Label-Free Liquid Chromatography–Mass Spectrometry Quantitation of Relative N- and O-Glycan Concentrations in Human Milk in Japan

Abstract

Human milk is abundant in carbohydrates and includes human milk oligosaccharides (HMOs) and N/O-glycans conjugated to proteins. HMO compositions and concentrations vary in individuals according to the maternal secretor status based on the fucosyltransferase 2 genotype; however, the profile of N/O-glycans remains uninvestigated because of the analytical complexity. Herein, we applied a label-free chromatography–mass spectrometry (LC–MS) technique to elucidate the variation in the composition and concentration of N/O-glycans in human milk. We used label-free LC–MS to relatively quantify 16 N-glycans and 12 O-glycans in 200 samples of Japanese human milk (1–2 months postpartum) and applied high performance anion exchange chromatography with pulsed amperometric detection to absolutely quantify the concentrations of 11 representative HMOs. Cluster analysis of the quantitative data revealed that O-glycans and several HMOs were classified according to the presence or absence of fucose linked to galactose while N-glycans were classified into a different group from O-glycans and HMOs. O-glycans and HMOs with fucose linked to galactose were more abundant in human milk from secretor mothers than from nonsecretor mothers. Thus, secretor status influenced the composition and concentration of HMOs and O-glycans but not those of N-glycans in human milk.