The study of glycopeptides is associated with challenges regarding the microheterogeneity of different isomeric glycans occupying the same glycosylation sites in glycoproteins. It is immensely valuable to perform both qualitative and quantitative site-specific glycosylation analysis of glycopeptide isomers due to their link to several diseases. Achieving isomeric separation of glycopeptides is particularly challenging due to the low abundance of glycopeptides as well as inefficient ionization. Although some methods have demonstrated the isomeric separation of glycopeptides, a more efficient nanoflow-based stationary phase is needed for the isomeric separation of both - and -glycopeptides. In this study, the separation of - and -glycopeptide isomers at 75 °C was achieved with an in-house packed 1 cm long mesoporous graphitized carbon (MGC) column. Different gradient compositions of the optimized mobile phase for separating permethylated glycans on MGC column were tested, and we observed efficient separation of - and -glycopeptide isomers at a gradient elution time of 120 min. After achieving the isomeric separation of sialylated glycopeptides from model glycoproteins derived from bovine fetuin, the separation of isomeric glycopeptides derived from asialofetuin, α-1 glycoprotein and human blood serum were also demonstrated. Furthermore, the developed method for the separation of isomeric - and -glycopeptide on MGC column showed high reproducibility over three months. We observed an average retention time shift of 1 min and consistent resolution of separated peaks throughout three months. MGC column can serve as an efficient tool for obtaining the isomeric separation of - and -glycopeptide from complex biological samples in future studies. This will enable a more profound understanding of the roles played by isomeric - and -glycopeptide in important biological processes and their correlations to various disease progressions.

中文翻译：

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介孔石墨化碳柱上异构 N-和 O-糖肽的 LC-MS/MS


糖肽的研究与糖蛋白中占据相同糖基化位点的不同异构聚糖的微观异质性挑战相关。由于糖肽异构体与多种疾病的联系，对糖肽异构体进行定性和定量位点特异性糖基化分析非常有价值。由于糖肽丰度低且电离效率低，实现糖肽异构体分离尤其具有挑战性。尽管一些方法已经证明了糖肽的异构体分离，但需要更有效的基于纳流的固定相来实现α和β糖肽的异构体分离。在本研究中，使用内部填充的 1 厘米长介孔石墨化碳 (MGC) 柱在 75 °C 下实现了 - 和 - 糖肽异构体的分离。对用于在 MGC 柱上分离全甲基化聚糖的优化流动相的不同梯度组成进行了测试，我们观察到在 120 分钟的梯度洗脱时间下，β-和-糖肽异构体的有效分离。在实现牛胎球蛋白模型糖蛋白中唾液酸化糖肽的异构体分离后，还证明了脱唾液酸胎球蛋白、α-1糖蛋白和人血清中异构糖肽的分离。此外，所开发的在 MGC 柱上分离异构体和糖肽的方法在三个月内表现出高重现性。我们观察到三个月内平均保留时间偏移为 1 分钟，分离峰的分辨率保持一致。在未来的研究中，MGC 柱可以作为从复杂生物样品中分离 α 和 β 糖肽异构体的有效工具。 这将使人们更深刻地了解异构体和糖肽在重要生物过程中所发挥的作用及其与各种疾病进展的相关性。