Quantitative Proteomics of Human Blood Exosomes

Abstract— Exosomes are extracellular membrane vesicles secreted by cells into biological fluids. The membrane of exosomes protects their contents from degradation and contains markers of the cells producing them. Almost all cells of the body produce exosomes, however, tumor cells secrete them more intensively. However, initial stages of the study of exosome functions and identification of tumor protein biomarkers in their composition meet a serious problem of isolating pure, characterized exosome preparations. In this study we have performed quantitative proteomic analysis of human serum exosomes isolated using differential ultracentrifugation, ultracentrifugation in a sucrose cushion, or sedimentation of a serum exosomal fraction by means of the commercial Exosome Isolation Kit (Invitrogen from ThermoFisher Scientific Baltics, UAB, Lithuania). The protein composition of the obtained exosome samples was determined by mass spectrometric methods of selected reactions monitoring (SRM) and shotgun proteomic analysis. The resultant preparations were characterized by the content of the main markers (CD9, CD82, HSPA8, CD63). In the exosomes isolated from serum of healthy volunteers samples by ultracentrifugation in the sucrose cushion, the content of the above mentioned markers was determined as 32.85, 15.59, 6.07 fmol/μg of total protein, respectively. It was shown that the centrifugation method with the sucrose cushion was optimal for the isolation of exosomes. The other methods, including the commercial kit, did not yield positive results. Thus, results of this study have shown that the centrifugation using the sucrose cushion is the most optimal for serum exosome isolation.