Site-specific glycosylation of donkey milk lactoferrin investigated by high-resolution mass spectrometry

A comprehensive monosaccharide composition of the N -glycans of donkey milk lactoferrin, isolated by ion exchange chromatography from an individual milk sample, was obtained by means of chymotryptic digestion, TiO 2 and HILIC enrichment, reversed-phase high-performance liquid chromatography, electrospray mass spectrometry, and high collision dissociation fragmentation. The results obtained allowed identifying 26 different glycan structures, including high mannose, complex and hybrid N -glycans, linked to the protein backbone via an amide bond to asparagine residues located at the positions 137, 281 and 476. Altogether, the N -glycan structures determined revealed that most of the N -glycans identified in donkey milk lactoferrin are neutral complex/hybrid. Indeed, 10 neutral non-fucosylated complex/hybrid N -glycans and 4 neutral fucosylated complex/hybrid N -glycans were found. In addition, two high mannose N -glycans, four sialylated fucosylated complex N -glycans and six sialylated non-fucosylated complex N -glycans, one of which containing N -glycolylneuraminic acid (Neu5Gc), were found. A comparison of the monosaccharide composition of the N -glycans of donkey milk lactoferrin with respect to that of human, bovine and goat milk lactoferrin is reported. Data are available via ProteomeXchange with identifier PXD004289.