Comprehensive review of MS-based studies on N-glycoproteome and N-glycome of extracellular vesicles.

Extracellular vesicles (EVs) are lipid bilayer-enclosed particles that can be released by all type of cells. Whereas, as one of the most common post-translational modifications, glycosylation plays a vital role in various biological functions of EVs, such as EV biogenesis, sorting, and cellular recognition. Nevertheless, compared with studies on RNAs or proteins, those investigating the glycoconjugates of EVs are limited. An in-depth investigation of N-glycosylation of EVs can improve the understanding of the biological functions of EVs and help to exploit EVs from different perspectives. The general focus of studies on glycosylation of EVs primarily includes isolation and characterization of EVs, preparation of glycoproteome/glycome samples and MS analysis. However, the low content of EVs and non-standard separation methods for downstream analysis are the main limitations of these studies. In this review, we highlight the importance of glycopeptide/glycan enrichment and derivatization owing to the low abundance of glycoproteins and the low ionization efficiency of glycans. Diverse fragmentation patterns and professional analytical software are indispensable for analysing glycosylation via MS. Altogether, this review summarises recent studies on glycosylation of EVs, revealing the role of EVs in disease progression and their remarkable potential as biomarkers.