title: Structural determination by negative-ion MALDI-QIT-TOFMSn after pyrene derivatization of variously fucosylated oligosaccharides with branched decaose cores from human milk.
abstract: We prepared neutral oligosaccharide fraction from milk of a woman (blood type A, Le(b+)) by anion-exchange column chromatography after the removal of lipids and proteins. Further fractionation was performed by means of Aleuria aurantia lectin-Sepharose column chromatography and reverse-phase HPLC after labeling with a pyrene derivative. This pyrene labeling allowed identification by negative-MALDI-TOFMS(n) analysis of 22 oligosaccharides with decaose cores, among which 21 had novel structures. Negative ions could not be produced from neutral oligosaccharides without labeling on MALDI. Mono-, di-, tri-, and tetrafucosylated decaose fractions contained three, nine, six, and four isomers, respectively. Our method enables easy determination of fucosylated structures on the N-acetyllactosamine branches of these isomers. On negative-MS(n) the fragment ions included several A and D ions, from which fucosylation on the branches could be elucidated. Other characteristic ions were also detected. Y-type cleavage at the reducing side of -3GlcNAc indicated the occurrence of type 1 chain. Specific fragment ions were produced from H, Le(a), and Le(x) antigens. Linkage-specific exoglycosidase digestion confirmed the structures. The results indicate that the diversity of the oligosaccharides is due to combinations of type 1 H, Le(a), Le(x), and Le(b)/Le(y) on branched decaose cores. In typical oligosaccharides, 6-branches always consist of type 2 chain, while 3-branches, such as beta and gamma chains, are fucosylated type 1 chains. From the viewpoint of biosynthesis, the presence of fucosylation and type 1 chain may halt elongation of the N-acetyllactosamine and promote formation of branched structures.
authors.affiliation: Laboratory of Glycobiology, The Noguchi Institute, Itabashi, Tokyo, Japan. amano@noguchi.or.jp
authors.forename: Junko
authors.initials: J
authors.lastname: Amano
authors.forename: Minako
authors.initials: M
authors.lastname: Osanai
authors.forename: Takahiro
authors.initials: T
authors.lastname: Orita
authors.forename: Daisuke
authors.initials: D
authors.lastname: Sugahara
authors.forename: Kenji
authors.initials: K
authors.lastname: Osumi
mesh_terms: D002240:Carbohydrate Sequence; D005591:Chemical Fractionation; D005260:Female; D005643:Fucose / Q000737:chemistry*; D006801:Humans; D007536:Isomerism; D008895:Milk, Human / Q000737:chemistry*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009844:Oligosaccharides / Q000737:chemistry*; D011721:Pyrenes / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D009844:Oligosaccharides; D011721:Pyrenes; D005643:Fucose; C030984:pyrene