Synthetic Disaccharide Standards Enable Quantitative Analysis of Stored Heparan Sulfate in MPS IIIA Murine Brain Regions

Heparan sulfate (HS) is a complex polysaccharide from the glycosaminoglycan (GAG) family that accumulates in tissues in several neurological lysosomal storage diseases known as mucopolysaccharidosis (MPS) disorders. The quantitation of HS in biological samples is important for studying MPS disorders but is very challenging because of its high molecular weight and heterogeneity. Recently, acid-catalyzed butanolysis followed by LC-MS/MS analysis has emerged as a promising method for the determination of HS. Butanolysis of HS produces fully desulfated disaccharide cleavage products which are detected by LC-MS/MS. Herein we describe the synthesis of butylated HS disaccharide standards and their use for determining the identity of major product peaks in LC-MS chromatograms from butanolysis of HS as well as the related GAGs heparin and heparosan. Furthermore, synthesis of a d 9-labeled disaccharide internal standard enabled the development of a quantitative LC-MS/MS assay for HS. The assay was utilized for the analysis of MPS IIIA mouse brain tissues, revealing significant differences in abundance and in the regional accumulation of the various HS disaccharides in affected mice.