Proximity labeling technologies for multiscale spatial proteomics: from organelle contact sites to intercellular and...

Proximity labeling (PL) converts molecular proximity into covalent tags that can be enriched and identified by mass spectrometry. Faster ligases and engineered peroxidases have broadened the applications of PL. Genetically encoded split systems add spatial control, helping extend bait-centered mapping to organelle interfaces, intercellular interactions and extracellular communication. This Review discusses how these approaches address two related questions: which proteins occupy a defined interface, and how proteins move from their site of origin to a recipient compartment, cell or tissue. We examine how interface-associated proteins are identified and how source-destination labeling follows protein movement. We then consider the further evidence needed to explain their mechanisms and biological functions. By relating experimental design to the biological boundary under study, this Review offers practical guidance for selecting PL strategies across multiple scales.