Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, 79409-1061, USA.
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, 79409-1061, USA.
Anal Chim Acta. 2024 Aug 15;1317:342907. doi: 10.1016/j.aca.2024.342907. Epub 2024 Jun 25.
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 N- and O-glycopeptides.
In this study, the separation of N- and O-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 N- and O-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 N- and O-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 N- and O-glycopeptide from complex biological samples in future studies. This will enable a more profound understanding of the roles played by isomeric N- and O-glycopeptide in important biological processes and their correlations to various disease progressions.
糖肽的研究与不同异构体糖占据糖蛋白中相同糖基化位点的微不均一性有关。由于其与多种疾病有关，对糖肽异构体进行定性和定量的糖基化分析是非常有价值的。由于糖肽的丰度低且离子化效率低，因此实现糖肽异构体的分离极具挑战性。尽管有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米流的固定相来分离 N-和 O-糖肽的异构体。
本研究在 75°C 下用自制的 1cm 长介孔石墨化碳（MGC）柱实现了 N-和 O-糖肽异构体的分离。测试了不同优化的流动相梯度组成，用于分离 MGC 柱上的全甲基化聚糖，我们观察到在梯度洗脱时间为 120min 时，N-和 O-糖肽异构体得到有效分离。在实现了从牛胎球蛋白衍生的模型糖蛋白中分离出唾液酸化糖肽的异构体之后，还证明了从脱唾液酸胎球蛋白、α-1 糖蛋白和人血清中分离出的异构体糖肽。此外，在 MGC 柱上分离异构体 N-和 O-糖肽的方法在三个月内显示出很高的重现性。我们观察到平均保留时间的变化为 1min，并且在三个月内分离出的峰的分辨率保持一致。
MGC 柱可作为未来研究中从复杂生物样品中获得 N-和 O-糖肽异构体分离的有效工具。这将使我们能够更深入地了解异构体 N-和 O-糖肽在重要生物学过程中所扮演的角色，以及它们与各种疾病进展的相关性。
