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Title: Decoding heparan sulfate structure using glycomics strategies
Author: Miller, Rebecca
ISNI:       0000 0004 2734 6597
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2011
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This thesis describes studies on the difficulties and solutions involved with the separation and analysis of heparin and heparan sulfate (HS) saccharides. Their repeating uronic acid and glucosamine nature means that they can only be distinguished or separated via their sulfates and acetates, making them a significant challenge using the high performance liquid chromatography (HPLC) techniques available. More comprehensive knowledge of the limited methods available, as well as new methods, is essential for optimal results. Size exclusion chromatography (SEC) separation of glycosaminoglycans (GAG) oligosaccharide mixtures is commonly used but the products have not previously been fully characterised, and studies here aimed at filling in some gaps in understanding of this complex process. Mass spectrometry is particularly useful for this task, as it can provide detailed molecular masses and structural information. Mass spectrometry analysis of SEC separated oligosaccharides showed that although these mixtures were separated via their hydrodynamic volume as expected, complex separations were observed. Increasingly poor resolution and overlapping of saccharide sizes was evident, particularly for hexasaccharides and larger, and indicated that previous suppositions about the nature of "size-defined" saccharides produced by SEC are unsound. Mass spectrometry also led to an unexpected identification of much higher proportions of odd-numbered oligosaccharides than previously described. High performance liquid chromatography (HPLC) techniques are commonly used in the glycosaminoglycan (GAG) field, but still have limitations, making the isolation of homogenous glycosaminoglycan (GAG) oligosaccharides of bioactive size (typically dp6-8+) an ambitious task. Here the development and application of porous graphitised carbon (PGC) chromatography columns was explored for preparative, analytical and micro- separations of heparin/HS saccharides based on a complex interplay of size, shape and charge. Graphitised carbon displays an additional advantage over most high performance liquid chromatography techniques used in the separation and analysis of heparin and heparan sulfate (HS) as it is compatible with mass spectrometry. Compatibility with electrospray mass spectrometry (ESI-MS) was confirmed, permitting development of an in- line method for the separation and characterisation of complex mixtures and pure saccharide species. Heterogeneous saccharide pools can be analysed rapidly, providing structural insights into multiple saccharide components, and detailed structural / information can be obtained on pure saccharide species. Finally, this thesis described studies used existing and novel high performance liquid chromatography techniques to separate and isolate single glycosaminoglycan oligosaccharides with the goal of sequencing these structures using electrospray mass spectrometry. Heparin and heparan sulfate (HS) disaccharide standards were initially studied by characterising internal fragmentation patterns unique to their sulfation patterns. This information was then used to explore sequencing of larger pure GAG oligosaccharides isolated from tissue HS. Compatibility of derived oligosaccharide sequences with disaccharide compositional analysis was confirmed. Verification that this electrospray mass spectrometry approach could sequence accurately was obtained by blind testing of chemically synthesised heparin / heparan sulfate oligosaccharides with known structures validated by different methods 'from other labs. The initial data suggest that the method has potential for sequencing of pure oligosaccharides, and by combining this method in future with bioactivity assays should assist in unravelling the structure-activity relationships of heparin / heparan sulfate saccharides.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available