An in situ ascorbate peroxidase labeling in secreted exosomes identifies a stress-induced exosome proteome alteration

Abstract The extracellular vesicle exosome mediates intercellular communication by transporting macromolecules such as proteins and ribonucleic acids. Determining cargo content 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. Here we report an in situ labeling approach for exosome proteome profiling, termed Exosome-Proxy APEX Labeling (EPAL), which bypasses the exosome isolation steps. In EPAL, proteins either in the exosome biogenesis vesicles in cells or in secreted exosomes in the conditioned medium can specifically be biotinylated with expressing a variant of the engineered ascorbic peroxidase APEX that is fused to an exosome cargo protein such as CD63. Mass spectrometry analysis of the proteins biotinylated in exosomes secreted from kidney proximal tubule-derived cells reveals that oxidative stress can induce an alteration in exosome protein contents, including accumulation of ribosomal proteins in exosomes.