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.牛乳来源外泌体（Bovine milk-derived exosomes, BMDEs）作为药物递送载体在制药工业中具备潜在应用前景。为阐明靶向递送的分子机制，对外泌体蛋白质的糖基化模式开展全面分析至关重要。本研究首先通过多种衍生化策略，对牛乳来源外泌体及乳清中的游离寡糖（free oligosaccharides, FOSs）、O-聚糖（O-glycans）与N-聚糖（N-glycans）进行了系统性表征。经分析，最终在牛乳中共鉴定出13种游离寡糖、44种O-聚糖以及94种N-聚糖。为解析糖蛋白的位点特异性糖基化特征，本研究采用一步法富集并表征完整糖肽，最终共鉴定得到1359种蛋白质，其中包含114种糖蛋白，且绝大多数蛋白定位于外泌体组分中。本研究初步发现了约95条糖肽，并在牛乳来源外泌体中验证了5个预测的糖基化位点。综上，本研究揭示了牛乳来源外泌体中聚糖与糖蛋白的特征及分布规律，为其在药物递送与食品科学领域的潜在应用提供了重要的理论依据与研究思路。