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Integration of <18/sup>O Labeling and Solution Isoelectric Focusing in a Shotgun Analysis of Mitochondrial Proteins

dc.contributor.advisorFenselau, Catherineen_US
dc.contributor.authorWang, Jinshanen_US
dc.description.abstractThe coupling of efficient separations and mass spectrometry instrumentation is highly desirable to provide global proteomic analysis. When quantitative comparisons are part of the strategy, separation and analytical methods should be selected, which optimize the isotope labeling procedure. Enzyme-catalyzed <18/sup>O labeling is considered to be the labeling method most compatible with analysis of proteins from tissue and other limited samples. The introduction of label at the peptide stage mandates that protein manipulation be minimized in favor of peptide fractionation post-labeling. In the present study, forward and reverse <18/sup>O labeling are integrated with solution isoelectric focusing and capillary LC-tandem mass spectrometry to study changes in mitochondrial proteins associated with drug resistance in human cancer cells. A total of 637 peptides corresponding to 278 proteins were identified in this analysis. Of these, twelve proteins have been demonstrated from the forward and reverse labeling experiments to have abundances altered by greater than a factor of two between the drug susceptible MCF-7 cell line and the MCF-7 cell line selected for resistance to mitoxantrone. Galectin-3 binding protein precursor was detected in the resistant cell line, but was not detected in the drug susceptible line. Such proteins are challenging to <18/sup>O and other isotope strategies and a solution is offered, based on reverse labeling. These twelve proteins play a role in several pathways including apoptosis, oxidative phosphorylation, fatty acid metabolism and amino acid metabolism. For some of these proteins, their possible functions in drug resistance have been proposed.en_US
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dc.titleIntegration of <18/sup>O Labeling and Solution Isoelectric Focusing in a Shotgun Analysis of Mitochondrial Proteinsen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.subject.pqcontrolledChemistry, Analyticalen_US
dc.subject.pquncontrolledcomparative proteomicsen_US
dc.subject.pquncontrolledreverse labelingen_US
dc.subject.pquncontrolledsolution isoelectric focusingen_US
dc.subject.pquncontrolledgalectin-3 binding proteinen_US
dc.subject.pquncontrolledhuman mitochondriaen_US

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