title: Development and application of label free quantitative proteomic methods
Creators: name: Cummings, Rebecca
Publisher: name: University of Liverpool Repository
Year: 2013

description: The aim of this Ph.D. was to develop advanced methods for quantitative proteomics and use these methods to investigate the presence of protein biomarkers of sperm performance, differential expression of sperm membrane proteins and differential expression of E.coli proteins. Quantitative analysis of E.coli generated analytical samples that were analysed with multiple mass spectrometers and with multiple software packages. Through these samples an optimal label free quantitative proteomic workflow was generated and software was thoroughly tested to determine the optimal software to be used for data analysis on varying biological questions. Identification of protein(s) that correlate with increased or decreased fertility would be economically beneficial. Currently semen samples are subject to quality control where general movement and morphological defects are studied, but this does not always correlate with the ejaculate passing a post cryopreservation quality control check or that specific bull generating offspring. Identification of a protein or set of proteins with abundance variation in bulls of known high or low fertility would allow lower fertility bulls to be removed from the breeding programme at an early age, reducing rearing costs, and would allow longitudinal health monitoring of individual bulls. Discovery of differentially expressed proteins in the membrane of sperm with the X or Y chromosome would allow the generation of a method to separate the two sperm populations. This will be beneficial as most livestock farmers would prefer offspring of a specific sex, either to sell or replenish animal stock. Quantitative analysis of proteins present in bovine seminal plasma led to the identification and quantitative comparison of the seminal plasma proteins present in two breeds of bull, Holstein and Belgian Blue and a quantitative comparison of the seminal plasma from two domestic farm animal species, bovine and porcine. Intra species comparisons determined no quantitative variation between the two breeds, while the inter species comparison determined variation between the proteins present in both species seminal plasma and the corresponding amounts of proteins present in both species. A quantitative comparison was performed to determine the expression of proteins from two strains of E.coli, a wild type strain (MG1655) and a genome depleted strain (MDS66), this led to the confirmation of gene deletions in the genome depleted strain due to their lack of protein products in mass spectrometric analysis, and the identification of proteins that were differentially expressed due to pleiotropic effects of these genome deletions. To investigate the proteins expressed in the sperm membrane a mass spectrometer compatible enrichment method was generated and membrane proteins were identified, quantified and compared between sperm expressing X and Y chromosomes. This study did not lead to the determination of any proteins with differential expression in the X or Y bearing sperm.