Microarrays enable high throughput analysis with minute amounts of analyte. They arewidely used in the 'omics' fields both as diagnostic and analytical tools. Their abilityto dramatically impact an entire field of research has focused our attention on the developmentof novel methods for the formation, analysis and applications of microarraysto study carbohydrate-protein interactions and the analysis of glycosylation patterns ofbiomolecules.Availability of appropriately modified ligands is often a limiting factor in the preparationof microarrays. To address this issue robust routes for the synthesis of nine aminoethylglycosides were developed that can be employed for microarray formation.The syntheses of more complex ligands typically deliver small quantities of materialdespite the requirements for special skills, equipment and long preparation times.Considering the number of complex oligosaccharides that are necessary for systematicmicroarray studies, the problem of availability of these complex structures is difficult toaddress 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 usedfor efficient chemoselective immobilisation of sugars and peptides carrying N-terminalcysteines. The reaction proceeds under physiological conditions and has the potential tobecome a valuable tool for immobilisation of N-terminal cysteine-containing moleculesfrom biological samples.The new NCL coupling methodology was developed on gold surfaces and analysedby MALDI-ToF MS. The majority of array systems, however, rely on secondary proteininteractions on glass or polystyrene surfaces. A direct, more accurate analytical toolcould ease the analysis and significantly improve the quality of data read-out from glassmicroarrays. MALDI-ToF MS that is applicable to gold microarrays cannot be used onsurfaces that do not provide the necessary electrical conductivity. The undertaken experimentsindicated that application of conductive tape to the back of glass or polystyreneslides 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 ourattention towards the application of microarrays to analyse carbohydrate-protein interactions.The tools for analysis of glycosylation of biomolecules are laborious and can onlybe used in specialised labs. As glycosylated biomolecules gain prominence in research,clinical and industrial settings, high throughput analysis of glycosylation patterns is becominga requirement for quality control. A technique for screening of glycosylationpatterns in glycopeptides on microarrays was developed based on biophotonic scattering.This technique enables the detection of glycosylation patterns by screening immobilisedglycoproteins with a range of lectins.To study the interactions between enzymes and carbohydrates, a chemoenzymatic synthesisof a mannopeptide, which consisted of four carbohydrate units, was shown in solutionand on chip. Three different glycosyltransferases were successfully employed.New methods for microarray formation and analysis were developed and applied tocarbohydrate-protein interaction studies. This yielded a new technique to determine proteinglycosylation patterns and to produce complex glycans by enzymatic synthesis.
|Date of Award
|1 Aug 2013
- The University of Manchester
|Sabine Flitsch (Supervisor)
- Glycoarray, Peptidearray, Native Chemical Ligation, non conductive, aluminium foil, MALDI on glass