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ABSTRACT Title of Dissertation: INTEGRATION OF ABSTRACT Title of Dissertation: INTEGRATION OF 18O LABELING AND SOLUTION ISOELECTRIC FOCUSING IN A SHOTGUN ANALYSIS OF MITOCHONDRIAL PROTEINS Jinshan Wang, Doctor of Philosophy, 2007 Dissertation Directed by: Professor Catherine Fenselau Department of Chemistry and Biochemistry The 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 18O 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 18O 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 18O 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. INTEGRATION OF 18O LABELING AND SOLUTION ISOELECTRIC FOCUSING IN A SHOTGUN ANALYSIS OF MITOCHONDRIAL PROTEINS by Jinshan Wang Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2007 Advisory Committee: Dr. Catherine Fenselau, Chair Dr. Neil Blough Dr. Jonathan D. Dinman Dr. David Fushman Dr. Douglas Julin © Copyright by Jinshan Wang 2007 Dedication This dissertation is dedicated to my parents Wenxiu Zhang and Mingsheng Wang. ii Acknowledgements I would first like to thank my advisor Dr. Catherine Fenselau for introducing me to the field of proteomics and for her encouragement, ideas and advice throughout the years. I have benefited greatly from her never-ending support and developed into a better and more independent scientist. I also want to thank the many former and current members of the Fenselau group for their warm help both in my research and life. Special thanks to Dr. Yanming An, Dr. Faith Hays, Dr. Rachael Strong, Ms. Natasha Smith for being such good friends and those happy hours we spent. I am thankful to Dr. Peter Gutierrez for his assistance in operation of the LCQ mass spectrometry instrument and database searching, Dr. Nathan Edwards for his kind help in bioinformatics. I would like to thank my parents and my sister for all their love, understanding and support throughout my life. They have encouraged me in every way imaginable, and I love them dearly. Finally, I would like to thank my husband, Wei and my daughter, Jialei for their love and support to make this possible. iii Table of Contents Chapter 1: Introduction ................................................................................. 1 Part A ........................................................................................................ 1 Drug resistance in cancer chemotherapy............................................ 1 Mitochondria and cancer chemotherapy ............................................. 4 MCF-7 cells as a model system .......................................................... 8 Part B ...................................................................................................... 10 Proteome and Proteomics................................................................. 10 Principles of mass spectrometry in proteomics ................................. 11 Protein identification and bioinformatics............................................ 19 Part C ...................................................................................................... 20 Separation of protein and peptide mixtures....................................... 20 1. Separation of a specific protein or a group of proteins............ 21 2. Multidimensional separations of proteins/peptides ................. 23 A. 2-D gel electrophoresis .................................................... 23 B. Multidimensional separations in-solution.......................... 24 Part D ...................................................................................................... 32 Quantitation....................................................................................... 32 1. Relative quantitation ............................................................... 33 A. Gel-based methods.......................................................... 33 B. Non-gel-based methods................................................... 34 2. Absolute quantitation ............................................................. 44 Part E ...................................................................................................... 44 Hypothesis and objectives ................................................................ 44 Chapter 2: Experimental ............................................................................. 46 Materials:................................................................................................. 46 Equipment ............................................................................................... 47 Methods .................................................................................................. 48 Cell culture and harvest .................................................................... 48 Isolation of mitochondria ................................................................... 49 Extraction of mitochondrial proteins .................................................. 50 Protein assay .................................................................................... 50 Digestion of Soluble Protein.............................................................. 51 Proteolytic 18O Labeling of mitochondrial soluble proteins ................ 51 Proteolytic 18O Labeling of lysozyme from chicken egg white........... 52 Forward and Reversed 18O labeling.................................................. 53 Solution Isoelectric Focusing (sIEF) Separation ............................... 53 Reversed-Phase HPLC (RPHPLC) analysis with UV Detector ......... 54 Peptide desalting .............................................................................. 54 MALDI-TOF Mass Spectrometry ....................................................... 55 NanoLC-QqTOF mass spectrometry and protein identification......... 55 µLC-ion trap mass spectrometry and protein identification ............... 56 Quantitation of the relative abundance of proteins............................ 57 iv Chapter 3: Results and Discussion ............................................................. 62 Mitochondrial isolation and protein extraction ......................................... 62 Peptide fractionation with solution isoelectric focusing as the first dimensional separation ........................................................................... 62 Peptide separation with reversed-phase LC/MS/MS as the second dimensional separation ........................................................................... 65 Peptide identification using tandem mass spectrometry.......................... 69 Protein identification and classification.................................................... 92 Integration of 18O labeling with solution isoelectric focusing (sIEF) ....... 107 Evalution of forward and reverse 18O labeling....................................... 119 Mitochondrial protein abundance profile in the MCF-7 cell line resistant to mitoxantrone ......................................................................................... 125 Biological implications of abundance changes ...................................... 128 Apoptosis ........................................................................................ 129 Fatty acid oxidation ......................................................................... 132 Oxidative phosphorylation............................................................... 134 Tricarboxylic acid cycle (TCA)......................................................... 136 Protein synthesis............................................................................. 137 Amino acid metabolism ................................................................... 138 Transport......................................................................................... 140 Chapter 4: Conclusions ............................................................................... 142 References .................................................................................................. 144 v List of Tables Table 1. List of peptides identified from mitochondrial
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