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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\u2010of\u2010the\u2010art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrep<jats:sup>TM<\/jats:sup> 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\u2010associated glycan sialyl\u2010Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. 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