title: Elucidation of a novel lacto-N-decaose core structure in human milk using nonlinear analytical technique combinations.
abstract: Detailed structural analysis of high molecular weight human milk oligosaccharides (HMOs) is still a challenging task. Here we present a modular strategy for a flexible de novo structural characterization of this class of molecules. The protocol combines established techniques such as separation by two-dimensional high-performance liquid chromatography with different types of mass spectrometry, exoglycosidase digestion, and linkage analysis in an individual glycan-based manner. As a proof of principle, this approach was applied to two distinct HMO isomers representing a difucosylated octaose core and a trifucosylated decaose core. Obtained data revealed the presence of one terminal Lewis A and one internal Lewis X epitope in the case of the octaose and led to the identification of this molecule as a difucosylated iso-lacto-N-octaose. The trifucosylated, doubly branched lacto-N-neo-decaose was shown to represent a new type of HMO core structure in which the branched antenna is linked to carbon atom 3 of the innermost galactosyl residue. Hence, using this analytical protocol a novel HMO structure could be defined. Our results further demonstrate that a combination of different techniques may be required for de novo structural analysis of these molecules.
authors.affiliation: Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University Giessen, Friedrichstrasse 24, 35392 Giessen, Germany.
authors.forename: Dennis
authors.initials: D
authors.lastname: Blank
authors.forename: Hildegard
authors.initials: H
authors.lastname: Geyer
authors.forename: Kai
authors.initials: K
authors.lastname: Maass
authors.forename: Katsuko
authors.initials: K
authors.lastname: Yamashita
authors.forename: Rudolf
authors.initials: R
authors.lastname: Geyer
mesh_terms: D002236:Carbohydrate Conformation; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008895:Milk, Human / Q000737:chemistry*; D009844:Oligosaccharides / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D009844:Oligosaccharides