Microarrays enable high throughput analysis with minute amounts of analyte. They are widely used in the ’omics’ fields both as diagnostic and analytical tools. Their ability to dramatically impact an entire field of research has focused our attention on the development of novel methods for the formation, analysis and applications of microarrays to study carbohydrate-protein interactions and the analysis of glycosylation patterns of biomolecules. Availability of appropriately modified ligands is often a limiting factor in the preparation of microarrays. To address this issue robust routes for the synthesis of nine amino ethylglycosides were developed that can be employed for microarray formation. The syntheses of more complex ligands typically deliver small quantities of material despite the requirements for special skills, equipment and long preparation times. Considering the number of complex oligosaccharides that are necessary for systematic microarray studies, the problem of availability of these complex structures is difficult to address solely with synthetic ligands. A modified native chemical ligation (NCL) strategy, in which a surface bound oxo-ester is used instead of a thioester, was optimised and used for efficient chemoselective immobilisation of sugars and peptides carrying N-terminal cysteines. The reaction proceeds under physiological conditions and has the potential to become a valuable tool for immobilisation of N-terminal cysteine-containing molecules from biological samples. The new NCL coupling methodology was developed on gold surfaces and analysed by MALDI-ToF MS. The majority of array systems, however, rely on secondary protein interactions on glass or polystyrene surfaces. A direct, more accurate analytical tool could ease the analysis and significantly improve the quality of data read-out from glass microarrays. MALDI-ToF MS that is applicable to gold microarrays cannot be used on surfaces that do not provide the necessary electrical conductivity. The undertaken experiments indicated that application of conductive tape to the back of glass or polystyrene slides made MALDI-ToF analysis on poorly conducting surfaces possible. Furthermore, the triphenylmethyl (trityl) groups attached to the surface-molecules were shown to act as ’internal-matrix’ and enable the direct MALDI analysis. Once the new array formation and analysis techniques were developed, we turned our attention towards the application of microarrays to analyse carbohydrate-protein interactions. The tools for analysis of glycosylation of biomolecules are laborious and can only be used in specialised labs. As glycosylated biomolecules gain prominence in research, clinical and industrial settings, high throughput analysis of glycosylation patterns is becoming a requirement for quality control. A technique for screening of glycosylation patterns in glycopeptides on microarrays was developed based on biophotonic scattering. This technique enables the detection of glycosylation patterns by screening immobilised glycoproteins with a range of lectins. To study the interactions between enzymes and carbohydrates, a chemoenzymatic synthesis of a mannopeptide, which consisted of four carbohydrate units, was shown in solution and on chip. Three different glycosyl transferases were successfully employed. New methods for microarray formation and analysis were developed and applied to carbohydrate-protein interaction studies. This yielded a new technique to determine protein glycosylation patterns and to produce complex glycans by enzymatic synthesis.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:566539 |
| Date | January 2012 |
| Creators | Weissenborn, Martin |
| Contributors | Flitsch, Sabine |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/development-and-application-of-peptide-and-glycoarrays(dfe37ee9-85be-4751-b491-de7b1eb759f2).html |
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