SOLUTION ISOELECTRIC FOCUSING AND ITS APPLICATION IN COMPARATIVE PROTEOMIC STUDIES OF NUCLEAR PROTEINS

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dc.contributor.advisorFenselau, Catherine Cen_US
dc.contributor.authorAn, Yanmingen_US
dc.contributor.departmentChemistryen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.date.accessioned2005-10-11T10:03:04Z
dc.date.available2005-10-11T10:03:04Z
dc.date.issued2005-05-27en_US
dc.description.abstractIn proteomic research, experimental and computational approaches are combined to provide global analysis of the entire proteomes of cells and tissues. The identification and quantification of multiple proteins, which constitute a specific biological system, are important for understanding complex problems in biology. The coupling of highly efficient separations and mass spectrometry instrumentation is evolving rapidly and is being widely applied to problems ranging from biological function to drug development. Development of rapid and high-resolution separation technology is an important field in proteomics. In this study, a solution isoelectric focusing apparatus was modified and built into a two-dimensional separation method for peptides. Newly commercialized isoelectric membranes, which carry immobilized ampholytes, were integrated to establish the pH boundaries in this apparatus. High-performance liquid chromatography was employed as the second dimension, integrated with mass spectrometry. An insoluble nuclear protein fraction was used for optimization and evaluation of this method. The insoluble nuclear proteins were recovered from the nuclei of human MCF-7 human cancer cells and cleaved enzymatically. The resulting peptides were analyzed by the two-dimensional separation method, which coupled solution isoelectric focusing with reversed-phase liquid chromatography interfaced with mass spectrometry. A total of 281 peptides corresponding to 167 proteins were identified by this experiment. The high sample capacity and concentration effect of isoelectric focusing make it possible to detect relatively low abundance proteins in a complex mixture. This two-dimensional separation method dramatically improves peptide detection and identification compared with a single dimension LC-MS analysis. This method has been demonstrated to provide efficient and reproducible separation of both protein and peptides. The two-dimensional separation method was combined with proteolytic isotopic labeling for comparative analysis of protein expression in different cells. Abundances of nuclear proteins from three different drug resistant MCF-7 cancer cell lines were compared to those from the drug susceptible parent cell line using this combined strategy. The abundances of 19 proteins were found to be significantly changed. Their functions are considered in relation to potential mechanisms of in drug resistance.en_US
dc.format.extent2633437 bytes
dc.format.mimetypeapplication/pdf
dc.identifier.urihttp://hdl.handle.net/1903/2869
dc.language.isoen_US
dc.subject.pqcontrolledChemistry, Analyticalen_US
dc.subject.pquncontrolledproteomicsen_US
dc.subject.pquncontrolledsolution isoelectric focusingen_US
dc.subject.pquncontrollednuclear proteinsen_US
dc.subject.pquncontrolledmass spectrometryen_US
dc.subject.pquncontrolleddrug resistanceen_US
dc.subject.pquncontrolledMCF-7 cancer cellsen_US
dc.titleSOLUTION ISOELECTRIC FOCUSING AND ITS APPLICATION IN COMPARATIVE PROTEOMIC STUDIES OF NUCLEAR PROTEINSen_US
dc.typeDissertationen_US

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