Rapid Isolation of Extracellular Vesicles from Blood Plasma with Size-Exclusion Chromatography Followed by Mass Spectrometry-Based Proteomic Profiling

Abstract Background: Circulating small extracellular vesicles (sEVs) and its associated proteins are of great interest in the early detection of many diseases. However, there is no gold standard for plasma sEVs isolation, especially for proteomic profiling which could be largely affected by contamination such as lipoproteins. Previous studies suggested combinations of different sEVs isolation methods could improve the purity of the isolated fractions. Nevertheless, there is no systematic evaluation of size-exclusion chromatography (SEC), ultracentrifugation (UC) and their combination in a proteomic perspective. Results: Here we exhibited that SEC+UC showed comparable recovery of sEVs with higher purity in contrast to single-step UC or SEC isolation. In our proteomic analysis, there are 992 protein species identified in the sEVs fractions isolated by SEC+UC, much more than the sEVs fractions isolated by UC (453) or SEC (682) alone. As compared to Vesiclepedia and Exocarta databases, SEC+UC kept 584 previously identified sEV-associated proteins and 360 other proteins. Furthermore, detailed analysis suggested that sEV-associated proteins, such as CD9, CD81 and ITGB1, showed the better protein rank in SEC+UC group than UC group and SEC group. Lipoproteins, the most common contamination in sEVs proteomic analysis, along with other free-floating proteins in the plasma, were largely removed in SEC+UC. Conclusions: We suggested that combining SEC with UC could significantly improve the performance of mass-spectrum (MS)-based proteomic profiling in analyzing plasma-derived sEVs.

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Human Serum Extracellular Vesicle Proteomic Profile Depends on the Enrichment Method Employed

International Journal of Molecular Sciences ◽

10.3390/ijms222011144 ◽

2021 ◽

Vol 22 (20) ◽

pp. 11144

Author(s):

Mikel Azkargorta ◽

Ibon Iloro ◽

Iraide Escobes ◽

Diana Cabrera ◽

Juan M. Falcon-Perez ◽

...

Keyword(s):

Human Serum ◽

Size Exclusion Chromatography ◽

Extracellular Vesicles ◽

Membrane Attack Complex ◽

Protein Detection ◽

Size Exclusion ◽

Blood Proteins ◽

Proteomic Profiling ◽

Serum Samples ◽

Exclusion Chromatography

The proteomic profiling of serum samples supposes a challenge due to the large abundance of a few blood proteins in comparison with other circulating proteins coming from different tissues and cells. Although the sensitivity of protein detection has increased enormously in the last years, specific strategies are still required to enrich less abundant proteins and get rid of abundant proteins such as albumin, lipoproteins, and immunoglobulins. One of the alternatives that has become more promising is to characterize circulating extracellular vesicles from serum samples that have great interest in biomedicine. In the present work, we enriched the extracellular vesicles fraction from human serum by applying different techniques, including ultracentrifugation, size-exclusion chromatography, and two commercial precipitation methods based on different mechanisms of action. To improve the performance and efficacy of the techniques to promote purity of the preparations, we have employed a small volume of serum samples (<100 mL). The comparative proteomic profiling of the enriched preparations shows that ultracentrifugation procedure yielded a larger and completely different set of proteins than other techniques, including mitochondrial and ribosome related proteins. The results showed that size exclusion chromatography carries over lipoprotein associated proteins, while a polymer-based precipitation kit has more affinity for proteins associated with granules of platelets. The precipitation kit that targets glycosylation molecules enriches differentially protein harboring glycosylation sites, including immunoglobulins and proteins of the membrane attack complex.

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