Fig. 2.

Summarized outline of the major published enzyme-catalyzed proximity labeling assays. A, in BioID (20), a bait protein fused to a promiscuous biotin protein ligase (blue) is expressed in cells. Biotin is added to initiate biotinylation of closely associated proteins (gray). B, EMARS (17). In this example, HRP-coupled cholera toxin (tan) is added to cells and binds lipid raft-associated gangliosides in the plasma membrane (blue). Fluorescein- or biotin-conjugated aryl azide with hydrogen peroxide initiates labeling of neighboring proteins (gray). C, SPPLAT (19). HRP-coupled antibody (tan) to a plasma membrane target protein (blue) is added to cells. Biotin-tyramide with hydrogen peroxide initiates labeling of neighboring proteins (gray). D, use of APEX for organelle-specific labeling. Here, APEX (tan) has been engineered to be selectively expressed in the mitochondrial matrix as described (18). Cells are briefly incubated with a biotin-tyramide derivative (biotin-phenol) with hydrogen peroxide to initiate labeling of matrix-associated proteins (gray). In all cases, cells are lysed, and labeled proteins are isolated by affinity pulldown using immobilized streptavidin (for biotinylated proteins) or anti-fluorescein antibody (for fluorescein-tagged proteins). Samples are analyzed by mass spectrometry.