An approach for separation and complete structural sequencing of heparin/heparan sulfate-like oligosaccharides

Abstract

As members of the glycosaminoglycan (GAG) family, heparin and heparan sulfate (HS) are responsible for mediation of a wide range of essential biological actions, most of which are mediated by specific patterns of modifications of regions of these polysaccharides. To fully understand the regulation of HS modification and the biological function of HS through its interactions with protein ligands, it is essential to know the specific HS sequences present. However, the sequencing of mixtures of HS oligosaccharides presents major challenges due to the lability of the sulfate modifications, as well as difficulties in separating isomeric HS chains. Here, we apply a sequential chemical derivatization strategy involving permethylation, desulfation, and trideuteroperacetylation to label original sulfation sites with stable and hydrophobic trideuteroacetyl groups. The derivatization chemistry differentiates between all possible heparin/HS sequences solely by glycosidic bond cleavages, without the need to generate cross-ring cleavages. This derivatization strategy combined with LC-MS/MS analysis has been used to separate and sequence five synthetic HS-like oligosaccharides of sizes up to dodecasaccharide, as well as a highly sulfated Arixtra-like heptamer. This strategy offers a unique capability for the sequencing of microgram quantities of HS oligosaccharide mixtures by LC-MS/MS.

Figure 1

Structures of 12 commercially available common HS disaccharides before and after the chemical derivatizations, with each corresponding molecular weight labeled. In the structures, the symbol Me, Ac and Ac’ represents for methyl group, acetyl group and trideuteroacetyl group respectively. GlcN, GlcNAc and GlcNS represents for free glucosamine, acetylated glucosamine and N-sulfated glucosamine respectively. 2S and 6S indicate the position of the sulfate group. ΔUA represents for unsaturated uronic acid.

Figure 2

MS/MS spectra from the LTQ analyzer of an LTQ-FT of [M+2Na]²⁺ for NS-decamer (A), NS6S-decamer (B), NS-dodecamer (C) and NS6S-dodecamer (D) after the chemical derivatizations. Sufficient sequential glycosidic bond cleavage fragments were observed for each synthesized HS-like oligosaccharides, which enable the accurate and complete structural sequencing.

Figure 3

LC separation of derivatized products of five synthesized HS-like oligosaccharides by reverse-phase C18 column packed with either traditional porous material (A) or fused core-porous shell material (B). Same colors in two chromatograms represent the same oligasaccharide as indicated on the right side. For porous C18 column (50mm, 200Å, 3μm): 4 of the 5 oligosaccharides were separated, with NS-hendecamer and NS-decamer co-eluted at 45.29min. For core-shell Halo C18 column (50mm, ~160Å, ~2.6μm): 5 oligosaccharides were able to be separated from each other or at least partially separated.

Figure 4

Structures of the synthesized Arixtra-like heptamer before (A) and after (B) the derivatizations. Two black arrows indicated the different behavior during the derivatization between GlcNS6S and GlcNS6S3S residues, which was interpreted from the MS/MS spectra and the comparison between theoretical and observed m/z values for each glycosidic bond cleavage fragment as obtained from a Synapt G2 HDMS Q-TOF (C).

Figure 5

Prediction for the structure of the derivatized product of 3-O-sulfated HS disaccharide ΔUA-GlcNS3S, and there is a 14Da mass difference between 3-O-sulfated and 6-O-sulfated isomeric disaccharides after derivatizations.