- Proximity labeling is a powerful and versatile biochemical approach used to identify and study protein–protein interactions and the molecular composition of local cellular environments with high spatial and temporal resolution.
- The technique relies on engineered enzymes that catalyze the covalent tagging of nearby proteins—typically within a range of 10–20 nanometers—with a biotin or similar chemical handle. These tagged proteins can then be purified and identified by mass spectrometry or Western blotting, allowing researchers to map protein interactomes, identify organelle-specific proteomes, or study dynamic interaction networks in living cells.
- There are two major classes of proximity labeling enzymes: peroxidase-based (e.g., APEX, APEX2) and biotin ligase-based (e.g., BioID, TurboID).
- In the BioID method, a mutated biotin ligase (BirA*) is fused to a protein of interest. Upon addition of biotin to the culture medium, BirA* activates biotin to a reactive intermediate that covalently attaches to lysine residues on proteins in close proximity.
- TurboID and miniTurbo are improved versions of BioID that label proteins faster and more efficiently, enabling labeling over shorter time frames and even in living organisms. In contrast, APEX-based labeling uses a modified ascorbate peroxidase that generates short-lived radicals from biotin-phenol in the presence of hydrogen peroxide. These radicals rapidly label proximal proteins with biotin but have a very short diffusion range, increasing spatial specificity.
- Proximity labeling is particularly useful for studying transient, weak, or spatially constrained interactions, which may be missed by traditional methods such as co-immunoprecipitation or yeast two-hybrid assays. It allows researchers to capture the protein environment of membrane-bound proteins, nuclear subcompartments, synapses, mitochondria, and other organelles. Furthermore, it can be applied in live cells and even in whole organisms, offering a near-physiological snapshot of interaction landscapes.
- Despite its strengths, proximity labeling requires careful experimental design. The enzyme tag must not disrupt the function or localization of the bait protein, and labeling time and expression levels must be optimized to minimize background. Controls, including inactive enzyme variants and appropriate spatial reference proteins, are essential to distinguish specific interactions from nonspecific background.
- In conclusion, proximity labeling has revolutionized the study of protein interactions and subcellular proteomes by enabling covalent capture of transient and spatially defined protein environments in living systems. It complements imaging and biochemical techniques and is increasingly integrated with mass spectrometry-based proteomics for high-throughput discovery applications.
