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"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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