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Serum Exosome Isolation by Size-Exclusion Chromatography for the Discovery and Validation of Preeclampsia-Associated Biomarkers

  • Rosana Navajas
  • Fernando J. Corrales
  • Alberto ParadelaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1959)

Abstract

Exosomes are extracellular nanovesicles of complex and heterogeneous composition that are released in biofluids such as blood. The interest in the characterization of exosomal biochemistry has increased over the last few years as they convey cellular proteins, lipids, and RNA that might reflect the biological or pathological condition of the source cell. In particular, association of changes of exosome proteins with specific pathogenic processes arises as a promising method to identify disease biomarkers as for the pregnancy-related preeclampsia. However, the overlapping physicochemical and structural characteristics of different types of extracellular vesicles have hindered the consolidation of universally accepted and standardized purification or enrichment protocols. Thus, it has been recently demonstrated that the exosomal protein profile resulting from in-depth proteomics analyses is highly dependent on the preparation protocol used, which will determine the particle type specificity and the presence/absence of contaminating proteins.

In this chapter, an isolation method of serum exosomes based on size-exclusion chromatography (SEC) using qEV columns (Izon) is described. We show that this method is fast and reliable, as the population of exosomes isolated is homogeneous in terms of size, morphology, and protein composition. This exosome enrichment method is compatible with downstream qualitative and quantitative proteomic analysis of the samples.

Key words

Biomarkers Serum/plasma Exosomes Size-exclusion chromatography Shotgun proteomics Biomedicine Preeclampsia 

Notes

Acknowledgments

CNB-CSIC lab is a member of Proteored, PRB2-ISCIII and is supported by grant PT13/0001, of the PE I + D + i 2013–2016, funded by ISCIII and FEDER. We thank the technical staff of the CNB-CSIC electron microscopy facility for advice and technical expertise.

References

  1. 1.
    Gerszten RE, Wang TJ (2008) The search for new cardiovascular biomarkers. Nature 451(7181):949–952. https://doi.org/10.1038/nature06802CrossRefPubMedGoogle Scholar
  2. 2.
    Hanash SM, Pitteri SJ, Faca VM (2008) Mining the plasma proteome for cancer biomarkers. Nature 452(7187):571–579. https://doi.org/10.1038/nature06916CrossRefPubMedGoogle Scholar
  3. 3.
    Mateos J, Carneiro I, Corrales F et al (2017) Multicentric study of the effect of pre-analytical variables in the quality of plasma samples stored in biobanks using different complementary proteomic methods. J Proteome 150:109–120. https://doi.org/10.1016/j.jprot.2016.09.003CrossRefGoogle Scholar
  4. 4.
    Anderson NL, Anderson NG (2002) The human plasma proteome: history, character, and diagnostic prospects. Mol Cell Proteomics 1(11):845–867. https://doi.org/10.1074/mcp.R200007-MCP200CrossRefPubMedGoogle Scholar
  5. 5.
    Cao Z, Tang HY, Wang H et al (2012) Systematic comparison of fractionation methods for in-depth analysis of plasma proteomes. J Proteome Res 11(6):3090–3100. https://doi.org/10.1021/pr201068bCrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Faca V, Pitteri SJ, Newcomb L et al (2007) Contribution of protein fractionation to depth of analysis of the serum and plasma proteomes. J Proteome Res 6(9):3558–3565. https://doi.org/10.1021/pr070233qCrossRefPubMedGoogle Scholar
  7. 7.
    Polaskova V, Kapur A, Khan A et al (2010) High-abundance protein depletion: comparison of methods for human plasma biomarker discovery. Electrophoresis 31(3):471–482. https://doi.org/10.1002/elps.200900286CrossRefPubMedGoogle Scholar
  8. 8.
    Mathivanan S, Ji H, Simpson RJ (2010) Exosomes: extracellular organelles important in intercellular communication. J Proteome 73(10):1907–1920. https://doi.org/10.1016/j.jprot.2010.06.006CrossRefGoogle Scholar
  9. 9.
    Arbelaiz A, Azkargorta M, Krawczyk M et al (2017) Serum extracellular vesicles contain protein biomarkers for primary sclerosing cholangitis and cholangiocarcinoma. Hepatology 66(4):1125–1143. https://doi.org/10.1002/hep.29291CrossRefPubMedGoogle Scholar
  10. 10.
    Boukouris S, Mathivanan S (2015) Exosomes in bodily fluids are a highly stable resource of disease biomarkers. Proteomics Clin Appl 9(3-4):358–367. https://doi.org/10.1002/prca.201400114CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Sodar BW, Kovacs A, Visnovitz T et al (2017) Best practice of identification and proteomic analysis of extracellular vesicles in human health and disease. Expert Rev Proteomics 14(12):1073–1090. https://doi.org/10.1080/14789450.2017.1392244CrossRefPubMedGoogle Scholar
  12. 12.
    Lobb RJ, Becker M, Wen SW et al (2015) Optimized exosome isolation protocol for cell culture supernatant and human plasma. J Extracell Vesicles 4:27031. https://doi.org/10.3402/jev.v4.27031CrossRefPubMedGoogle Scholar
  13. 13.
    Goulopoulou S, Davidge ST (2015) Molecular mechanisms of maternal vascular dysfunction in preeclampsia. Trends Mol Med 21(2):88–97. https://doi.org/10.1016/j.molmed.2014.11.009CrossRefPubMedGoogle Scholar
  14. 14.
    Powe CE, Levine RJ, Karumanchi SA (2011) Preeclampsia, a disease of the maternal endothelium: the role of antiangiogenic factors and implications for later cardiovascular disease. Circulation 123(24):2856–2869. https://doi.org/10.1161/CIRCULATIONAHA.109.853127CrossRefPubMedGoogle Scholar
  15. 15.
    Venkatesha S, Toporsian M, Lam C et al (2006) Soluble endoglin contributes to the pathogenesis of preeclampsia. Nat Med 12(6):642–649. https://doi.org/10.1038/nm1429CrossRefPubMedGoogle Scholar
  16. 16.
    Maynard SE, Min JY, Merchan J et al (2003) Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest 111(5):649–658. https://doi.org/10.1172/JCI17189CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Kleinrouweler CE, Wiegerinck MM, Ris-Stalpers C et al (2012) Accuracy of circulating placental growth factor, vascular endothelial growth factor, soluble fms-like tyrosine kinase 1 and soluble endoglin in the prediction of pre-eclampsia: a systematic review and meta-analysis. BJOG 119(7):778–787. https://doi.org/10.1111/j.1471-0528.2012.03311.xCrossRefPubMedGoogle Scholar
  18. 18.
    Aebersold R, Mann M (2016) Mass-spectrometric exploration of proteome structure and function. Nature 537(7620):347–355. https://doi.org/10.1038/nature19949CrossRefPubMedGoogle Scholar
  19. 19.
    Lange V, Picotti P, Domon B et al (2008) Selected reaction monitoring for quantitative proteomics: a tutorial. Mol Syst Biol 4:222. https://doi.org/10.1038/msb.2008.61CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Rosana Navajas
    • 1
  • Fernando J. Corrales
    • 1
  • Alberto Paradela
    • 1
    Email author
  1. 1.Functional Proteomics FacilityCentro Nacional de Biotecnología (CNB-CSIC), ProteoRed-ISCIIIMadridSpain

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