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Reverse Polarity Capillary Electrophoresis-Tandem Mass Spectrometry Analysis of Sulfated Glycosaminoglycans

‐ Sep 29, 2020 7:55am

"Sulfated glycosaminoglycan (GAG) carbohydrates are ubiquitous linear, acidic polysaccharide chains

abundant on the cell surface, with non-template driven modifications that effect many biological functions

through protein-binding interactions. The complex nature of GAG oligomers remains a significant analytical

challenge. Capillary electrophoresis-mass spectrometry (CE-MS) presents a fast and efficient approach for

separating complex GAG mixtures. Negative electron transfer dissociation (NETD) is a powerful

fragmentation method that can be performed on mass spectrometry platforms capable of ion-ion reactions.

Electron based fragmentation methods like NETD are capable of fragmenting GAGs without decomposing

sensitive modifications. By combining a CE-MS platform with NETD we can differentiate isoforms within

complex biological mixtures and assign precise structural characterization to identify biologically relevant

protein binding motifs present on GAGs.

The biological activity of GAGs is generated by sulfo-modifications throughout the molecule and isomeric

stereochemistry of the C5 carboxyl group on the uronic acid backbone. A background electrolyte with high

organic solvent content (70% MeOH) and 25 mM ammonium acetate enables reproducible separation

conditions for reverse polarity CE and negative ion mode MS detection of GAG samples. CE-MS has been

shown to be capable of separating isolated GAGs and simple mixtures of GAGs with lengths up to 27

degrees of polymerization, which vary in extent and position of informative sulfo-modifications. Complex

biological mixtures of GAGs, such as Enoxaparin (a low molecular weight heparin pharmaceutical) have

been separated into multiple structures and isoforms over time in a reproducible way. NETD performed with

fluoranthene has been demonstrated to effectively fragment GAG oligomers producing both glycosidic and

cross-ring cleavages necessary for characterization of sulfo-modification. NETD provides informative

fragmentation on a millisecond time scale that is suitable for online experiments. In-house software has been

developed to aid data analysis throughput of structural assignments."


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