Comparison Of N-glycans In Bovine Milk Lactoferrin At Different Lactation Periods

Bovine lactoferrin is a functional glycoprotein,and its various biological functions are related to N-glycans.However,there are few studies on the comparison of N-glycans of bovine lactoferrin in different lactation periods and the distinction between ?2,3-and?2,6-linked isomers of sialylated N-glycans.In this study,the N-glycans of bovine lactoferrin in different lactation stages were compared qualitatively and quantitatively based on mass spectrometry technology,and the linked isomers of its sialylated N-glycans were further identified.This research includes the separation and purification of bovine lactoferrin,the derivatization of bovine lactoferrin N-glycans and the differentiation of isomers,and the specific derivation of sialylated N-glycans ?2,3-and ?2,6-linked isomers.The specific conclusions are as follows:1.The optimized styrene matrix-based strong cation chromatography was used to separate and purify bovine lactoferrin at different lactation periods.SDS-PAGE identified the purity of bovine lactoferrin>95%.Using matrix-assisted laser analysis and ionization time-of-flight mass spectrometry technology,it was identified that the molecular weight of bovine lactoferrin was about 80 k Da,the largest in transitional milk(83018 Da),followed by colostrum(82732 Da)and mature milk(82690 Da),which may be related to The glycosylation level of different lactation period proteins is related.2.Based on mass spectrometry and online liquid mass spectrometry technology,qualitative and quantitative comparison and analysis of the differences in N-glycans of bovine lactoferrin in different lactation stages.The results showed that the N-glycans of milk lactoferrin was mainly composed of high mannose type throughout the lactation stage.As the lactation period prolongs,the types and content of total N-glycans in milk lactoferrin gradually decrease.The content of sialylated N-glycans in transitional milk reached 21.8%,which was 2 and 10 times that of colostrum and mature milk,respectively;the content of fucosylated N-glycans in transitional milk reached 25.2%,which were colostrum,respectively And mature milk 2.6 and 4.8 times.Among them,the content of complex N-glycans(H4N5A1F1)modified by monosialic acid and monofucose changed most significantly,accounting for 14.5% in transitional milk,1.3% and 0.9% in colostrum and mature milk,respectively.At the same time,two pairs of isomers(H4N4 and H7N2)were identified in the N-glycans of bovine lactoferrin.3.Using the specific derivatization method of sialic acid linked isomers to derivatize the sialylated N-glycans of bovine lactoferrin in different lactation stages with ?2,3-and?2,6-linkage specific derivatization,and use mass spectrometry and online liquid mass spectrometry Technical qualitative and quantitative comparison and analysis of sialylated N-glycans linking isomers.The results showed that with the prolonged lactation period,the types of bovine lactoferrin sialylated N-glycans ?2,3-and ?2,6-linked isomers gradually decreased,and the content showed a trend of first increasing and then decreasing,of which?2,3-The content of linked isomers is higher than that of ?2,6-linked isomers,and the content of ?2,3-linked isomers with the composition of H4N5F1A1 is the highest in transition milk,which is 3.8 times that of ?2,6-linked isomers.4.There are significant differences in the composition and content of N-glycans of bovine lactoferrin in different lactation periods,and bovine lactoferrin contains hydroxyacetylneuraminic acid and rich ?2,3-linked sialylated N-glycans.Follow-up studies can explore the biological function differences caused by glycosylation differences based on cell or animal model,and provide theoretical reference for the development of lactoferrin as a functional biological resource.