The approach reported by Rhee and colleagues* promises to address a major deficiency of current proteomics platforms—their inability to report on protein distribution within the very small confines of discrete cell organelles and compartments. Here, the team uses a genetically targetable enzyme that catalyzes the generation of short-lived, highly reactive radicals in live cells; in turn, these radicals covalently react with electron-rich amino acids such as Tyr, Trp, His, and Cys. The enzyme selected was ascorbate peroxidase (APEX), chosen for its stability and activity in all cellular compartments.
In initial studies, APEX was targeted to the mitochondrial matrix of human embryonic kidney (HEK) cells and labeling was started by adding biotin-phenol and H2O2 to the cell medium; the phenol portion of the reagent served as the source of radicals to tag the nearby proteins, while the biotin unit was intended for use in streptavidin-mediated microscopy analysis. Fixing the cells 1 min after labeling initiation served to also stop the APEX reaction (see Figure). Analysis of the experiment revealed that the radicals produced did indeed have a very short range of action (few tens of nanometers), allowing for good spatial resolution of the labeling reaction, and that labeling occurred in a strict APEX- and H2O2-dependent manner. The proof-of-concept work was extended to include various cytosol-oriented APEX fusions, as well as a mitochondrial matrix facing variant. Analysis of the proteome labeled in the latter case revealed a number of proteins previously unknown to be associated with the mitochondria.
The new technique appears to be well-validated through the present work and should be extendable to nearly all systems in which fusing APEX to an organelle- or compartment-targeting signal is available.