Complete mass spectral characterization of a synthetic ultralow-molecular- weight heparin using collision-induced dissociation

Glycosaminoglycans (GAGs) are a class of biologically important molecules, and their structural analysis is the target of considerable research effort. Advances in tandem mass spectrometry (MS/MS) have recently enabled the structural characterization of several classes of GAGs; however, the highly sulfated GAGs, such as heparins, have remained a relatively intractable class due their tendency to lose SO₃ during MS/MS, producing few sequence-informative fragment ions. The present work demonstrates for the first time the complete structural characterization of the highly sulfated heparin-based drug Arixtra. This was achieved by Na⁺/H⁺ exchange to create a more ionized species that was stable against SO₃ loss, and that produced complete sets of both glycosidic and cross-ring fragment ions. MS/MS enables the complete structural determination of Arixtra, including the stereochemistry of its uronic acid residues, and suggests an approach for solving the structure of more complex, highly sulfated heparin-based drugs.