Assessment of vault particles in cancer cell line‐derived extracellular vesicle preparations

Extracellular vesicles (EVs) may contain a variety of molecular cargo, including proteins and nucleic acids. Vault particle components have been repeatedly reported in the literature as EV cargo. Here, we demonstrated that vault RNA (vtRNA) and major vault protein (MVP) were highly abundant in EV pellets enriched by differential centrifugation (DC) by qPCR and western blotting, respectively. RNase and proteinase treatment of DC-derived pellets demonstrated that most vtRNA and MVP were not enclosed and protected within an EV membrane. Vault-like particles were visualised in 100k DC pellets by cryo-transmission electron microscopy. EVs were enriched by size exclusion chromatography, and western blotting of individual fractions showed co-elution of EV markers and vault particle proteins. Immunocapture of EVs post-ultracentrifugation (100k DC pellet) showed co-purification of MVP, whereas EVs isolated by direct immunocapture from conditioned medium were MVP-negative. The current study highlights the importance of determining the topology of putative EV-associated components to determine if they are EV cargo or contaminants that have been co-purified.