Methylation in ambient ion source enables direct isomer differentiation of oligosaccharides by tandem mass spectrometry

Oligosaccharides are carbohydrate polymers with prebiotic, immunomodulatory, and antimicrobial activities. However, their structural complexity makes it challenging for characterization. In this work, we demonstrated a powerful instant in situ methylation approach to derivatize oligosaccharides with tetramethylammonium hydroxide (TMAH) in an ambient ion source known as direct analysis in real time (DART). The reaction occurs instantaneously when the sample and TMAH mixture is bombarded by excited-state helium from the DART source, and the methylated species are simultaneously charged through the Penning process. Mass spectrometry (MS) showed the formation of partial, full, and per-methylation products, along with methylated saccharide fragments. Unique fragmentation patterns can be generated because of the enhancement on the structural differences of isomers when fully or partially methylated. By using accurate mass and tandem MS (MS/MS), we demonstrated that trisaccharide and pentasaccharide isomers with subtle structural variations produced distinctively characteristic fragment ions. Isomeric saccharides in mixtures were qualitatively identified, and trisaccharide isomers in real samples were quantified. Varying degrees of polymerization of oligosaccharides, and their stereochemical and other isomeric differences were directly characterized. This method is significantly simpler compared to currently used workflow, and the time-consuming chromatographic separation step was eliminated.