LC‐MS/MS of Permethylated O‐Glycans, Free Oligosaccharides, and Glycosphingolipid Glycans Using Mesoporous Graphitized Carbon Column

ABSTRACT 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.