Preparation, separation, and conformational analysis of differentially sulfated heparin octasaccharide isomers using ion mobility mass spectrometry

Abstract

Heparin is a linear sulfated polysaccharide widely used in medicine because of its anticoagulant properties. The various sulfation and/or acetylation patterns on heparin impart different degrees of conformational change around the glycosidic bonds and subsequently alter its function as an anticoagulant, anticancer, or antiviral drug. Characterization of these structures is important for eventual elucidation of its function but presents itself as an analytical challenge due to the inherent heterogeneity of the carbohydrates. Heparin octasaccharide structural isomers of various sulfation patterns were investigated using ion mobility mass spectrometry (IMMS). In addition to distinguishing the isomers, we report the preparation and tandem mass spectrometry analysis for multiple sulfated or acetylated oligosaccharides. Herein, our data indicate that heparin octasaccharide isomers were separated on the basis of their structural conformations in the ion mobility cell. Subsequent to this separation, isomers were further distinguished using product ions resulting from tandem mass spectrometry. Overall, IMMS analysis was used to successfully characterize and separate individual isomers and subsequently measure their conformations.

Figure 1

Heparin octasaccharide structures. The number of carbon atom is indicated at each structure.

Figure 2

The arrival time distributions for eight sulfated heparin octasaccharide isomers (a) in the 4⁻ charge state and (b) in the 5⁻ charge state and (c) for sodium ion coordinated eight sulfated heparin octasaccharide isomers in the 4⁻ charge state. The arrival times are from three individual measurements and standard deviation is ±0.03msec

Figure 3

MS/MS spectra of (a) de-N-sulfated, (b) de-6,O-sulfated, and (C) de-2,O-sulfated heparin octasaccharides. The products ions are labeled using the Domon and Costello nomenclature. The precursor ion at m/z=496.0 is subject to 27V collision energy in the transfer cell.