Proteomics analysis of plasma-derived exosomes extracted by thrombin pre-treated ExoQuick isolation

Exosomes are very small nanosized, membrane-bound vesicles, ranging from 30 to 150 nm in size, are originated and released from a variety of cells from body fluids including blood plasma. The isolation of pure exosome from blood plasma is challenging since it is rich in various contaminants from diverse origins. We applied ExoQuick with thrombin for purification of plasma-derived exosome from healthy individual. Immunoassays confirmed the presence of proteins that are classically associated to exosomes (CD9, CD63 and CD81) but these proteins have not been detected by MS. We found several common proteins enriched in our data set compared with plasma-derived exosome markers from databases. Moreover, proteomics analysis and external database integration revealed an enrichment of those extracellular vesicles. The Gene Ontology data analyses determined the presence of proteins that are involved in catalytic, transporter functions and protein metabolism activities. These results allowed the detection of tetraspanin i.e. TSPAN14 in plasma where proteins were undetected by other studies. Furthermore, this study confirmed the detection of CD5L and LGALS3BP proteins. Although the undouble presence of plasma contaminants hamper MS analysis, still proteins with biomarker potential (TSPAN14, CD5L and LGALS3BP) are accessible in a reasonable sample processing frame that outperforms high-purity focused extraction methods e.g. SEC. The study suggests the use of TSPAN14, CD5L and LGALS3BP as new exosomal markers of plasma-derived exosomes in quantitative proteomics analysis.