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

Mitsuhiro Kinoshita, Hiroko Ohta, Kanata Higaki, Yoko Kojima, Tadasu Urashima, Kazuki Nakajima, Minoru Suzuki, Kit M. Kovacs, Christian Lydersen, Takao Hayakawa, Kazuaki Kakehi

Research output: Contribution to journalArticle

14 Citations (Scopus)

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 ∼ 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 (Galβ1-4Glc) 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, Galβ1-4GlcNAcβ1-6[Galβ1-4GlcNAcβ1-3]Galβ1-4Glc) as the common core structure, and most of them contained Fucα1-2 residues at the nonreducing ends. Furthermore, the oligosaccharides from both samples contained multibranched oligosaccharides having two Galβ1-4GlcNAc (N-acetyllactosamine, LacNAc) residues on the Galβ1-4GlcNAcβ1-3 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.

Original languageEnglish
Pages (from-to)242-253
Number of pages12
JournalAnalytical Biochemistry
Volume388
Issue number2
DOIs
Publication statusPublished - 15-05-2009

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Glycoside Hydrolases
High performance liquid chromatography
Oligosaccharides
Ionization
Seals
Mass spectrometry
Digestion
Desorption
Mass Spectrometry
Milk
Lasers
High Pressure Liquid Chromatography
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Colostrum
Glycoconjugates
Fucose
Molecular mass
Lactose
Complex Mixtures
Elongation

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Kinoshita, Mitsuhiro ; Ohta, Hiroko ; Higaki, Kanata ; Kojima, Yoko ; Urashima, Tadasu ; Nakajima, Kazuki ; Suzuki, Minoru ; Kovacs, Kit M. ; Lydersen, Christian ; Hayakawa, Takao ; Kakehi, Kazuaki. / 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. In: Analytical Biochemistry. 2009 ; Vol. 388, No. 2. pp. 242-253.
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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. / Kinoshita, Mitsuhiro; Ohta, Hiroko; Higaki, Kanata; Kojima, Yoko; Urashima, Tadasu; Nakajima, Kazuki; Suzuki, Minoru; Kovacs, Kit M.; Lydersen, Christian; Hayakawa, Takao; Kakehi, Kazuaki.

In: Analytical Biochemistry, Vol. 388, No. 2, 15.05.2009, p. 242-253.

Research output: Contribution to journalArticle

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AU - Kinoshita, Mitsuhiro

AU - Ohta, Hiroko

AU - Higaki, Kanata

AU - Kojima, Yoko

AU - Urashima, Tadasu

AU - Nakajima, Kazuki

AU - Suzuki, Minoru

AU - Kovacs, Kit M.

AU - Lydersen, Christian

AU - Hayakawa, Takao

AU - Kakehi, Kazuaki

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