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Proximity Labeling (PL) is a revolutionary proteomics technology that enables in situ capture of protein composition and interaction networks within defined spatial ranges in living cells or tissues. By fusing an engineered enzyme to a protein of interest (POI), short-lived reactive intermediates are generated upon substrate addition to covalently tag proximal biomolecules. These tagged proteins are then enriched by streptavidin beads and identified by high-sensitivity mass spectrometry, revealing the “molecular neighborhood” of the target protein in its native cellular environment. PL overcomes fundamental limitations of traditional co-immunoprecipitation and affinity purification mass spectrometry, which depend on stable protein associations and detergent-resistant complexes, providing a transformative solution for studying weak, transient, and membrane protein interactions. The core of PL lies in the fusion of a POI with an engineered enzyme. Upon addition of a specific substrate, the enzyme catalyzes the formation of reactive intermediates—such as biotinoyl-5’-AMP generated by biotin ligases (BioID series), or biotin-phenoxyl radicals generated by peroxidases (APEX series). These intermediates possess extremely short half-lives (microsecond to millisecond scale) and become inactivated after diffusing approximately 10-20 nm, ensuring that only proteins in spatial proximity to the target are covalently labeled. Following labeling, biotinylated proteins are efficiently enriched using streptavidin-coated magnetic beads, digested with trypsin, and analyzed by high-resolution mass spectrometry for identification and quantification.

Chemical Crosslinking Mass Spectrometry (XL-MS) introduces covalent crosslinks between proteins or protein complexes, followed by mass spectrometry analysis to identify protein-protein interactions and elucidate macromolecular complex architectures. Core Principle: Bifunctional chemical crosslinkers (e.g., BS3, DSS, glutaraldehyde) react with specific amino acid residues (primarily lysine ε-amino groups, but also cysteine, tyrosine, etc.) on two proteins, forming a “molecular bridge”. After enzymatic digestion (typically trypsin), the crosslinked peptides are identified by high-resolution MS, revealing spatial proximity and interaction sites.

PLA is an antibody-based, ultra-sensitive in situ technique for detecting protein-protein interactions. Two target proteins are recognized by specific primary antibodies, followed by binding of secondary antibodies conjugated with DNA oligonucleotide probes. When the two targets are within <40 nm, the probes are ligated into a circular template by a DNA ligase. Rolling circle amplification (RCA) generates long DNA products, which are detected by fluorescent oligonucleotide hybridization, with each interaction event appearing as a countable, discrete fluorescent focus.