The extracellular vesicle exosome mediates intercellular communication by transporting macromolecules such as proteins and ribonucleic acids (RNAs). Determining cargo contents with high accuracy will help decipher the biological processes that exosomes mediate in various contexts. Existing methods for probing exosome cargo molecules rely on a prior exosome isolation procedure. Researchers at the Medical College of Georgia, Augusta University have developed an in situ labelling approach for exosome cargo identification, which bypasses the exosome isolation steps. In this methodology, a variant of the engineered ascorbate peroxidase APEX, fused to an exosome cargo protein such as CD63, is expressed specifically in exosome-generating vesicles in live cells or in secreted exosomes in the conditioned medium, to induce biotinylation of the proteins in the vicinity of the APEX variant for a short period of time. Mass spectrometry analysis of the proteins biotinylated by this approach in exosomes secreted by kidney proximal tubule-derived cells reveals that oxidative stress can cause ribosomal proteins to accumulate in an exosome subpopulation that contains the CD63-fused APEX variant.
An in situ strategy for labelling exosome proteins by using
the engineered ascorbate peroxidase APEX
An APEX2 variant with an annexed exosome-targeting signal is expected to localize to the key exosome biogenesis subcellular structure, namely ILVs inside MVEs, and eventually to be released in exosomes. When biotin-phenol and hydrogen peroxide are added to live cells, the APEX variant could induce biotinylation of proximate proteins specifically in ILVs (intracellular labelling). Alternatively, APEX2-mediated biotinylation could also be induced in the collected conditioned medium of the APEX-producing cell culture (extracellular labelling). The resulting biotinylated proteins would be captured with Streptavidin affinity matrix under a denaturing condition and identified with mass spectrometry analysis. The exosome-targetable APEX variants were delivered to cells by transducing either recombinant adeno-associated viral or lentiviral vectors in this study.