CHARACTERIZATION OF HEPARIN OLIGOSACCHARIDE MIXTURES AS AMMONIUM-SALTS USING ELECTROSPRAY MASS-SPECTROMETRY

Among glycosaminoglycans, polysulfated heparin chains provide the grea test challenge to characterization due to high polarity, structural di versity, and sulfate lability. The present report demonstrates how ele ctrospray mass spectrometry (ESMS) can be used to derive compositional information from pure and mixed fractions of heparin tetra- to decasa ccharide fragments prepared by controlled digestion of heparin with he parinase 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 mix ed components. In the mass spectrum of a hexasaccharide fraction, the identification of six major mass peaks that represent the known hexasa ccharide structures confirms that ESMS analysis of heparin oligosaccha ride fragments gives a close representation of their constituent compo sition. In addition to the previously identified components, several u nreported oligosaccharides were detected in the spectra of octa- and d ecasaccharide fractions. The ESMS identification of the three major sp ecies in a decasaccharide fraction was confirmed after HPLC subfractio nation and reanalysis . ESMS detection sensitivity of low picomole amo unts of oligosaccharides can be readily achieved.