Proteome Profiling of Exosomes Purified from a Small Amount of Human Serum: The Problem of Co-Purified Serum Components

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.

Small Extracellular Vesicles Isolated from Serum May Serve as Signal-Enhancers for the Monitoring of CNS Tumors

“…However, serum is still the preferred sample form for blood-based clinical diagnoses and it is a practical choice for future clinical developments. It should be noted that co-purification of proteins [46] and lipoprotein particles [47] in EV isolation methods is a common and well known challenge [31]. The presence of protein aggregates [48] and lipoproteins in sEV isolates may provide additional explanation for the lack of increase in the concentration of enriched sEV particles in cancer patients' serum, contrary to literature data on plasma [49] or serum samples [40].…”

Proteomics of Extracellular Vesicles: Update on Their Composition, Biological Roles and Potential Use as Diagnostic Tools in Atherosclerotic Cardiovascular Diseases

“…SEC ensures exosomes with high purity that can be used to measure levels of potential biomarkers in small-volume clinical samples [ 69 ]. SEC is often used in combination with other techniques to ameliorate the separation procedure, and sometimes it can be applied as the last step of the differential ultracentrifugation [ 102 ]. A commercial SEC column, known as qEV, is also available to obtain rapid and effective EV isolation, exosomes with higher purity than those obtained using differential ultracentrifugation or precipitation methods, but also reliable and reproducible results.…”

An optimized method for plasma extracellular vesicles isolation to exclude the copresence of biological drugs and plasma proteins which impairs their biological characterization

“…To investigate the functional role of plasma EVs (pEVs) accurately the purity of pEVs is highly important. The presence of immunoglobulins (Ig) in pEV samples is a general finding using ultracentrifugation (UC), polyethylene glycol precipitation (PEG) or size exclusion chromatography (SEC) as isolation methods, but is likely a co-isolated protein contaminating pEVs [9]. UC is based on sedimentation of solutes including EVs at a high centrifugation force.…”