A new method for purifying N-Glycans released from milk glycoprotein

Human milk is the first source of nutrition for infants, which delivers an array of unique bioactive components to offspring. Modern bovine-milk-based infant formulas are good substitutes when mother's milk is not available. As the third most abundant component in human milk, human free oligosaccharides (HMOs) may interference the analysis of total N-glycans on the glycoproteins in human milk. Herein, we combined acetone precipitation protein with the filter aided sample preparation method (FASP) to thoroughly remove HMOs and purify N- glycans. Furthermore, we also compared both N-glycosylation and glycoproteins between human and bovine milk, which may provide new ideas for the composition adjustment of infant formula in the food industry. Significance: We described a new method, which can successfully remove HMOs, further extract and purify the N- glycans on glycoproteins from pooled human milk for MALDI-TOF MS analysis by applying acetone precipitation and FASP together. We applied the new method to purify the N-glycans from whey proteins in pooled bovine milk and compared the N-glycosylation differences between pooled human and bovine milk by MALDI-TOF MS. We first reported the difference of N-glycan pattern of glycoproteins between pooled bovine and human milk by lectin blotting, and found significant differences in types and abundance of glycoproteins between the two sourced milk.