Liu, Fang2011-06-152011-06-152011-06-15http://hdl.handle.net/10222/13791Protein 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.enProteome, Prefractionation, GeLC, GELFrEE, RPLC, Mass Spectrometry, Protein IdentificationAssessment of Universal Approaches to Proteome Prefractionation