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Free oligosaccharides are abundant macronutrients in milk and involved in prebiotic functions and antiadhesive binding of viruses and pathogenic bacteria to colonocytes. Despite the importance of these oligosaccharides, structural determination of oligosaccharides is challenging, and milk oligosaccharide biosynthetic pathways remain unclear. Oligosaccharide structures are conventionally determined using a combination of chemical reactions, exoglycosidase digestion, nuclear magnetic resonance spectroscopy, and mass spectrometry. Most reported free oligosaccharides are highly abundant and have lactose at the reducing end, and current oligosaccharide biosynthetic pathways in human milk are proposed based on these oligosaccharides. In this study, a new mass spectrometry technique, which can identify linkages, anomericities, and stereoisomers, was applied to determine the structures of free oligosaccharides in human, bovine, and caprine milk. Oligosaccharides that do not follow the current biosynthetic pathways and are not synthesized by any discovered enzymes were found, indicating the existence of undiscovered biosynthetic pathways and enzymes., (© 2022. The Author(s).)

Carbohydrates have various functions in biological systems. However, the structural analysis of carbohydrates remains challenging. Most of the commonly used methods involve derivatization of carbohydrates or can only identify part of the structure. Here, we report a de novo method for completely structural identification of underivatised oligosaccharides. This method, which can provide assignments of linkages, anomeric configurations, and branch locations, entails low-energy collision-induced dissociation (CID) of sodium ion adducts that enable the cleavage of selective chemical bonds, a logical procedure to identify structurally decisive fragment ions for subsequent CID, and the specially prepared disaccharide CID spectrum databases. This method was first applied to determine the structures of four underivatised glucose oligosaccharides. Then, high-performance liquid chromatography and a mass spectrometer with a built-in logical procedure were established to demonstrate the capability of the in situ CID spectrum measurement and structural determination of the oligosaccharides in chromatogram. This consolidation provides a simple, rapid, sensitive method for the structural determination of glucose oligosaccharides, and applications to oligosaccharides containing hexoses other than glucose can be made provided the corresponding disaccharide databases are available.