title: Sequence characterization and glycosylation sites identification of donkey milk lactoferrin by multiple enzyme digestions and mass spectrometry.
abstract: Lactoferrin, a protein showing an array of biochemical properties, including immuno-modulation, iron-binding ability, as well as antioxidant, antibacterial and antiviral activities, but which may also represent a potential milk allergen, was isolated from donkey milk by ion exchange chromatography. The characterization of its primary structure, by means of enzymatic digestions, SPITC derivatization of tryptic digest, reversed-phase high performance liquid chromatography, electrospray and matrix-assisted laser desorption/ionization mass spectrometry, is reported. Our results allowed the almost complete characterization of donkey lactoferrin sequence, that, at least for the covered sequence, differs from the horse genomic deduced sequence (UniProtKB Acc. Nr. O77811) by five point substitutions located at positions 91 (Arg → His), 328 (Thr → Ile/Leu), 466 (Ala → Gly), 642 (Asn → Ser) and 668 (Ser → Ala). Analysis of the glycosylated protein showed that glycans in donkey lactoferrin are linked to the protein backbone via an amide bond to asparagine residues located at the positions 137, 281 and 476.
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: 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: 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: 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.
authors.forename: Antonella
authors.initials: A
authors.lastname: Di Francesco
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: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense, Denmark.
authors.forename: Andrea Maria
authors.initials: AM
authors.lastname: Lorenzten
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense, Denmark.
authors.forename: Peter
authors.initials: P
authors.lastname: Roepstorff
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018397:Equidae; D006031:Glycosylation; D007781:Lactoferrin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008892:Milk / Q000737:chemistry* / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D011134:Polysaccharides; D007781:Lactoferrin
keywords: Donkey milk; Glycosylation sites; Lactoferrin; Mass spectrometry; Sequence characterization