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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.
Highlights
Heparin and heparan sulfate (Hp/HS) are glycosaminoglycans (GAGs), highly anionic unbranched polysaccharides found on the surface of essentially all mammalian cells
As the amount of acetic acid increased from 10% to 40%, the main product was shifted from conjugation through the aliphatic amine (IISAEAB1) to conjugation through the arylamine (IIS-AEAB2, Fig. 2)
At lower pH, the aliphatic amine was protonated and its nucleophilicity would decrease significantly, leaving the unprotonated arylamine to react with the reducing end of heparin disaccharide II-S to form II-S-AEAB2 conjugate
Summary
Heparin and heparan sulfate (Hp/HS) are glycosaminoglycans (GAGs), highly anionic unbranched polysaccharides found on the surface of essentially all mammalian cells. Different cell types will often display different HS structures, and these structures can change as part of the cell’s physiological response3 This structural diversity mediates a wide range of protein-GAG interactions of varying specificity and affinity. Size exclusion chromatography (SEC) is the first separation method following partial depolymerization of the Hp/HS polysaccharide. Hyphenation of IPRP with SEC and time of flight mass spectrometry have greatly improved separation and provided more complete and important structural information of low molecular weight heparin (LMWH). Reverse-phase chromatography without the use of strong ion pairing reagents (RP) has shown strong separation ability for heavily derivatized chondroitin sulfate isomers and heparan sulfate, but the derivatization process leaves the oligosaccharides in non-native conditions not suitable for further functional analysis. The use of microarrays of fractionated Hp/HS will substantially improve the understanding of Hp/ HS-protein interactions
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