RESUMO
There is a long-held consensus that several proteins are unique to small extracellular vesicles (EVs), such as exosomes. However, recent studies have shown that several of these markers can also be present in other subpopulations of EVs to a similar degree. Furthermore, few markers have been identified as enriched or uniquely present in larger EVs, such as microvesicles. The aim of this study was to address these issues by conducting an in-depth comparison of the proteome of large and small EVs. Large (16,500g) and small EVs (118,000g) were isolated from three cell lines using a combination of differential ultracentrifugation and a density cushion and quantitative mass spectrometry (tandem mass tag-liquid chromatography-tandem mass spectrometry) was used to identify differently enriched proteins in large and small EVs. In total, 6493 proteins were quantified, with 818 and 1567 proteins significantly enriched in small and large EVs, respectively. Tetraspanins, ADAMs and ESCRT proteins, as well as SNAREs and Rab proteins associated with endosomes were enriched in small EVs compared with large EVs, whereas ribosomal, mitochondrial, and nuclear proteins, as well as proteins involved in cytokinesis, were enriched in large EVs compared with small EVs. However, Flotillin-1 was not differently expressed in large and small EVs. In conclusion, our study shows that the proteome of large and small EVs are substantially dissimilar. We validated several proteins previously suggested to be enriched in either small or large EVs (e.g., ADAM10 and Mitofilin, respectively), and we suggest several additional novel protein markers.