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

Protocol
First Online:
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 
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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.

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

Authors and Affiliations

  1. 1.Functional Proteomics FacilityCentro Nacional de Biotecnología (CNB-CSIC), ProteoRed-ISCIIIMadridSpain

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