The development of proteomic techniques to study the Australian paralysis Tick, Ixodes holocyclus : the application of proteomic technology to an organism with poor bioinformatic information
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The Australian paralysis tick, Ixodes holocyclus, is representative of the majority of organisms studied in biology in that the bioinformatic information available (genome sequence, annotated coding regions and protein sequences) are far from complete. The study of well characterised model organisms has shown that proteomics and its associated technologies are able to isolate, identify and characterise individual protein isoforms at femto to attomole amounts of sample. With these model organisms, this can be achieved in either unpurified or partially purified samples (shotgun proteomics) or by high resolution separations using isoelectric fractionation and two-dimensional gel electrophoresis. In a poorly characterised organism, this is not the case. The work presented in this thesis applies proteomic technologies to characterising the tick proteome in a hypothesis and non-hypothesis driven manner. In the non-hypothesis driven approaches, fractionation and separation methodologies were applied to determine which method or combination of methods provided the greatest number of protein identifications. The results of these studies showed that the resolution of protein isoforms provided by 2-DGE is invaluable for characterising proteins from I. holocyclus. This is because the homogenous protein spot can be excised from the gel and characterised by de novo sequencing of MS/MS spectra with the knowledge that all peptides are from the same protein. However, successful de novo sequencing is reliant on good quality MS/MS spectra, which is partly reliant on intensely stained gel spots, which is determined by the amount of sample loaded onto the gel. It is well documented and demonstrated in this study that overloading of 2-D gels with samples containing high abundance proteins, tick cytoskeletal proteins in this case, can cause spot resolution problems. Fractionation of the sample using a Multi-compartment Electrolyser and equalisation with Proteominer partially addresses this issue, but further refinement is necessary. The optimised sample preparation methods were then applied in hypothesis driven experiments to characterise specific protein subtypes using Western blots and a novel fluorescent zymogram approach. The analysis identified a number of proteins that will need further characterisation, using molecular biological and recombinant protein expression techniques, to determine their suitability as vaccine candidates.
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