Donkey milk IgG was probed for the site-specific N-glycosylation pattern through RP-UHPLC-MS/MS. The affinity-purified milk IgG was subjected to SDS-PAGE and proteomic analysis, which revealed the presence of subtypes. Multiple N-glycopeptides arising from the predicted donkey IgG1, IgG2, IgG3, IgG5, IgG6, and IgG7 subtypes' heavy-chain constant region were shown to contain glycans at the highly conserved glycosylation site NST in the CH2 domain. Differences in the peptide backbone with the NST site among subtypes generated after trypsin digestion resulted in the evaluation of the subtype-specific glycan pattern. Glycan sequence analysis indicated predominantly biantennary complex types with core fucosylation at the site NST. Interestingly, an additional site NQT in the CH1 domain of the heavy-chain constant region of IgG5 was found to possess mainly sialylated biantennary complex glycans with NeuAc and NeuGc. Structural diversity of glycans was mainly observed in the predicted donkey IgG1, IgG5, and IgG7, whereas IgG2, IgG3, and IgG6 resulted in the glycopeptides that are of low abundance in the analyzed samples. These findings would pave the way for a better understanding of donkey milk functional properties.

中文翻译：

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驴奶中免疫球蛋白 G 亚型的定点特异性 N 糖分析


通过 RP-UHPLC-MS/MS 探测驴奶 IgG 的位点特异性 N-糖基化模式。对亲和纯化的牛奶 IgG 进行 SDS-PAGE 和蛋白质组学分析，结果显示存在亚型。由预测的驴 IgG1、IgG2、IgG3、IgG5、IgG6 和 IgG7 亚型的重链恒定区产生的多个 N-糖肽在 CH2 结构域中高度保守的糖基化位点 NST 处含有聚糖。胰蛋白酶消化后产生的亚型之间肽骨架与 NST 位点的差异导致了亚型特异性聚糖模式的评估。聚糖序列分析表明，主要是双触角复合物类型，在位点 NST 处具有核心岩藻糖基化。有趣的是，发现 IgG5 重链恒定区 CH1 结构域中的另一个位点 NQT 主要具有与 NeuAc 和 NeuGc 的唾液酸化双触角复合物聚糖。聚糖的结构多样性主要在预测的驴 IgG1 、 IgG5 和 IgG7 中观察到，而 IgG2 、 IgG3 和 IgG6 导致分析样品中糖肽丰度较低。这些发现将为更好地理解驴奶的功能特性铺平道路。