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.

Integrated Approach to Identify Heparan Sulfate Ligand Requirements of Robo1

“…In this respect, previous studies have shown that a Z 2 /Y 2 ratio of ~0.3 of tetrasaccharides is indicative of IdoA, while a Z 2 /Y 2 ratio of ~0.9 is indicative of GlcA. 20b …”

“…Therefore, the Robo1-binding octasaccharides were further analyzed by sequential permethylation, desulfation, and pertrideuteroacetylation followed by online separation and structural analysis by MS/MS. 20 By replacing the polar and labile sulfates with much more stable and hydrophobic trideuteroacetyl groups, oligosaccharides were obtained that could be separated by reverse-phase capillary HPLC and fragmented by MS/MS without loss of information on sites of modification. The structural complexity of HS oligosaccharides increases exponentially as oligosaccharides become larger, and therefore manual interpretation of MS/MS spectra and identification of isomeric sequences is challenging.…”

“…Previous studies using synthetic oligosaccharide standards have shown that β -elimination can occur during the permethylation step, generating shorter sequences. 20a The generated tetrasaccharides represent partial sequences of the original octasaccharides, which are valuable for structure identification.…”

“…20a Briefly, the HS octasaccharides were first converted into their triethylammonium (TEA) salt form and lyophilized. The dried HS octasaccharide TEA salts were permethylated using sodium hydroxide and methyl iodide in dimethyl sulfoxide (DMSO), followed by desalting using a C18 Sep-Pak cartridge (Waters Co.) and conversion to pyridinium salts.…”

The function of heparan sulfate during branching morphogenesis

“…What often remains to be determined is biochemical analysis of the HS structures that are made in the absence of one enzyme. This has been hampered in the past by the requirement of large amounts of HS to be analyzed, recently more sensitive techniques such as LC-MS/MS have been used to sequence complex mixtures of HS tetrasacharides (65). …”