title: Structural characterization of multibranched oligosaccharides from seal milk by a combination of off-line high-performance liquid chromatography-matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and sequential exoglycosidase digestion.
abstract: A complex mixture of diverse oligosaccharides related to the carbohydrates in glycoconjugates involved in various biological events is found in animal milk/colostrum and has been challenging targets for separation and structural studies. In the current study, we isolated oligosaccharides having high molecular masses (MW approximately 3800) from the milk samples of bearded and hooded seals and analyzed their structures by off-line normal-phase-high-performance liquid chromatography-matrix-assisted laser desorption/ionization-time-of-flight (NP-HPLC-MALDI-TOF) mass spectrometry (MS) by combination with sequential exoglycosidase digestion. Initially, a mixture of oligosaccharides from the seal milk was reductively aminated with 2-aminobenzoic acid and analyzed by a combination of HPLC and MALDI-TOF MS. From MS data, these oligosaccharides contained different numbers of lactosamine units attached to the nonreducing lactose (Galbeta1-4Glc) and fucose residue. The isolated oligosaccharides were sequentially digested with exoglycosidases and characterized by MALDI-TOF MS. The data revealed that oligosaccharides from both seal species were composed from lacto-N-neohexaose (LNnH, Galbeta1-4GlcNAcbeta1-6[Galbeta1-4GlcNAcbeta1-3]Galbeta1-4Glc) as the common core structure, and most of them contained Fucalpha1-2 residues at the nonreducing ends. Furthermore, the oligosaccharides from both samples contained multibranched oligosaccharides having two Galbeta1-4GlcNAc (N-acetyllactosamine, LacNAc) residues on the Galbeta1-4GlcNAcbeta1-3 branch or both branches of LNnH. Elongation of the chains was observed at 3-OH positions of Gal residues, but most of the internal Gal residues were also substituted with an N-acetyllactosamine at the 6-OH position.
authors.affiliation: Faculty of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-osaka, Japan.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Kinoshita
authors.forename: Hiroko
authors.initials: H
authors.lastname: Ohta
authors.forename: Kanata
authors.initials: K
authors.lastname: Higaki
authors.forename: Yoko
authors.initials: Y
authors.lastname: Kojima
authors.forename: Tadasu
authors.initials: T
authors.lastname: Urashima
authors.forename: Kazuki
authors.initials: K
authors.lastname: Nakajima
authors.forename: Minoru
authors.initials: M
authors.lastname: Suzuki
authors.forename: Kit M
authors.initials: KM
authors.lastname: Kovacs
authors.forename: Christian
authors.initials: C
authors.lastname: Lydersen
authors.forename: Takao
authors.initials: T
authors.lastname: Hayakawa
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Kakehi
mesh_terms: D000818:Animals; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D008892:Milk / Q000737:chemistry*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry*; D046048:Seals, Earless / Q000502:physiology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D009844:Oligosaccharides; D006026:Glycoside Hydrolases