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Mass Spectrometric Method Development and Applications for Comprehensive Proteome Analysis Open Access


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Type of item
Degree grantor
University of Alberta
Author or creator
Supervisor and department
Li, Liang (Chemistry Department)
Examining committee member and department
Yeung, Ken (Chemistry and Biochemistry, University of Western Ontario)
Lucy, Charles (Chemistry)
Schultz, Michael (Biochemistry)
Vederas, John (Chemistry)
Serpe, Michael (Chemistry)
Department of Chemistry

Date accepted

Graduation date
Doctor of Philosophy
Degree level
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.
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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