Protein prefractionation is a popular and effective strategy for improved MS analysis of complex proteome mixtures. A challenge of prefractionation is the even partitioning with high recovery of all components of the mixture, particularly hydrophobic proteins. This thesis assesses various proteome prefractionation platforms, with a goal of comprehensive proteome analysis. A more reliable dataset of 1136 S. cerevisiae transmembrane proteins was computationally generated, and used to assess two gel-based platforms (GeLC/MS and GELFrEE/MS). These platforms were determined to be comparable for proteome analysis. The requirement for high-throughput, automated fractionation demands a gel-free separation workflow. Here, a LC-based workflow was optimized, relying on SDS-assisted yeast extraction, organic solvent protein precipitation, and reversed phase separation in a formic acid/isopropanol solvent system. Though this workflow afforded improvements over conventional LC strategies to proteome fractionation, the gel-based platforms were demonstrated to be superior, in terms of their unbiased separation of hydrophobic vs hydrophilic proteins.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:NSHD.ca#10222/13791 |
| Date | 13 May 2011 |
| Creators | Liu, Fang |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
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