Proximity Labeling of Biomolecular Condensates: Methods and Interpretation

Biomolecular condensates are dynamic and compositionally heterogeneous assemblies that organize transcription, RNA metabolism, stress adaptation, and proteostasis. Their weak interactions and rapid molecular exchange make conventional isolation prone to loss of spatial context. Proximity labeling (PL) creates covalent records of molecules surrounding a targeted enzyme or photocatalyst before lysis, but the recovered signal depends on chemistry, labeling duration, molecular abundance, accessibility, and dense–dilute phase partitioning. This narrative review provides a condensate-centered framework for method selection and interpretation. We compare peroxidase, biotin ligase, and photocatalytic strategies, including quantitative parameters and the distinction between direct RNA labeling, protein profiling, and whole-animal applications. Applications to stress granules, fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43) assemblies, transcriptional condensates, multiphase germ granules, and nuclear compartments illustrate condition-dependent composition. A cross-study comparison of protein and RNA maps highlights the effects of bait selection, stress, sampling time, and chemistry. We also discuss contact capture and directional trafficking as complementary measurements. Substrate partitioning, residue bias, bait fusion effects, reaction-induced stress, and enrichment background require explicit controls. Because proximity alone does not establish direct binding, liquid–liquid phase separation (LLPS), stable residence, or causality, quantitative imaging, appropriate models, and functional validation are needed to connect candidate lists with mechanisms.