In our previous work, we have invented a spatially-restricted protein labeling method for mapping subcellular proteome. Traditional proteomic approach requires serial physical isolations of cellular components, which are often tedious and sometimes impossible to achieve. Our new method utilizes an engineered peroxidase, APEX, to covalently tag the relevant proteome in living cells with a small molecule handle. Thereafter, we identify the labeled proteome via mass spec analysis.
APEX catalyzes the one-electron oxidation of phenol substrates into highly reactive and short-lived phenoxyl free radicals. These free radicals readily react with biomolecules such as proteins to form covalent adduct labels. Due to their short life-time in the aqueous solution, phenoxyl radicals have a diffusion radius on the order of tens of nanometers, which restricts the labeling reaction to the vicinity of the APEX enzyme. We applied this highly spatial-specific enzyme-mediated protein labeling strategy to map the proteome of mitochondrial sub-compartments in living cells. We are currently extending this approach to label ribosomes at the neuronal synapse. Combined with the high sensitivity of RNA sequencing, this would enable us to investigate local protein synthesis via ribosome profiling.