Comprehensive Comparison of Bioactive N-Glycans among Seven Species of Livestock and Poultry Plasma Using a Relative Quantification Strategy.

Glycans have been proven to play special roles in keeping human health as a class of nutritional and bioactive ingredients in many food materials. However, their broad use in the food industry is hindered by the lack of comprehensive analytical methods for high-quality food glycomics studies and large-quantity raw materials for their production. This study focuses on structural identification and quantitative comparison of bioactive N-glycans in seven species of livestock and poultry plasma as potential natural glycan resources by a novel comprehensive relative quantification strategy based on stable isotope labeling with nondeuterated and deuterated 4-methyl-1-(2-hydrazino-2-oxoethyl)-pyridinium bromide (d0/d7-HMP) in combination with linkage-specific derivatization of sialic acid residues. Methodological validation of the method in terms of detection sensitivity, signal resolution, quantification linearity, precision, and accuracy on model neutral and complicated sialylated glycans demonstrated its advantages over the existing methods. Based on this method, a series of bioactive N-glycans were found in seven species of livestock and poultry plasma, and their differences in structure, abundance percentages, and relative contents of N-glycans were revealed, demonstrating their excellent applicability for comprehensive food glycomics analysis and great exploitation potential of these plasma samples as large-quantity raw materials in producing bioactive N-glycans for application in food and pharmaceutical industries.

Improved Glycoqueuing Strategy Reveals Novel α2,3-Linked Di-/Tri-Sialylated Oligosaccharide Isomers in Human Milk.

Sialylated human milk oligosaccharides (SHMOs) possess unique biological activities. Qualitative and quantitative analyses of SHMOs at different lactation stages are limited by interference from neutral oligosaccharides, glycan structural complexity, and low detection sensitivity. Herein, our previously developed glycoqueuing strategy was improved and applied to enable an isomer-specific quantitative comparison of SHMOs between colostrum milk (CM) and mature milk (MM). A total of 49 putative structures were determined, including 1 α2,6-linked and 13 α2,3-linked isomers separated from seven newly discovered SHMO compositions. The content of most oligosaccharides was more than 50% lower in MM than in CM, and α2,3-sialylation was observed in 43.74% of SHMOs from CM and 22.95% of SHMOs from MM. Finally, the fucosylation level of the SHMOs increased from 16.45 to 22.28% with prolonged lactation. These findings provide the basis for further studies on the structure-activity relationship of SHMOs and a blueprint to improve infant formula.

A preliminary study on isomer-specific quantification of sialylated N-glycans released from whey glycoproteins in human colostrum and mature milk using a glycoqueuing strategy.

Sialylated N-glycans are an integral component of whey proteins in human milk and play an irreplaceable role in infant growth and development. Currently, there are few studies on quantitative comparison of sialylated N-glycans in milk obtained at different lactation stages. Here, a preliminary isomer-specific quantification of whey sialylated N-glycans of human colostrum milk (CM) and mature milk (MM) was performed by using our recently developed glycoqueuing strategy. Such a preliminary comparison revealed that the whey sialylated N-glycan content was 86.4% lower in MM than in CM. Twenty-three α2,6-linked sialylated N-glycan isomers were detected with no α2,3-linked isomer observed. For the first time, three mono-sialylated and four bi-sialylated glycan isomers were reported. With the prolongation of lactation, the relative abundance of mono-sialylated glycans increased, whilst the relative abundance of bi-sialylated glycans decreased significantly. These findings contribute to the understanding of the structure-function relationship of sialylated N-glycans in the human whey fraction.

Purification of N- and O-glycans and their derivatives from biological samples by the absorbent cotton hydrophilic chromatographic column.

Mass spectrum (MS) is one of the most commonly used tools for qualitative and quantitative analysis of glycans. However, due to the complexity of biological samples and the low ionization efficiency of glycans, these need to be purified and derivatized prior to MS analysis. Existing purification strategies require a combination of multiple methods and are cumbersome to operate. Here, we propose a new method for the purification of glycoprotein N/O-glycans and their derivatives using a hand-packed absorbent cotton hydrophilic interaction chromatography column (HILIC). The method's reliability and applicability were verified by purifying N/O-glycans and the derivatives of standard glycoproteins, such as chicken albumin and porcine stomach mucin. Stable isotope labelling was used to compare the glycans' recovery following different purification methods. Absorbent cotton HILIC was also successfully applied for the analysis of human serum and fetal bovine serum glycoprotein N-glycans. Finally, testing revealed high binding capacity (9 mg/g-1 maltohexaose/absorbent cotton) and good recovery (average recovery was 91.7%) of glycans. Compared with traditional procedures, the proposed purification method offers considerable advantages, such as simplicity, high efficiency, economy, universality, and broad applicability for the pretreatment of glycans and their derivatives in biological samples prior to MS analysis.