Abstract
Untargeted proteomics analysis of extracellular vesicles (EVs) isolated from human serum or plasma remains a technical challenge due to the contamination of these vesicles with lipoproteins and other abundant serum components. Here we aimed to test a simple method of EV isolation from a small amount of human serum (<1 mL) using the size-exclusion chromatography (SEC) standalone for the discovery of vesicle-specific proteins by the untargeted LC–MS/MS shotgun approach. We selected the SEC fraction containing vesicles with the size of about 100 nm and enriched with exosome markers CD63 and CD81 (but not CD9 and TSG101) and analyzed it in a parallel to the subsequent SEC fraction enriched in the lipoprotein vesicles. In general, there were 267 proteins identified by LC–MS/MS in exosome-containing fraction (after exclusion of immunoglobulins), yet 94 of them might be considered as serum proteins. Hence, 173 exosome-related proteins were analyzed, including 92 proteins absent in lipoprotein-enriched fraction. In this set of exosome-related proteins, there were 45 species associated with the GO cellular compartment term “extracellular exosome”. Moreover, there were 31 proteins associated with different immune-related functions in this set, which putatively reflected the major role of exosomes released by immune cells present in the blood. We concluded that identified set of proteins included a bona fide exosomes components, yet the coverage of exosome proteome was low due to co-purified high abundant serum proteins. Nevertheless, the approach proposed in current work outperformed other comparable protocols regarding untargeted identification of exosome proteins and could be recommended for pilot exploratory studies when a small amount of a serum/plasma specimen is available.
Highlights
Extracellular vesicles (EVs) have gained a large interest in the recent decade due to their role in cell-to-cell communication
Total protein amount and levels of specific proteins considered as exosome markers (CD63, CD81, CD9, and TSG101 [2,17,18,19]) were monitored in the subsequent size-exclusion chromatography (SEC) fractions
The average size of the major type of vesicles measured in fraction 7 (F7) by the dynamic light scattering (DLS) was approx. 95 nm, while the average size of the major type of vesicles detected in fraction 9 (F9) was approx. 25 nm (Figure 1C); fraction 8 represented a mixture of both types of vesicles
Summary
Extracellular vesicles (EVs) have gained a large interest in the recent decade due to their role in cell-to-cell communication. Exosomes are the smallest virus-sized EVs (30–150 nm). They derive from the inward budding of the endosomal membrane to form the multivesicular body that fuses with the plasma membrane to release exosomes to the extracellular space [1]. The exosomal cargo consists of precisely selected molecules located inside these vesicles or associated with their membrane [2]. Exosomes could reach recipient cells in the local environment (paracrine mode) or could be transported to distant tissues via the circulation system (endocrine mode). Numerous investigations revealed an important role of exosomes in intercellular communication under both normal and pathological
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
