title: Site-specific glycosylation of donkey milk lactoferrin investigated by high-resolution mass spectrometry.
abstract: 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, TiO2and 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.
authors.affiliation: Organic Mass Spectrometry Laboratory, Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125, Catania, Italy.
authors.forename: Serafina
authors.initials: S
authors.lastname: Gallina
authors.affiliation: Organic Mass Spectrometry Laboratory, Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125, Catania, Italy.
authors.forename: Rosaria
authors.initials: R
authors.lastname: Saletti
authors.affiliation: Organic Mass Spectrometry Laboratory, Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125, Catania, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Cunsolo
authors.affiliation: Organic Mass Spectrometry Laboratory, Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125, Catania, Italy.
authors.forename: Vera
authors.initials: V
authors.lastname: Muccilli
authors.affiliation: Organic Mass Spectrometry Laboratory, Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125, Catania, Italy. sfoti@unict.it.
authors.forename: Salvatore
authors.identifier: 0000-0002-4007-6582
authors.initials: S
authors.lastname: Foti
authors.affiliation: Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark.
authors.forename: Peter
authors.initials: P
authors.lastname: Roepstorff
authors.affiliation: Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark.
authors.forename: Morten I
authors.initials: MI
authors.lastname: Rasmussen
mesh_terms: D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D018397:Equidae; D006031:Glycosylation; D006041:Goats; D006801:Humans; D007781:Lactoferrin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013058:Mass Spectrometry; D008892:Milk / Q000737:chemistry* / Q000378:metabolism; D009005:Monosaccharides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D009005:Monosaccharides; D011134:Polysaccharides; D007781:Lactoferrin
keywords: Donkey milk; Lactoferrin glycosylation; Mass spectrometry; N-Glycan structures; TiO2 and HILIC enrichment