Quintuple BRET Platform for Light-Free, Compartment-Resolved Proximity Proteomics in Neurons

We present the first lightfree, genetically minimal platform for spatiotemporally resolved proximity labeling across five neuronal compartments—synaptic, nuclear, endosomal, mitochondrial, and activitydependent microdomains. A covalent “catalyst–luciferase sandwich” architecture sitespecifically conjugates five distinct catalysts (Ce6, Ru(bpy)₃²⁺, Ir(III), CuEosin, Coporphyrin) to luciferases (NanoLuc, RLuc8, AkaLuc). Key features include Ca²⁺gated CalBRET tagging of active synapses with 500 ms precision, proximitygated catalysis via splitluciferase reconstitution or BRETtriggered quencher removal for sub10 nm resolution, and a dualradical strategy combining singlet oxygen and carbon radicals for microenvironmentadaptive labeling. Validated in primary neurons and organoids, the platform achieves compartmentexclusive labeling (5.1–10.4 nm radius), deeptissue compatibility (>3 mm penetration), and a 4.2× efficiency gain under hypoxia (1% O₂). By eliminating phototoxicity and integrating spatial, temporal, and functional dimensions, this system establishes a new standard for livecell neuronal proteomics.