Quantitative proteomics of human blood exosomes

Exosomes are extracellular membrane vesicles secreted by cells into biological fluids. The outer membrane of exosomes protects their content from degradation and contains markers of the parent cell. Almost all cells of the body produce exosomes, however, tumor cells secrete them more intensively. Due to fact that exosomes contain proteins of cells secreting them, these vesicles could be a valuable source for biomarkers discovery. Currently, a number of studies prove the participation of exosomes in carcinogenesis. However, there is a problem of isolating pure and characterized exosomes for further use in investigation of functions or identification of tumor protein biomarkers. In this work, we have performed experiments on exosomes isolation from human plasma by three methods: differential ultracentrifugation, ultracentrifugation in sucrose cushion, sedimentation of the exosomal fraction from serum by using a commercial kit. The protein composition of the obtained samples was determined by mass spectrometric methods of selected reactions monitoring (SRM) and shotgun proteomic analysis. The obtained exosomal samples were searched for the presence of exosomal markers (CD9, CD82, HSPA8, CD63). In the samples of exosomes isolated by ultracentrifugation with the sucrose cushion, the content of the above markers was determined as 32.85, 15.59, 6.07 fmol/mg of total protein, correspondently. It was shown that the centrifugation method with the sucrose cushion was optimal for the isolation of exosomes.