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

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.

Large-scale preparation and glycan characterization of sialylglycopeptide from bovine milk glycomacropeptide and its bifidogenic properties.

Bovine glycomacropeptide (GMP) is a 7,000-Da glycopolypeptide released from κ-casein during cheese making. The O-glycan chains linked to GMP have many biological activities, but their utilization for nutraceutical products is limited due to their low content. To concentrate the functional glycan chains of GMP, we prepared sialylglycopeptide concentrate (SGC) from GMP-containing whey protein concentrate via proteolytic digestion of peptide chains and concentration of sialylglycopeptide by ultrafiltration using membranes with a molecular weight cut-off of 1,000 Da. The abundant saccharides detected in the prepared SGC were N-acetylneuraminic acid (Neu5Ac: 32.3% wt/wt), N-acetylgalactosamine (11.3%), and galactose (10.2%), which constitute O-glycans attached to GMP. The Neu5Ac content in SGC was found concentrated at approximately 4.8-fold of its content in GMP-containing whey protein concentrate (6.8%). Structural analysis of O-glycopeptides by liquid chromatography tandem mass spectrometry identified 88 O-glycopeptides. Moreover, O-acetylated or O-diacetylated Neu5Ac was detected in addition to the previously characterized O-glycans of GMP. Quantitative analysis of O-glycan in SGC by fluorescence labeling of chemically released O-glycan revealed that a disialylated tetrasaccharide was the most abundant glycan (76.6% of the total O-glycan). We further examined bifidogenic properties of SGC in vitro, which revealed that SGC served as a more potent carbon source than GMP and contributes to the growth-promoting effects on certain species of bifidobacteria. Overall, our study findings indicate that SGC contains abundant O-glycans and has a bifidogenic activity. Moreover, the protocol for the preparation of SGC described herein is relatively simple, providing a high yield of glycan, and can be used for large-scale preparation.

Preparation and reactivity of iron complexes bearing anionic carbazole-based PNP-type pincer ligands toward catalytic nitrogen fixation.

Iron-chloride, -dinitrogen, and -methyl complexes bearing anionic carbazole-based PNP-type pincer ligands are designed, prepared and characterized by X-ray analysis. Some iron complexes are found to work as catalysts toward nitrogen fixation under mild reaction conditions.