Characterization of heparin oligosaccharide mixtures as ammonium salts using electrospray mass spectrometry

Among glycosaminoglycans, polysulfated heparin chains provide the greatest challenge to characterization due to high polarity, structural diversity, and sulfate lability. The present report demonstrates how electrospray mass spectrometry (ESMS) can be used to derive compositional information from pure and mixed fractions of heparin tetra- to decasaccharide fragments prepared by controlled digestion of heparin with heparinase I. It also describes an improved procedure for fractionation of heparin oligosaccharides up to octadecasaccharides. Ammonium salts prove to be superior to sodium salts, particularly for analysis of mixed components. In the mass spectrum of a hexasaccharide fraction, the identification of six major mass peaks that represent the known hexasaccharide structures confirms that ESMS analysis of heparin oligosaccharide fragments gives a close representation of their constituent composition. In addition to the previously identified components, several unreported oligosaccharides were detected in the spectra of octa- and decasaccharide fractions. The ESMS identification of the three major species in a decasaccharide fraction was confirmed after HPLC subfractionation and reanalysis. ESMS detection sensitivity of low picomole amounts of oligosaccharides can be readily achieved.