Method development and proteomic profiling of boar seminal plasma using mass spectrometry-based workflows
| dc.contributor.advisor | Lehloenya, K. C. Stoychev, S. Ramukhithi, F. V. R. | |
| dc.contributor.author | Mokwena, Pateswana Wilson | |
| dc.date.accessioned | 2024-07-04T07:31:38Z | |
| dc.date.available | 2024-07-04T07:31:38Z | |
| dc.date.issued | 2023 | |
| dc.description | A Thesis submitted to the Faculty of Science, Agriculture and Engineering in fulfilment of the requirements for the Degree of Master in the Department of Agriculture at the University of Zululand, South Africa [2023]. | en |
| dc.description.abstract | Artificial insemination is an important technology in the swine industry; it is of great significance in the preservation and improvement of elite breeds. Conventional methods used in assessing semen lack precision, and the use of low fertile boars in breeding systems is prominent. Using mass spectrometry in fertility studies has gained momentum in the race for new markers of fertility and semen cryopreservation. Therefore, the aim of this study was to develop and optimise a mass spectrometry-based proteomic method suitable for profiling boar seminal plasma. To accomplish this aim, gel-based and in-solution methods were evaluated for depth-of-coverage, reproducibility and throughput. For the gel-based method, one-dimensional sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis was coupled to in-gel digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS). For the in-solution methods, acetone precipitation was evaluated by either SDS or urea re-solubilisation, followed by semi-automated on-bead protein capture, clean-up, digestion and LC-MS/MS analysis. The gel-based method data were acquired through data-dependant acquisition and protein identifications were generated through a database search (ParagonTM Algorithm of the ProteinPilotTM Software, v5.0.2). The in-solution data were obtained via the sequential window acquisition of all theoretical mass-data independent acquisition method and protein identification; relative protein quantification was performed using Spectronaut® Software v16. Regarding in-solution analysis, the precipitated and non-precipitated methods performed similarly in terms of the depth of coverage (number of peptides and proteins detected) and reproducibility. Hence, direct analysis of seminal plasma without up-front precipitation was possible. The gel-based method did not improve the identification of peptides and proteins compared with the in-solution workflow. Novel peptide level fractionation was incorporated into the in-solution method to increase protein coverage during a pilot study of eight boars from three breeds (two Large White, three each Kolbroek and Windsnyer). Five hundred and eighty-seven proteins from 233 protein groups were identified across all runs. Thirty-nine proteins were differentially expressed between the Kolbroek and Windsnyer boars. In gene ontology terms, most differentially expressed proteins showed catalytic functions; a very small proportion were identified as directly involved in reproductive functions. | en |
| dc.identifier.uri | https://uzspace.unizulu.ac.za/handle/10530/2527 | |
| dc.language.iso | en | |
| dc.publisher | University of Zululand | en |
| dc.title | Method development and proteomic profiling of boar seminal plasma using mass spectrometry-based workflows | en |
| dc.title.alternative | Swine preservation | en |
| dc.type | Thesis | en |