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J Extracell Vesicles. 2019 Jun 3;8(1):1621131. doi: 10.1080/20013078.2019.1621131. eCollection 2019.

Different isolation approaches lead to diverse glycosylated extracellular vesicle populations.

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i3S-Institute for Research and Innovation in Health, University of Porto, Porto, Portugal.
IPATIMUP -Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal.
Instituto de Ciências Biomédicas Abel Salazar (ICBAS), University of Porto, Porto, Portugal.
Faculty of Medicine of the University of Porto, Porto, Portugal.
Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.


Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates.


Extracellular vesicles; OptiPrep density gradient; glycosylation; isolation protocols; size exclusion chromatography; total exosome isolation; ultracentrifugation

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