Peracylation Coupled with Tandem Mass Spectrometry for Structural Sequencing of Sulfated Glycosaminoglycan Mixtures without Depolymerization

Abstract

The structures of glycosaminoglycans (GAGs), especially the patterns of modification, are crucial to modulate interactions with various protein targets. It is very challenging to determine the fine structures using liquid chromatography-mass spectrometry (LC-MS) due in large part to the gas-phase sulfate losses upon collisional activation. Previously, our group reported a method for fine structure analysis that required permethylation of the GAG oligosaccharide. However, uncontrolled depolymerization during the permethylation process due to esterification of uronic acid lowers the reliability of the method to resolve structures of GAGs, especially for larger oligosaccharides. Here, we describe a simplified derivatization method using propionylation and desulfation. The oligosaccharides have all hydroxyl and amine groups protected with propionyl groups and then have sulfate groups removed to generate unprotected hydroxyl and amine groups at all sites that were previously sulfated. This derivatized oligosaccharide generates informative fragments during collision-induced dissociation that resolve the original sulfation patterns. This method is demonstrated to enable accurate determination of sulfation patterns of even the highly sulfated pentasaccharide fondaparinux by MS² and MS³. Using a mixture of dp6 from porcine heparin, we demonstrate that this method allows for structural characterization of complex mixtures, including clear chromatographic separation and sequencing of structural isomers, all at high yields without evidence of depolymerization. This represents a marked improvement in the reliability to structurally characterize GAG oligosaccharides over permethylation-based derivatization schemes.

Keywords: LC-MS/MS; carbohydrates; derivatization; glycosaminoglycans; heparin.

Figure 1.

Chemical derivatization workflow. To enhance solubility in THF, Arixtra was first converted to its TEA salt by passing through a cation exchange resin, followed by propionylation with propionic anhydride. After propionylation, derivatized Arixtra was converted to its pyridinium salt. Sulfates were removed with solvolytic desulfation, leaving sites of sulfation marked by free hydroxyl or amine groups.

Figure 2.

SEC chromatograms of a. reaction blank with no HA dp10, b. unmodified HA dp10, and c. fully propionylated HA dp10 with UV detection at 232 nm, respectively. After propionylation, there was only one GAG peak with a similar peak shape and retention time as the unmodified HA dp10. The SEC chromatogram demonstrated no depolymerization of HA dp10 during propionylation.

Figure 3.

Sulfation sequencing analysis of Arixtra by acylation/desulfation and MS/MS. a. The structure and fragmentation path for derivatized Arixtra; b. Tandem MS spectrum of [M+2H]²⁺=602.735, corresponding to fully derivatized Arixtra. -P represents loss of a propionyl group (56.026 Da) and -OMe represents loss of methanol (32.026 Da) from the reducing end. The mass accuracy for each assigned product ion was below 10 ppm.

Figure 4

Sulfation sequencing analysis of the non-reducing end of derivatized Arixtra by MS³. a. Fragmentation path of B₃ ion, [B₃+H] ⁺; b. MS³ spectrum of [B₃+H] ⁺ of fully acylated, desulfated Arixtra. The mass accuracy for each assigned product ion was below 5 ppm.

Figure 5

Sulfation sequencing analysis of the reducing end of derivatized Arixtra by MS³. a. fragmentation path of Y₁-OMe ion, [Y₁-OMe +H] ⁺; b. MS³ of [Y₁-OMe + H] ⁺. The mass accuracy for each assigned product ion was below 5 ppm.

Figure 6

LC separation of the derivatized heparin dp6 mixture. a. TIC of the derivatized dp6 mixture; b. EIC corresponding to a fully derivatized octasulfated dp6, doubly protonated (m/z = 730.763); c. MS/MS pseudo-MRM trace of 730.763 → 274.128, corresponding to the [Z₁+H]⁺ product of a disulfated reducing end glucosamine as shown in Figure 7; d. MS/MS pseudo-MRM trace of 730.763 → 330.1541, corresponding to the [Z₁+H]⁺ product of a monosulfated reducing end glucosamine as shown in Figure 8.

Figure 7

Sulfation sequencing analysis of Structure 1 by acylation/desulfation and MS/MS. a. The structure and observed product ions for Structure 1; b. Averaged MS/MS spectrum of [M+2H]²⁺ = 730.763 obtained between 21.90–22.26 min, corresponding to fully derivatized oligosaccharide illustrated in Structure 1. –PA represents the loss of propionic acid (74.037 Da). The mass accuracy for each assigned product ion was below 10 ppm.

Figure 8

Sulfation sequencing analysis of Structure 2 by acylation/desulfation and MS/MS. a. The structure and observed product ions for Structure 2; b. Averaged MS/MS spectrum of [M+2H]²⁺ = 730.763 obtained between 16.04–16.39 min, corresponding to fully derivatized oligosaccharide illustrated in Structure 2. The mass accuracy for each assigned product ion was below 10 ppm.