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Title: Stable isotope labelling in the analysis of bacterial antigen processing
Author: Thomas, Victoria J.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2007
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The mechanism by which antigens are processed and presented to T cells exerts a powerful influence on all immune responses, including those directed against pathogens. Knowledge of the peptides produced by antigen presenting cells for presentation to T cells is important in understanding why individuals have different abilities to combat infection and may prove critical in the design of new vaccines. The aim of this project was to attempt to use a novel metabolic labelling technique to enable biochemical identification of potential T cell epitopes in bacterial antigens. The technique is based upon labelling antigens with the stable isotope carbon-13, and subsequent identification of antigen-derived peptides using mass spectrometric isotopic composition analysis. The ultimate goal of the work was to pulse antigen presenting cells with labelled bacteria and identify MHC class II-associated bacteriaderived peptides with the objective of identifying potential vaccine candidates. Towards this goal, outer membrane proteins were prepared and analysed by polyacrylamide gel electrophoresis and immunoblotted with serum from healthy individuals. The results showed that the serum antibodies bound to a number of outer membrane proteins, confirming that they contained B cell epitopes. As a first step to demonstrate that the outer membrane proteins also contained T cell epitopes, it was shown that whole bacteria and bacterial antigens can be successfully labelled with carbon-13 such that all derived peptide fragments may be distinguished by isotopic analysis. However, due to the complexity of the peptide mixture generated by a tryptic digest of the outer membrane proteins, it was critical to combine isotope analysis with excellent chromatographic resolution in order to identify carbon-13 labelled peptides. To address this problem, a new high sensitivity 2D nanoflow liquid chromatography system was developed and its capabilities compared with conventional ID reverse phase chromatographic techniques. Two dimensional separation prior to mass spectral analysis substantially improved the separation of complex biological mixtures leading to an improvement in the identification of carbon-13 labelled peptides in such mixtures. Two dimensional chromatography and carbon-13 labelling was then applied to investigate the processing of tetanus toxin C fragment (TTCF) by lysosomes isolated from EBV transformed B lymphoblastoid cell lines. Whilst large TTCF fragments were demonstrably generated during processing, small (< 3kd) peptides suitable for binding to MHC class II molecules were extremely scarce. Further experiments suggested that this was unlikely to be a consequence of insufficient sensitivity of the carbon-13 technique, but probably a consequence of rapid destruction of the smaller fragments by lysosomal enzymes and high stability of the larger digestion fragments of TTCF. It was concluded from these studies that the metabolic labelling of bacterial proteins with carbon-13 is applicable to the analysis of processed products of bacterial antigens in complex peptide mixtures. Small peptides, with sizes in the range bound to MHC class II molecules, appear to be very sparse in processing compartments, presumably because of rapid proteolysis. Peptides bound to MHC class II are known to be relatively resistant to proteolysis so in future work it is proposed to examine TTCF-derived peptides bound to MHC class II molecules rather than free within lysosomes.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available