Quantitative Proteomics of Human Blood Exosomes

  • N. A. Shushkova
  • N. E. Vavilov
  • S. E. Novikova
  • T. E. Farafonova
  • O. V. Tikhonova
  • P.-C. Liao
  • V. G. ZgodaEmail author


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.


exosome biomarkers methods of isolation ultracentrifugation mass-spectrometry 



  1. 1.
    Popesko, B., Novák, P., Papadaki, S., and Hrabec, D., Transformations in Business and Economics, 2015, vol. 14, pp. 373−388.Google Scholar
  2. 2.
    Shen, H., Che, K., Cong, L., Dong, W., Zhang, T., Liu, Q., and Du, J., Oncotarget, 2017, vol. 8, pp. 36 812–36 823. Google Scholar
  3. 3.
    Logozzi, M., De Milito, A., Lugini, L., Borghi, M., Calabrò, L., Spada, M., Perdicchio, M., Marino, M.L., Federici, C., Iessi, E., Brambilla, D., Venturi, G., Lozupone, F., Santinami, M., Huber, V., Maio, M., Rivoltini, L., and Fais, S., PLoS One, 2009, vol. 4, e5219. CrossRefGoogle Scholar
  4. 4.
    Rolfo, C., Castiglia, M., Hong, D., Alessandro, R., Mertens, I., Baggerman, G., Zwaenepoel, K., Gil-Bazo, I., Passiglia, F., Carreca, A.P., Taverna, S., Vento, R., Santini, D., Peeters, M., Russo, A., and Pauwels, P., Biochim. Biophys. Acta, 2014, vol. 1846, pp. 539–546. Google Scholar
  5. 5.
    Carretero-González, A., Otero, I., Carril-Ajuria, L., de Velasco, G., and Manso, L., Cancer Microenviron., 2018, vol. 11, pp. 13–21. CrossRefGoogle Scholar
  6. 6.
    Pultz, D.A.B., Cordero da Luz, A.F., Faria, S.S., Ferreira de Souza, P.F.L., Tavares, C.B.P., Goulart, A.V., Fontes, W., Goulart, R.L., and Barbosa Silva, J.M., Int. J. Cancer, 2017, vol. 140, pp. 2397–2407. CrossRefGoogle Scholar
  7. 7.
    InvitrogenTM. User Guide: Total Exosome Isolation (from serum), 2012. (June). https://www.invitrogen.comGoogle Scholar
  8. 8.
    Hood, C.A., Fuentes, G., Patel, H., Page, K., Menakuru, M., and Park, J.H., J. Pept. Sci., 2008, vol. 14, pp. 97–101.CrossRefGoogle Scholar
  9. 9.
    Vaudel, M., Barsnes, H., Berven, F.S., Sickmann, A., and Martens, L., Proteomics, 2011, vol. 11, pp. 996–999. CrossRefGoogle Scholar
  10. 10.
    Momen-Heravi, F., Balaj, L., Alian, S., Mantel, P.Y., Halleck, A.E., Trachtenberg, A.J., Soria, C.E., Oquin, S., Bonebreak, C.M., Saracoglu, E., Skog, J., and Kuo, W.P., Biol. Chem., 2013, vol. 394, pp. 1253–1262. CrossRefGoogle Scholar
  11. 11.
    Mayer, M.P. and Bukau, B., Cell Mol. Life Sci., 2005, vol. 62, pp. 670–684.CrossRefGoogle Scholar
  12. 12.
    Kopylov, A.T., Lisitsa, A.V., and Zgoda, V.G., Biomedical Chemistry: Research and Methods, 2018, vol. 1, 119. Google Scholar
  13. 13.
    Clark, D.J., Fondrie, W.E., Yang, A., and Mao, L., J. Proteomics, 2016, vol. 133, pp. 161–169. CrossRefGoogle Scholar
  14. 14.
    Lenassi, M., Cagney, G., Liao, M., Vaupotic, T., Bartholomeeusen, K., Cheng, Y., Krogan, N.J., Plemenitas, A., and Peterlin, B.M., Traffic, 2010, vol. 11, pp. 110–122. CrossRefGoogle Scholar
  15. 15.
    Bobrie, A., Colombo, M., Krumeich, S., Raposo, G., and Théry, C., J. Extracell Vesicles, 2012, vol. 1, pp. 1–11. CrossRefGoogle Scholar
  16. 16.
    Caradec, J., Kharmate, G., Hosseini-Beheshti, E., Adomat, H., Gleave, M., and Guns, E., Clin. Biochem., 2014, vol. 47, pp. 1286–1292. CrossRefGoogle Scholar
  17. 17.
    Helwa, I., Cai, J., Drewry, M.D., Zimmerman, A., Dinkins, M.B., Khaled, M.L., Seremwe, M., Dismuke, W.M., Bieberich, E., Stamer, W.D., Hamrick, M.W., and Liu, Y., PLoS One, 2017, vol. 12, e0170628. CrossRefGoogle Scholar
  18. 18.
    Ioachim, E., Michael, M.C., Salmas, M., Damala, K., Tsanou, E., Michael, M.M., Malamou-Mitsi, V., and Stavropoulos, N.E., BMC Cancer, 2006, vol. 6, 140.CrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2019

Authors and Affiliations

  • N. A. Shushkova
    • 1
  • N. E. Vavilov
    • 1
  • S. E. Novikova
    • 1
  • T. E. Farafonova
    • 1
  • O. V. Tikhonova
    • 1
  • P.-C. Liao
    • 2
  • V. G. Zgoda
    • 1
    Email author
  1. 1.Institute of Biomedical Chemistry (IBMC)MoscowRussia
  2. 2.National Cheng-Kung UniversityTainanTaiwan

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