Unusual free trisaccharides in caprine colostrum discovered by logically derived sequence tandem mass spectrometry

Free oligosaccharides in human milk have many biological functions for infant health. The reducing end of most human milk oligosaccharides is lactose, and caprine milk was reported to contain oligosaccharides structurally similar to those present in human milk. The structures of oligosaccharides were traditionally determined using nuclear magnetic resonance spectroscopy or enzyme digestion followed by various detection methods, e.g., liquid. Mass spectrometry has much higher sensitivity than nuclear magnetic resonance spectroscopy and enzyme digestion. However, conventional mass spectrometry methods only determine part of the structures of oligosaccharides, i.e., compositions and linkage positions. In this study, we used the latest developed mass spectrometry method, namely logically derived sequence tandem mass spectrometry, to determine the complete structures (i.e., composition, linkage positions, anomericities, and stereoisomers) of free neutral trisaccharides in caprine colostrum and mature milk. The high sensitivity of mass spectrometry enables us to discover oligosaccharides of low abundance. Isomers of (Hex)₂HexNAc, (Hex)₃, and (Hex)₂Fuc which have not been reported before were identified. Many of them do not have lactose at the reducing end. Instead, the reducing end is either Glcβ-(1-4)-Glc or Glcβ-(1-4)-GlcNAc. These unusual oligosaccharides are higher in concentration and more structurally diverse in caprine colostrum than that in caprine mature milk and human milk. The structural diversity indicates more complicated biosynthetic pathways of caprine milk compared to that of human milk.