Daramola Oluwatosin, Gautam Sakshi, Bennett Andrew I, Goli Mona, Guiterrez-Reyes Cristian D, Wang Junyao, Mechref Yehia
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas, USA.
J Sep Sci. 2025 Jun;48(6):e70187. doi: 10.1002/jssc.70187.
Glycosylation, a common post-translational modification, is crucial in various biological processes and diseases. While N-linked glycans have been widely studied, O-linked glycans and other small glycan structures, such as glycosphingolipid (GSL) glycans and free oligosaccharides, have been less explored due to their structural complexity and low abundance. To address the challenge of isomeric separation for these small glycans, we employed a mesoporous graphitized carbon (MGC) column, which offers an effective alternative to the nano-porous graphitized carbon (PGC) column. Previously, the MGC column demonstrated efficient isomeric separation of N-glycans and glycopeptides. In this study, we optimized chromatographic conditions using standard samples to extend the application of the MGC column to O-glycans, free oligosaccharides, and GSL glycans. These optimizations resulted in improved retention and isomeric separation. The method was validated through successful isomeric separation of O-glycans, free oligosaccharides, and GSL glycans derived from human milk, showcasing the MGC column's potential for comprehensive glycomics analysis of biological samples. This study underscores the robustness and reproducibility of the MGC column, highlighting its suitability for advanced glycomics, particularly in biological samples where O-glycans, GSL glycans, or free oligosaccharides play significant roles in disease progression.
糖基化是一种常见的翻译后修饰，在各种生物过程和疾病中起着至关重要的作用。虽然N-连接聚糖已得到广泛研究，但O-连接聚糖以及其他小聚糖结构，如糖鞘脂（GSL）聚糖和游离寡糖，由于其结构复杂性和低丰度，研究较少。为应对这些小聚糖的异构体分离挑战，我们采用了介孔石墨化碳（MGC）柱，它为纳米多孔石墨化碳（PGC）柱提供了一种有效的替代方案。此前，MGC柱已证明能有效分离N-聚糖和糖肽的异构体。在本研究中，我们使用标准样品优化了色谱条件，以将MGC柱的应用扩展到O-聚糖、游离寡糖和GSL聚糖。这些优化提高了保留率和异构体分离效果。该方法通过成功分离人乳来源的O-聚糖、游离寡糖和GSL聚糖的异构体得到验证，展示了MGC柱在生物样品全面糖组学分析中的潜力。本研究强调了MGC柱的稳健性和可重复性，突出了其适用于先进糖组学，特别是在O-聚糖、GSL聚糖或游离寡糖在疾病进展中起重要作用的生物样品中。
