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Abstract No.: JMS-Awards-2
Speaker: Kerryn Mason
Session: JMS Awards
Presentation date: Thu, Aug 31, 2006
Presentation time: 13:30 – 15:00

Characterisation of Sulphated Oligosaccharides in Mucopolysaccharidosis type IIIA by ESI-MS

Kerryn E. Mason1,2, Peter J. Meikle1,2, John J. Hopwood1,2, Maria Fuller1,2

1 Department of Genetic Medicine, Children, Youth and Womens Health Service, Adelaide, Australia
2 Department of Paediatrics, University of Adelaide, Adelaide, Australia

Correspondence address: Kerryn Mason, Child, Youth and Women's Health Service, LDRU, Genetic Medicine, King William Rd, North Adelaide, 5006 Australia.

Keywords: Biomarkers; Carbohydrates; Ionization, Electrospray; MS/MS, Structure Determination.

Novel aspect: Sulphated oligosaccharides as potential biochemical markers for disease activity for MPS IIIA.


Heparan sulphate (HS) is a glycosaminoglycan with considerable structural diversity that binds a myriad of growth factors and proteins which play pivotal roles in a variety of biological processes. HS degradation begins with endo-degradation of the long chain polymer to HS oligosaccharides that are further degraded from their non-reducing end by the sequential action of lysosomal exoenzymes to monosaccharides and inorganic sulphate for transport out of the lysosome and reutilization by the cell. A block in catabolism caused by a deficiency of sulphamidase leads to the accumulation of partially degraded HS fragments in the lysosomes of affected cells and their excretion in the urine leading to the clinical phenotype of mucopolysaccharidosis (MPS) IIIA.
Sulphated oligosaccharides isolated from the urine of a MPS IIIA patient using anion exchange and gel filtration chromatography were derivatised with 1-phenyl-3-methyl-5-pyrazolone, analysed by electrospray ionisation-mass spectrometry (ESI-MS) and identified based on a mass to charge (m/z) ratio. Reducing end monosaccharides were then characterised using collision activated dissociation in negative ion mode. A number of sulphated oligosaccharides, ranging from di- to hexadecasaccharide were identified and shown to have non-reducing end glucosamine N-sulphate with reducing end uronic acid or N-acetylglucosamine. Using nitrous acid digestion (at pH 1.5) combined with separation by reverse-phase high performance liquid chromatography (HPLC) and ESI-MS we have identified multiple forms of these oligosaccharides containing both N-sulphated and N-acetylated glucosamine residues with one to three sulphates per disaccharide. Furthermore, using reverse phase HPLC ESI-MS we have demonstrated that the different sulphated forms of each oligosaccharide were not mass spectrometric artifacts. Many structural isomers were present suggesting a complex mixture of oligosaccharides present in the urine as a consequence of a defect in heparan sulphate degradation. Analysis of a cohort of MPS IIIA and control urines by electrospray ionisation-tandem mass spectrometry (ESI-MS/MS) for di- to hexasaccharides showed that many of these sulphated oligosaccharides were significantly elevated in the MPS IIIA group.
We consider these sulphated oligosaccharides as potential biochemical markers for disease activity for MPS IIIA and anticipate that these findings will lead to improvements in the evaluation of therapy currently under investigation for this disorder.

This research was supported by the University of Adelaide (Australia), NH&MRC (Australia) and the Wellcome Trust (United Kingdom) grant reference number 060104Z/00/Z.