Directed evolution of APOX for proximity labeling using phenols with high redox potentials.

Proximity labeling (PL) can identify endogenous proteins in specific subcellular regions. APEX2 (enhanced ascorbate peroxidase 2) enables PL with high temporal resolution by oxidizing biotin phenol (BP) into a radical that promiscuously tags nearby proteins, followed by the isolation and identification of the tagged biomolecules. However, the BP radical has a relatively large diffusion radius, limiting spatial resolution. Replacing the phenol in BP with nitrophenol (NP) could shorten the labeling radius by shortening the radical lifetime, but existing PL heme peroxidases cannot efficiently oxidize substrates with high redox potentials. We report the ultrahigh-throughput directed evolution of APOX (APEX with high redOX potential), a quadruple mutant of APEX2 with a higher reduction potential. Using APOX with a membrane-permeable alkyne-NP probe, we demonstrate PL in living mammalian cells, including proteomic mapping with excellent subcellular compartment specificity.