High performance liquid chromatography-mass spectrometry for mapping and sequencing glycosaminoglycan-derived oligosaccharides

Glycosaminoglycans (GAGs) have proven to be extremely difficult to analyze because of high negative charge, polydispersity, and sequence heterogeneity. Since the specificity of the interactions between GAGs and proteins results from the structure of these polysaccharides, an understanding of GAG structure is essential in developing a structure-activity relationship. Electrospray ionization mass spectrometry (MS) is particularly promising for the analysis of GAG-derived oligosaccharides due to its relative soft ionization capacity. Furthermore, on-line high performance liquid chromatography (HPLC)-MS greatly enhances the analysis of complex mixtures of GAG-derived oligosaccharides providing important structural information and affording their disaccharide composition. A detailed protocol for producing oligosaccharides from various GAGs, using controlled, specific enzymatic or chemical depolymerisation, is presented together with their HPLC separation using volatile reversed phase ion-pairing reagents and on-line electrospray ionisation MS structural identification. This analysis provides an oligosaccharide map together with sequence from a reading frame beginning at the non-reducing end of the GAG chains. The preparation of oligosaccharides can be done in 10 h with subsequent HPLC analysis in 1-2 h and HPLC-MS analysis taking 2 h.