Figure 1.

Schematic workflow of peroxidase and biotin ligase based in vivo proximity labeling for mapping molecular interactions. Proximity labeling enzymes fused to a targeting protein are incubated with biotin or a biotin conjugated compound and convert it to a reactive biotin intermediate. Peroxidases oxidize biotin–phenol to reactive phenoxyl radicals using hydrogen peroxide while biotin ligases utilize ATP and biotin to catalyze the formation of reactive biotin-5′-AMP. The biotin intermediate is released from the enzyme and covalently biotinylates proteins in the proximity but not distal proteins. After lysis, the biotinylated proteins are enriched by a streptavidin-pulldown followed by proteolysis and are analyzed by mass spectrometry.