Proximity labeling: spatially resolved proteomic mapping for neurobiology

Abstract

Understanding signaling pathways in neuroscience requires high-resolution maps of the underlying protein networks. Proximity-dependent biotinylation with engineered enzymes, in combination with mass spectrometry-based quantitative proteomics, has emerged as a powerful method to dissect molecular interactions and the localizations of endogenous proteins. Recent applications to neuroscience have provided insights into the composition of sub-synaptic structures, including the synaptic cleft and inhibitory post-synaptic density. Here we compare the different enzymes and small-molecule probes for proximity labeling in the context of cultured neurons and tissue, review existing studies, and provide technical suggestions for the in vivo application of proximity labeling.

Figure 1.

Workflow and mechanism of proximity labeling. (a) An engineered enzyme (green) is genetically targeted to the subcellular region of interest (e.g., the iPSD) and covalently tags proximal endogenous proteins with a biotin handle. The gray shapes are endogenous proteins residing inside and outside the region of interest. Following cell lysis, biotinylated proteins are enriched with streptavidin beads, digested to peptides on-bead, then analyzed by liquid chromatography and tandem MS. (b) For peroxidase-based labeling using APEX, H₂O₂ is added for 1 minute to cells preloaded with biotin-phenol (BP; red B=biotin) to initiate labeling. APEX oxidizes BP into a phenoxyl radical, which covalently tags proximal endogenous proteins at electron-rich side chains such as tyrosine. (c) For biotin ligase-based labeling using BioID, exogenously-supplied free biotin is utilized together with endogenous ATP for 18–24 hours of labeling. BioID converts biotin to reactive bioAMP, which is released from the enzyme’s active site to react with lysine residues on proximal proteins.