De Novo Sequencing of Complex Mixtures of Heparan Sulfate Oligosaccharides

Abstract

Here, we describe the first sequencing method of a complex mixture of heparan sulfate tetrasaccharides by LC-MS/MS. Heparin and heparan sulfate (HS) are linear polysaccharides that are modified in a complex manner by N- and O-sulfation, N-acetylation, and epimerization of the uronic acid. Heparin and HS are involved in various essential cellular communication processes. The structural analysis of these glycosaminoglycans is challenging due to the lability of their sulfate groups, the high heterogeneity of modifications, and the epimerization of the uronic acids. While advances in liquid chromatography (LC) and mass spectrometry (MS) have enabled compositional profiling of HS oligosaccharide mixtures, online separation and detailed structural analysis of isomeric and epimeric HS mixtures has not been achieved. Here, we report the development and evaluation of a chemical derivatization and tandem mass spectrometry method that can separate and identify isomeric and epimeric structures from complex mixtures. A series of well-defined synthetic HS tetrasaccharides varying in sulfation patterns and uronic acid epimerization were analyzed by chemical derivatization and LC-MS/MS. These synthetic compounds made it possible to establish relationships between HS structure, chromatographic behavior and MS/MS fragmentation characteristics. Using the analytical characteristics determined through the analysis of the synthetic HS tetrasaccharide standards, an HS tetrasacharide mixture derived from natural sources was successfully sequenced. This method represents the first sequencing of complex mixtures of HS oligosaccharides, an essential milestone in the analysis of structure-function relationships of these carbohydrates.

Figure 1

Structures of the synthetic tetrasaccharide GlcA-GlcNS-GlcA2S-GlcNS6S-(CH₂)₅NH₂ (compound 4d) before and after chemical derivatization. Permethylation protects the unsulfated groups, as well as allows the assignment of sulfation sites within the GlcN based on the effects of sulfation on the extent of permethylation. Sulfates are then gently removed by solvolysis, and the sites of sulfation are labeled with trideuteroacetyl groups. 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.

Figure 2

CID tandem MS spectra for compounds 3a–f with m/z of 1148.6 for precursor ion M⁺. These six tetrasaccharides could be divided into three isomeric sets: [3a], [3b, 3d, 3f], and [3c, 3e], where tetrasaccharides within each set were epimers. Sequential Y ions for each tetrasaccharide were observed that could be used as diagnostic ions to identify sulfation positional isomers. Differences of the relative intensities ratio of certain fragment ions were also observed within epimers. For example, compounds 3c–f containing terminal GlcA had Y₃/Y₂ ratio < 0.8, whereas compounds 3a and 3b containing terminal IdoA shown Y₃/Y₂ ratio > 0.8. On the other hand, Z₂ ions were observed for compounds 3a–f containing internal GlcA residue, but not for compounds 3c and 3d containing internal IdoA instead.

Figure 3

Reversed phased LC separation for derivatized synthetic tetrasaccharide mixture with alkyl linkers. Extracted ion charomatograms (EIC) for each of the four precursor ions were presented, with a total of 21 tetrasaccharides being separated. Peaks are labeled with the compound # of the eluting tetrasaccharide. Refer to Table 1 for tetrasaccharides structures.

Figure 4

LC chromatogram (A) and MS/MS spectra of the tetrasaccharide standards GlcA-GlcNAc6S-GlcA-GlcNAc6S (B) and GlcA-GlcNAc6S-IdoA-GlcNAc6S (C) of parent ion [M + Na]⁺. A series of B ions and Y ions were observed for sequencing of sulfation positions, whereas Z₂ ion with m/z of 547.4 was only observed for GlcA-GlcNAc6S-GlcA-GlcNAc6S with internal GlcA residue.

Figure 5

LC separation and sequencing of one postderivatized composition (with MW of 990. 490) of the native HS tetrasaccharide mixture. The TIC of MS/MS of parent ion [M + 2H]²⁺ (m/z of 496.25) and a series of EICs of diagnostic Y ions (left). EIC were extracted using the indicated m/z value with a window of 1 Da. Three major peaks (1, 2, and 4) and two minor peaks (3 and 1′) were observed. A list of total 12 biosynthetically possible theoretical structures for this specific MW is shown (top right), with identified structures shown in bold. The structural assignment(s) of each fragment ion mass is also shown (bottom right).