Background: Exosomes and other types of extracellular vesicles present an important component of circulating plasma. Exosomes released by endothelial and blood cells account for majority of plasma exosomal population; exosomes secreted by other cells might cross tissue-plasma barrier and reach circulating plasma as well. Definitely, exosomes of different cellular origins are different by content and function. However, exosomal surface membrane interacts with plasma components. This interaction may alter composition of exosomal surface and hence, provide these vesicles with new functional properties. This study was aimed to estimate composition and possible functional role of proteins attached on the surface of plasma exosomes. Methods: Here, extracellular vesicles from human plasma were isolated by ultracentrifugation and treated by trypsin. Trypsinized and native exosomes were analyzed by nanoparticle tracking analysis, Western blotting and quantitative high-resolution mass spectrometry. Results: Surface-attached proteins were removed from exosomes isolated from plasma of healthy donors by incubation with serine protease (trypsin). Treatment did not impact exosomes integrity while slightly reduced hydrodynamic radius. Mass spectrometry revealed 259 exosomal proteins; among them 79 proteins were completely removed and more than half of the proteins were partially removed by trypsinization. Gene ontology functional annotation revealed mostly extracellular locations of proteins cleaved from a surface of the plasma exosomes. Moreover, proteins cleaved from the exosome surface are supposed to be implicated into integrin-linked kinase (ILK), focal adhesion kinase (FAK) and other pathways connecting cell surface with intracellular signaling cascades. Conclusion: Taken together, our results demonstrate that a surface of circulating exosomes is decorated by plasma proteins, and these proteins can mask tissue-specific characteristic of the exosomal surface membrane and provide exosomes with new and uniform properties.

Extracellular vesicles (EV) are secreted by cells of multicellular organisms. EV mediate specific mode of intercellular communication by "horizontal" exchange of substances and information. This phenomenon seems to have an essential biological significance and became a subject of intensive research. Biogenesis, structural and functional features of the EV is being commonly studies in in vitro condition. Several methods of EV isolation from cell culture medium are established, however selection of method might influence on obtained results. The choice of the optimal method depends usually from the amount of medium and the aims of the research while is still challenging issue. We performed a comparative analysis of four different methods of EV isolation from cell culture medium: differential ultracentrifugation, ultracentrifugation with a 30% sucrose/D2O "cushion", precipitation with plant proteins and immune-affinity capturing. EV isolated by different approaches were compared in terms of following parameters: size, concentration, morphology of EV, contamination by non-vesicular particles, content of exosomal tetraspanins on the EV surface, content of total proteins, RNA, and several glioma-associated miRNAs. Applied methods included nano-patricle tracking analysis (NTA), dynamic light scattering (DLS), cryo-electron microscopy, flow cytometry and RT-qPCR. On the base of obtained results, we developed practical recommendations that may help researchers to make a best choice of EV isolation method.