Figure 2. Proximity labeling methods.

HRP and APEX are peroxidases that, when activated by H₂O₂, are able to turn biotin-phenol substrates into highly-reactive radicals that covalently tag neighboring proteins on electron-rich amino acids. HRP is inactive in a reducing environment, such as the cytosol, but functions extracellularly. BioID, a mutant form of the biotin ligase BirA, can convert biotin into radicals that can covalently tag neighboring proteins on lysine residues. PafA is a ligase that can covalently tag neighboring proteins with the small protein Pup onto lysine residues.