Improved de novo sequencing of heparin/heparan sulfate oligosaccharides by propionylation of sites of sulfation

Abstract

The structure of heparin and heparan sulfate (Hep/HS) oligosaccharides, as determined by the length and the pattern of sulfation, acetylation, and uronic acid epimerization, dictates their biological function through modulating interactions with protein targets. But fine structural determination is a very challenging task due to the lability of the sulfate modifications and difficulties in separating isomeric HS chains. Previously, we reported a strategy for chemical derivatization involving permethylation, desulfation, and trideuteroperacetylation, combined with standard reverse phase LC-MS/MS that enables the structural sequencing for heparin/HS oligosaccharides of sizes up to dodecasaccharide by positionally replacing all sulfates with more stable trideuteroacetyl groups, allowing for robust MS/MS sequencing. However, isomeric oligosaccharides that contain both N-sulfation and N-acetylation become isotopomers after labeling, differing only in the sites of deuteration. This prevents chromatographic separation of these different mixed domain sequences post-derivatization, and makes sequencing by MS/MS difficult due to co-fragmentation of the isotopomers leading to chimeric product ion spectra. In order to improve chromatographic separation of mixed domain oligosaccharides, we have introduced a propionylation step in place of trideuteroacetylation for labeling of sites of sulfation. HS standard disaccharides have been used to evaluate the efficiency of this improved chemical derivatization. The results show that we can quantitatively replace sulfation with propionyl groups with the same high efficiency as the previously reported trideuteroacetylation. After derivatization, we demonstrate the ability to chromatographically separate two mixed domain tetrasaccharide isomers differing solely by the order of N-sulfation and N-acetylation, allowing for full sequencing of each by MS/MS. These results represent a marked improvement in the ability of our previously reported derivatization strategy to analyze complex mixtures of Hep/HS oligosaccharides without a decrease in sensitivity.

Keywords: Chemical derivatization; Glycosaminoglycans; Heparin/heparan sulfate; LC-MS/MS.

Figure 1

Tetrasaccharides with one N-acetyl group and one N-sulfo group before and after chemical derivatization using the previously reported trideuteroactylation method. Dp4-a and Dp4-b have a different sequence, but after derivatization differ only by the location of three deuterium atoms. These isotopomers are impractical to chromatographically separate, resulting in ambiguous chimeric MS/MS spectra.

Figure 2

Improved chemical derivatization scheme for differentiating mixed domain isomers. Permethylation protects the unsulfated groups (magenta), as well as allows the assignment of sulfation sites within the GlcN based on the effects of sulfation on the extent of permethylation. Sulfates (blue) are then gently removed by solvolysis, and the sites of sulfation are labeled with propionyl group (red).

Figure 3

(A) MS spectrum and (B) EIC profiles for derivatized I-S: ΔUA2S-GlcNS6S (m/z 576.253). The two peaks in the EIC profile represent reducing end anomerization. The EIC profiles of two detected minor mass peaks, m/z 544.225 and 512.200 (C and D, respectively) show the same retention time as m/z 576.253 (B), indicating they are both source fragmentation products.

Figure 4

(A) MS spectrum and (B) EIC profile for derivatized I-A: ΔUA2S-GlcNAc6S. Derivatized I-A (m/z 562.250) yielded two EIC peaks, due to anomerization at the reducing end. The EIC profiles of two detected minor mass peaks, m/z 530.222 and 520.238 (C and D, respectively) show the same RT as m/z 562.25 (B), indicating they are both source fragmentation products.

Figure 5

Structures of the synthetic tetrasaccharide T₁: IdoA-GlcNS-IdoA-GlcNAc-O(CH₂)₅-NH₂ (A) and T₂: IdoA-GlcNAc-IdoA-GlcNS-O(CH₂)₅-NH₂ (B), before and after chemical derivatization. The primary amine on the reducing end linker is converted to a quaternary amine during the permethylation, providing a permanent fixed charge on the reducing end. An averaged MS spectrum taken from the elution window of the tetrasaccharides and their under-derivatized versions showed an abundant peak for the fully derivatized tetrasaccharide. β-elimination products were detected (not shown), as well as underpermethylation and in-source fragmentation products. However, no evidence of underpropionylation was observed.

Figure 6

LC-MS/MS spectra of the derivatized mixture of the two tetrasaccharide isomers. (A) EIC of derivatized dp4 (m/z 1100.563) from the LC-MS experiment. CID MS/MS spectra of the precursor ion [M]⁺ (m/z 1100.563) resulted in two qualitatively different product ion spectra: (B) one dominated by the 609.360 product ion, and (C) one dominated by the 623.376 product ion. (D) Theoretical fragmentation of the two derivatized isomers indicate that the Y₂ product ion would differentiate between the two isomers, and EICs of the two Y₂ ions show clear chromatographic separation of the isomeric mixture, with the product ion of m/z 609.360 diagnostic for T₁: IdoA-GlcNS-IdoA-GlcNAc-O(CH₂)₅-NH₂, and the product ion of m/z 623.376 is diagnostic for T_2: IdoA-GlcNAc-IdoA-GlcNS-O(CH₂)₅-NH₂.