Mass spectrometry based quantitative and qualitative analyses reveal N-glycan changes of bovine lactoferrin at different stages of lactation

Bovine lactoferrin is a functional N-glycoprotein whose glycan moieties play an important role in its biological activities. Although bovine lactoferrin is rich in sialylated N-glycans, there are no reports on the differentiation between α2,3-linked and α2,6-linked sialylated N-glycan isomers. In this study, bovine lactoferrin was separated and purified by cation exchange chromatography, and >95% pure protein was obtained. Following this, N-glycan derivatives of lactoferrin at different stages of lactation were qualitatively and quantitatively analyzed by MALDI-TOF-MS and HILIC-MS/MS. With prolonged lactation, the relative content of the sialylated and fucosylated bi-antenna complex N-glycan (Hex1Man3GlcNAc4GalNAc1Fuc1Neu5Ac1) changed the maximum, with 14.5%, 1.3%, and 0.9% contents in transitional milk, colostrum, and mature milk, respectively. Meanwhile, the contents of sialylated and fucosylated N-glycans in transitional milk were the highest. More importantly, the sialylated N-glycans-linked isomers could be distinguished by linkage-specific derivatization of sialic acid. There were 11 α2,3- and 13 α2,6-linked sialyation N-glycans isomers in the colostrum, which transformed to 4 and 3 N-glycan isomers, respectively, in the mature milk. These findings provide a foundation for an in-depth research on the structure–function relationship of bovine lactoferrin.