptibility towards oxidative species generated concomitantly during PPL. Leveraging this framework, we identified BODIPY-FL as a novel photocatalyst that enables efficient PPL with reduced oxidative byproducts. Confocal imaging and pull-down proteomics data further demonstrated that it has comparable performance with µMap method for antibody-targeted cell-surface interactome mapping. Attributed to the favorable physicochemical properties, BODIPY-FL also allows ligand-directed intracellular PPL. Collectively, this work establishes a standardized and mechanistically informative platform for evaluating and advancing PPL, which expands the chemical toolkit for spatially resolved interactome interrogation in living systems.</p>