Proximity labeling: A powerful tool for mapping protein interactions.

Protein-protein interactions (PPIs) are fundamental to cellular regulation, and their dysregulation contributes to numerous diseases. Conventional approaches for studying PPIs often lack sufficient spatial and temporal resolution and are limited in capturing weak, transient, or context-dependent molecular associations. Proximity labeling (PL) technologies address these limitations by using labeling enzymes or photocatalysts targeted to specific proteins, subcellular compartments, membranes, or other biological locations to generate reactive intermediates that covalently label nearby biomolecules in living systems, thereby providing an unbiased view of protein neighborhoods and molecular proximity landscapes. This review summarizes recent advances in enzyme-based and photocatalytic proximity labeling (PPL) platforms. We discuss whole-enzyme systems, with emphasis on their engineering optimization and applications in dynamic interactome mapping, organelle organization, and signaling regulation. Split-enzyme strategies are further reviewed as conditional labeling approaches that improve spatial specificity for studying inducible interactions and membrane contact sites. In addition, emerging PPL platforms based on metal complexes, organic chromophores, and genetically encoded photocatalysts are highlighted for their light-controlled activation and expanded applicability in genetically inaccessible systems. Finally, we compare the advantages, limitations, and suitable applications of different PL technologies and discuss future challenges, including improving biological compatibility, expanding tissue applications, and integrating PL with advanced omics and computational approaches.