Show simple item record

dc.contributor.advisorHulme, Alison
dc.contributor.advisorUhrin, Dusan
dc.contributor.authorThomas, Sarah Jane
dc.date.accessioned2016-08-29T11:29:11Z
dc.date.available2016-08-29T11:29:11Z
dc.date.issued2015-06-30
dc.identifier.urihttp://hdl.handle.net/1842/16215
dc.description.abstractGlycosaminoglycans (GAGs) are linear polysaccharides found on most animal cell surfaces and in extracellular matrices. Their key biological roles include cell signalling, cell-to-cell recognition, bacterial and viral adhesion, and antibody production. They are composed of disaccharide repeating units, containing uronic acid and amino sugar residues, and may be highly heterogeneously sulfated. Protein-GAG complexes are thought to play an important role in a number of aspects of cancer development, but are an under-studied area due to a lack of enabling tools to facilitate their analysis as discussed in Chapter 1. To obtain homogenous GAG structures, a range of conditions for the separation of GAG oligosaccharides by Zwitterionic Hydrophilic Interaction Liquid Chromatography (ZIC-HILIC) were developed using commercial chondroitin sulfate standards; these are discussed in Chapter 2. Mixed-modal separation mechanisms were explored across a different buffer compositions and elution programs to optimise the conditions to suit individual classes of native and modified glycosaminoglycans. These methods were then applied to the separation of chondroitin sulfate mixtures and heparin oligosaccharides. ZIC-HILIC may also be coupled to Mass Spectrometry to produce an online analytical method for GAG mixtures. A range of optimised conditions for the analysis of low molecular weight glycosaminoglycan oligosaccharides by Electrospray Mass Spectrometry were developed using commercial chondroitin sulfate disaccharide standards; as discussed in Chapter 3. These conditions were then employed in the tandem mass spectrometry (MS/MS) of chondroitin sulfate to identify diagnostic fragmentation patterns and this was applied in the characterisation of chondroitin sulfate disaccharides derived from enzymatic cleavage. The position of ring substituents can also be identified using this methodology, which is desirable in the analysis of chemically-labelled GAG structures. To allow for high resolution EPR and NMR studies of protein-GAG interactions, synthetic procedures for the incorporation of paramagnetic centres into GAG oligosaccharides and proteins were developed and these are discussed in Chapter 4. A propargyl-modified lysine was synthesised for recombinant expression into myoglobin. Copper-Catalysed Azide-Alkyne Cycloaddition (CuAAC) was employed in the spin labelling of myoglobin, however traditional experimental conditions were found to reduce the TEMPO-based spin labels used. Alternative conditions were developed using glutathione to stabilise the copper (II) catalyst without reducing the spin label. Spin labelling of the modified lysine was monitored by EPR. Modification of the non-reducing end was explored for the spin labelling of GAG oligosaccharides, to avoid the ring opening that typically occurs in reducing end labelling, and make use of the unsaturated uronic acid that is derived when obtaining GAGs through enzymatic depolymerisation. A hydrazide labelling of uronic acids was initially adopted, however due to concerns regarding selectivity and availability of hydrazide spin labels, an alternative Thiol-Ene Click (TEC) method was developed. TEC labelling of monosaccharides and unnatural amino acids was found to proceed efficiently under aqueous conditions and was monitored by NMR and HPLC. Preliminary studies in the TEC labelling of chondroitin sulfate disaccharides proved promising, however further optimisation is required for this method to be utilised in the study of protein-GAG interactions by NMR.en
dc.contributor.sponsorEngineering and Physical Sciences Research Council (EPSRC)en
dc.language.isoenen
dc.publisherThe University of Edinburghen
dc.subjectcarbohydratesen
dc.subjectbiomarkersen
dc.subjectmass spectrometryen
dc.subjectchromatographyen
dc.titleNew analytical and synthetic tools for the study of protein-gycosaminoglycan interactionsen
dc.typeThesis or Dissertationen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD Doctor of Philosophyen
dc.rights.embargodate2100-12-31en
dcterms.accessRightsRestricted Accessen


Files in this item

This item appears in the following Collection(s)

Show simple item record