Unusual free oligosaccharides in human bovine and caprine milk

Abstract

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.

Glycoprofiling of Oligosaccharides of Regular and Lactose-Free Milk by Mass Spectrometry

“…The negative mode ESI spectra provide useful information (Figure 3) about sialic acid attached glycans because of the anionic nature of this molecule. Although conventional mass spectrometry is known to yield less information about anomericity and monosaccharide stereoisomers [41], some of fragment ions (Figure 3) in ion trap mass spectrometry that was used in our study provide useful information on linkage positions. Chromatographic separation of glycoforms could be performed by using specific columns and isomeric separation could be done either by state-of-art mass spectrometers.…”

Identification of the High Mannose N-Glycan Isomers Undescribed by Conventional Multicellular Eukaryotic Biosynthetic Pathways

“…We recently developed a mass spectrometry method, namely, logically derived sequence (LODES) tandem mass spectrometry (MS n ), for oligosaccharide structural determination. − LODES/MS n involves sequential low energy collision induced dissociation (CID) of oligosaccharides in multistage tandem mass spectrometry. LODES/MS n does not require the mass spectrum database of oligosaccharides or N -glycan standards, and it can determine the linkage positions, anomericities, and stereoisomers of monosaccharides in oligosaccharides.…”

A simple tandem mass spectrometry method for structural identification of pentose oligosaccharides

“…[47][48][49][50] Because it does not require a mass spectrum library of oligosaccharide standards, LODES/MS n is particularly useful for oligosaccharides with a structure that has not been identified previously. 49,50 LODES/MS n uses intact oligosaccharides, reducing the sample loss in permethylation/ reduction and simplifying the sample preparation process. The dissociation mechanisms of Xyl, 51 Ara, and Rib 52 have been studied recently, and extending LODES/MS n to pentose oligosaccharides is a natural progression.…”

“…[43][44][45][46] LODES/MS n has been used to determine the linkage positions, anomeric configurations, and stereoisomers of three common hexoses (glucose, galactose, and mannose) and two common N-acetylhexosamines (N-acetylglucosamine and N-acetylgalactosamine) in oligosaccharides. [47][48][49][50] Because it does not require a mass spectrum library of oligosaccharide standards, LODES/MS n is particularly useful for oligosaccharides with a structure that has not been identified previously. 49,50 LODES/MS n uses intact oligosaccharides, reducing the sample loss in permethylation/ reduction and simplifying the sample preparation process.…”