Salt-free fractionation of complex isomeric mixtures of glycosaminoglycan oligosaccharides compatible with ESI-MS and microarray analysis

Abstract

Heparin and heparan sulfate (Hp/HS) are linear complex glycosaminoglycans which are involved in diverse biological processes. The structural complexity brings difficulties in separation, making the study of structure-function relationships challenging. Here we present a separation method for Hp/HS oligosaccharide fractionation with cross-compatible solvent and conditions, combining size exclusion chromatography (SEC), ion-pair reversed phase chromatography (IPRP), and hydrophilic interaction chromatography (HILIC) as three orthogonal separation methods that do not require desalting or extensive sample handling. With this method, the final eluent is suitable for structure-function relationship studies, including tandem mass spectrometry and microarray printing. Our data indicate that high resolution is achieved on both IPRP and HILIC for Hp/HS isomers. In addition, the fractions co-eluted in IPRP could be further separated by HILIC, with both separation dimensions capable of resolving some isomeric oligosaccharides. We demonstrate this method using both unpurified reaction products from isomeric synthetic hexasaccharides and an octasaccharide fraction from enoxaparin, identifying isomers resolved by this multi-dimensional separation method. We demonstrate both structural analysis by MS, as well as functional analysis by microarray printing and screening using a prototypical Hp/HS binding protein: basic-fibroblast growth factor (FGF2). Collectively, this method provides a strategy for efficient Hp/HS structure-function characterization.

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

“…38 Together with a high yield and no depolymerization, propionylation shows advantage over permethylation. LC-MS 2 and MS 3 Analyses of Derivatized Arixtra. Compared to N-sulfation and 6-O-sulfation on GlcNAc, it is very rare to have 3-O-sulfated glucosamines naturally, occurring in about 1/20 disaccharides in heparin and less than 1/100 disaccharides in HS.…”

“…The uronic acid (UA) can be 2-Osulfated and epimerized, while the GlcN can be 3-O-sulfated, 6-O-sulfated, N-deacetylated, and/or N-sulfated. 3 While Hp and HS share the same backbone repeat, they differ by both the protein core (HS is found on many proteins and virtually all mammalian cells, while Hp is solely found on serglycin in mast cells and some hematopoietic cells) and the extent of modification (Hp is typically more heavily sulfated with more iduronic acid, while HS is more heterogeneous). 4 These modifications are incomplete and untemplated during biosyn-thesis, making Hp/HS the most structurally complex member of the GAG family.…”

“…After solvolytic desulfation, the positional information on the original sulfation pattern will be represented by the newly formed hydroxyl or amine groups. The derivatized analyte can be well-retained on C18 RPLC and sequenced via MS 2 and MS 3 . This methodology provides a more robust option for the fine structure analysis of longer GAG oligosaccharides using commonly available commercial LC-MS systems.…”

“…The disaccharide motif of Hp/HS is comprised of either β- d -glucuronic or α- l -iduronic acid (GlcA and IdoA, respectively) and α- d - N -acetyl- d -glucosamine (GlcNAc) connected by 1→ 4 linkages. The uronic acid (UA) can be 2- O -sulfated and epimerized, while the GlcN can be 3- O -sulfated, 6- O -sulfated, N -deacetylated, and/or N -sulfated . While Hp and HS share the same backbone repeat, they differ by both the protein core (HS is found on many proteins and virtually all mammalian cells, while Hp is solely found on serglycin in mast cells and some hematopoietic cells) and the extent of modification (Hp is typically more heavily sulfated with more iduronic acid, while HS is more heterogeneous) .…”