- Mass Spectrometric Method Development and Applications for Comprehensive Proteome Analysis
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- In the field of proteomics research, it is desirable to detect the entire proteome or all the proteins present in a sample. However, due to the complexity of most samples, this task is challenging. My thesis work is mainly focused on the development of new protein solubilization and fractionation techniques to increase the identification efficiency in shotgun proteomic studies, namely to detect as many proteins as possible at high sample handling throughput. Several techniques have been developed or optimized in this thesis. First, protein level fractionation by using sequential protein precipitation and solubilization was effective in simplifying a complex sample and enhancing the proteome coverage. Second, microwave-assisted sequential protein solubilization (MAPS) was developed to speed up the protein solubilization process, increase protein solubility and protein digestion efficiency. As a result, by using MAPS combined with two dimensional-liquid chromatography (2D-LC) tandem mass spectrometry (MS/MS) analysis, peptide and protein identification efficiency was improved. Third, a faster and better resolution in protein fractionation was achieved by a macro-porous C18 reversed-phase liquid chromatography column (mRP-C18). The mRP-C18 fractionation method was then optimized and applied to the analysis of the phosphoproteome of MDA-MB-231 cells. Finally, sequential phosphopeptide enrichment by metal ion affinity chromatography (IMAC) and metal oxide affinity chromatography (TiO2) combined with strong cation exchange (SCX)-reverse phase liquid chromatography (RPLC) MS/MS method was applied to the analysis of human breast cancer tissues. 297 phosphoproteins were found possibly related to the metastasis of breast tumor and 875 phosphoproteins were found possibly related to the genesis of the breast tumor. The techniques developed or optimized in my thesis work improved sample preparation and fractionation efficiency, and therefore enhanced the overall efficiency of proteome identification. The newly identified phosphoproteins in MDA-MB-231 cells and human breast cancer tissues may generate novel insight into breast cancer biology. These approaches also hold great potential for profiling a wider range of proteomes in a more comprehensive and efficient way.
- Doctor of Philosophy
- Department of Chemistry
- Spring 2012
- Li, Liang (Chemistry Department)
Lucy, Charles (Chemistry)
Vederas, John (Chemistry)
Serpe, Michael (Chemistry)
Schultz, Michael (Biochemistry)
Yeung, Ken (Chemistry and Biochemistry, University of Western Ontario)
Theses and Dissertations Spring 2009 to present
Department of Chemistry
Apr 30, 2014 9:46 PM
Mar 6, 2012 12:30 PM
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