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A new sequencing approach for N-unsubstituted heparin/heparan sulfate oligosaccharides.

Liang, Qun Tao; Xiao, Xiao Mao; Lin, Jian Hui; Wei, Zheng.
Glycobiology; 25(7): 714-25, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25677303
The rare N-unsubstituted glucosamine (GlcNH(3)(+)) residues in heparan sulfate (HS) have important biological and pathophysiological roles. Because of their low natural abundance, the use of chemically generated, structurally defined, N-unsubstituted heparin/HS oligosaccharides can greatly contribute to the investigation of their natural role in HS. However, the sequencing of mixtures of chemically generated oligosaccharides presents major challenges due to the difficulties in separating isomers and the available detection methods. In this study, we developed and validated a simple and sensitive method for the sequence analysis of N-unsubstituted heparin/HS oligosaccharides. This protocol involves pH 4 nitrous acid (HNO(2)) degradation, size-exclusion HPLC and ion-pair reversed-phase liquid chromatography-ion trap/time-of-flight mass spectrometry (IPRP-LC-ITTOF MS). We unexpectedly found that absorbance at 232 nm (normally used for specific detection of C4-C5 unsaturated oligosaccharides) was, in most cases, still sufficiently sensitive to also simultaneously detect saturated oligosaccharides during HPLC, thus simplifying the positional analysis of GlcNH(3)(+)) residues. The IPRP-LC-ITTOF MS system can supply further structural information leading to full sequence determination of the original oligosaccharide. This new methodology has been used to separate and sequence a variety of chemically generated, N-unsubstituted dp6 species containing between 1 and 3 GlcNH(3)(+)) residues per oligosaccharide in different positional combinations. This strategy offers possibilities for the sequencing of natural N-unsubstituted oligosaccharides from HS and should also be applicable, with minor modification, for sequencing at N-sulfated residues using alternative pH 1.5 HNO(2) scission.
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