Glycoproteomic Analysis of Human Urinary Exosomes

Exosomes represent a class of secreted biological vesicles, which have recently gained attention due to their function as intertissue and interorganism transporters of genetic materials, small molecules, lipids, and proteins. Although the protein constituents of these exosomes are often glycosylated, a large-scale characterization of the glycoproteome has not yet been completed. This study identified 3144 unique glycosylation events belonging to 378 glycoproteins and 604 unique protein sites of glycosylation. With these data, we investigated the level of glycan microheterogeneity within the urinary exosomes, finding on average 5.9 glycans per site. The glycan family abundance on individual proteins showed subtle differences, providing an additional level of molecular characterization compared to the unmodified proteome. Finally, we show protein site-specific changes in regard to the common urinary glycoprotein, uromodulin. While uromodulin is an individual case, these same site-specific analyses provide a way forward for developing diagnostic glycoprotein biomarkers with urine as a noninvasive biological fluid. This study represents an important first step in understanding the functional urinary glycoproteome.外泌体（Exosomes）是一类分泌型生物囊泡，近年来因其作为遗传物质、小分子、脂质与蛋白质的组织间及生物体间转运载体的功能而受到广泛关注。尽管此类外泌体的蛋白质组分通常存在糖基化修饰，但目前尚未完成对其糖蛋白质组（glycoproteome）的大规模表征。本研究共鉴定得到378种糖蛋白对应的3144个独特糖基化事件，以及604个独特的蛋白质糖基化位点。借助上述数据，本研究对尿液外泌体中的聚糖微异质性水平展开分析，结果显示每个位点平均存在5.9种聚糖。相较于未修饰的蛋白质组，单个蛋白质的聚糖家族丰度差异较为细微，却为分子表征提供了额外维度。最后，我们针对常见尿液糖蛋白——尿调蛋白（uromodulin）展示了其位点特异性变化。尽管尿调蛋白仅为单个案例，但这类位点特异性分析为开发以尿液作为非侵入性生物体液的诊断用糖蛋白生物标志物指明了可行路径。本研究为解析功能性尿液糖蛋白质组迈出了重要的第一步。