In the last decade omics technologies enabled detailed and system-wide analysis of complex biological samples. Genomics, transcriptomics and metabolomics all benefited tremendously from technological advances in their respective fields. Proteomics was revolutionised by mass spectrometry, which allowed simultaneous identification of thousands of proteins in cells, tissues and organisms. And this mainly qualitative revolution, quickly turned quantitative. This work had two main objectives. Firstly, to apply the state of the art instrumentation, data analysis and bioinformatics methods to better our understanding of basic cell biology in a model organism Saccharomyces cerevisiae. Specifically, to quantitatively describe the effects of perturbations, such as adverse environmental conditions or chaperone gene deletions, on protein abundances in the cell. Additionally, it was aimed to demonstrate and evaluate the ability of a new timeof-flight mass spectrometer to perform large-scale absolute quantification. First, it was found that yeast cells are remarkably robust to deletions of major chaperone hub proteins (Ssa1p or Ssb1p deletions). This ability was attributed to network structure and redistribution of folding workload among other related chaperones rather than simple functional redundancy. Second, to build on the first set of results, a detailed time resolved description of yeast proteome dynamics in response to heat stress was provided for the wild type and Ssb1p chaperone mutant strains. In this study, for the first time in the literature, temporal expression patterns of many hallmark heat shock proteins were elucidated. Globally, a slow and sustained proteome remodelling or 'buffering' was revealed in both strains. However, it was also shown that the cells knocked out for the Ssb1p chaperone respond to heat in a distinctly different manner to the wild type strain. Finally, consistent and reproducible absolute quantification of multiple yeast proteomes was demonstrated using a new commercial time-of-flight mass spectrometer with ion mobility separation capabilities. The data obtained revealed global differences in cellular protein content between various chaperone prefoldin mutants as well as differential expression of a set of proteins promising to be interesting targets for further investigations.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:764359 |
| Date | January 2015 |
| Creators | Jarnuczak, Andrew |
| Contributors | Hubbard, Simon ; Grant, Christopher ; Schwartz, Jean-Marc |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/mass-spectrometrybased-quantitative-proteomics-applied-to-the-analysis-of-saccharomyces-cerevisiae-heat-stress-response-and-chaperone-deletion-strains(c653915b-70fa-44d7-9bb7-6e7965349ff0).html |
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