Comprehensive Analysis of the Glycome and Glycoproteome of Bovine Milk-Derived Exosomes

Bovine milk-derived exosomes (BMDEs) have potential applications in the pharmaceutical industry as drug delivery carriers. A comprehensive analysis of protein glycosylation in exosomes is necessary to elucidate the process of targeted delivery. In this work, free oligosaccharides (FOSs), O-glycans, and N-glycans in BMDEs and whey were first analyzed through multiple derivation strategies. In summary, 13 FOSs, 44 O-glycans, and 94 N-glycans were identified in bovine milk. To analyze site-specific glycosylation of glycoproteins, a one-step method was used to enrich and characterize intact glycopeptides. A total of 1359 proteins including 114 glycoproteins were identified and most of these were located in the exosomes. Approximately 95 glycopeptides were initially discovered and 5 predicted glycosites were confirmed in BMDEs. Collectively, these findings revealed the characterization and distribution of glycans and glycoproteins in BMDEs, providing insight into the potential applications of BMDEs in drug delivery and food science.