Elucidation of a novel lacto-N-decaose core structure in human milk using nonlinear analytical technique combinations. Blank, D., Geyer, H., Maass, K., Yamashita, K., & Geyer, R. Anal Biochem, 421(2):680–690, 2012.
doi  abstract   bibtex   
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.
@Article{blank12elucidation,
  author    = {Dennis Blank and Hildegard Geyer and Kai Maass and Katsuko Yamashita and Rudolf Geyer},
  title     = {Elucidation of a novel lacto-{N}-decaose core structure in human milk using nonlinear analytical technique combinations.},
  journal   = {Anal Biochem},
  year      = {2012},
  volume    = {421},
  number    = {2},
  pages     = {680--690},
  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.},
  doi       = {10.1016/j.ab.2011.11.030},
  keywords  = {glycans; glycan sequencing; glycan MS; mass spectrometry},
  owner     = {Sebastian},
  pmid      = {22197416},
  timestamp = {2012.04.25},
}

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