Electron detachment dissociation of synthetic heparan sulfate glycosaminoglycan tetrasaccharides varying in degree of sulfation and hexuronic acid stereochemistry

Glycosaminoglycan (GAG) carbohydrates provide a challenging analytical target for structural determination due to their polydisperse nature, non-template biosynthesis, and labile sulfate modifications. The resultant structures, although heterogeneous, contain domains which indicate a sulfation pattern or code that correlates to specific function. Mass spectrometry, in particular electron detachment dissociation Fourier transform ion cyclotron resonance (EDD FT-ICR MS), provides a highly sensitive platform for GAG structural analysis by providing cross-ring cleavages for sulfation location and product ions specific to hexuronic acid stereochemistry. To investigate the effect of sulfation pattern and variations in stereochemistry on EDD spectra, a series of synthetic heparan sulfate (HS) tetrasaccharides are examined. Whereas previous studies have focused on lowly sulfated compounds (0.5–1 sulfate groups per disaccharide), the current work extends the application of EDD to more highly sulfated tetrasaccharides (1–2 sulfate groups per disaccharide) and presents the first EDD of a tetrasaccharide containing a sulfated hexuronic acid. For these more highly sulfated HS oligomers, alternative strategies are shown to be effective for extracting full structural details. These strategies include sodium cation replacement of protons for determining the sites of sulfation, and desulfation of the oligosaccharides for the generation of product ions for assigning uronic acid stereochemistry.

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► Application of electron detachment dissociation to increasingly sulfated GAGs.
► Development of a two-step method for sulfation and C5 stereochemistry determination.
► Determination of hexuronic acid stereochemistry in highly sulfated tetrasaccharides.
► Desulfation of sulfated GAG tetrasaccharides to determine C5 stereochemistry.