Precise and In Vivo-Compatible Spatial Proteomics via Bioluminescence-Triggered Photocatalytic Proximity Labeling

Protein function is closely tied to its localization and interactions, which can be mapped using proximity labeling (PL). Traditional PL methods, such as peroxidases and biotin ligases, suffer from toxicity or high background. While visible-light-triggered photocatalytic labeling offers great potential, it is limited by light-induced background and restricted in vivo applications. Here we present BRET-ID, an in vivo-compatible PL technology for precise mapping of membraneless organelles and transient protein-protein interactions with subminute temporal resolution. BRET-ID combines a genetically encoded photocatalyst and NanoLuc luciferase, locally generating blue light to activate the photocatalyst via bioluminescence resonance energy transfer (BRET). This activation produces singlet oxygen, which oxidizes nearby proteins for analysis with a streamlined chemoproteomic workflow. BRET-ID enables precise mapping of ER membrane proteins, exhibiting high spatial specificity. Leveraging its high temporal resolution, BRET-ID provides 1 min snapshots of dynamic GPCR interactions during ligand-induced endocytosis. Additionally, BRET-ID identifies G3BP1-interacting proteins in arsenite-stressed cells and tumor xenografts, uncovering novel stress granule components, including the mTORC2 subunit RICTOR. BRET-ID serves as a powerful genetically encoded tool for studying protein localization and molecular interactions in living organisms.