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NMR-Based Metabolic Profiling: Methods and Application in Cancer

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NMR-Based Metabolic Profiling: Methods and Application in Cancer NMR-Based Metabolic Profiling: Methods and Application in Cancer Biomarker Discovery by Wencheng Ge A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemistry) in the University of Michigan 2014 Doctoral Committee: Professor Ayyalusamy Ramamoorthy, Chair Professor Zhan Chen Professor Ari Gafni Professor Michael D. Morris © Wencheng Ge 2014 ACKNOWLEDGEMENTS I would like to thank my advisor Professor Ayyalusamy Ramamoorthy for his guidance and advice throughout my graduate studies. I would like to express my deepest appreciation to Dr. Bagganahalli Somashekar, Dr. Neil MacKinnon and Dr. Pratima Tripathi for their suggestions and help in my research. They continually discussed with me about how to improve my research and encourage me to pull me through the difficulties. In addition, I want to thank Dr. Thekkelnaycke Rajendiran for giving me exciting projects related to prostate cancer research. His suggestions have always enlightened me and helped me to think in different perspectives. Thanks to my committee members, Professor Michael Morris, Professor Ari Gafni and Professor Zhan Chen for their thoughtful input and encouragement. Their support has continuously motivated me in my research. I would like to thank my friends here in Ann Arbor, especially Jing Lu for sharing ideas in statistics with me. Also, many thanks to my wonderful family for their support and patience. Lastly, I’m grateful to my brother Zicheng Ge and Branden DeRoche for their dedication. They bring sunshine to my life. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................ii LIST OF TABLES .......................................................................................................... viii LIST OF FIGURES ..........................................................................................................ix LIST OF ABBREVIATIONS ............................................................................................ xii CHAPTER 1 Introduction to metabolomics ..................................................................... 1 1.1 Metabolic profiling by NMR .................................................................................... 2 1.1.1 NMR experiments for different sample types.......................................................... 3 1.1.2 Data preprocessing ................................................................................................ 4 1.1.3 NMR spectral signal assignment ............................................................................ 5 1.2 Data processing and statistical analysis................................................................. 5 1.2.1 Data pretreatment .................................................................................................. 5 1.2.2 Univariate analysis ................................................................................................. 7 1.2.3 Multivariate analysis ............................................................................................... 8 1.3 Pathway analysis in disease biomarker discovery ............................................... 10 1.4 References ........................................................................................................... 11 CHAPTER 2 Deproteinization and Delipidation of Blood serum for Quantitative NMR- based Metabolomics ..................................................................................................... 18 iii 2.1 Abstract ................................................................................................................ 18 2.2 Introduction .......................................................................................................... 19 2.3 Experimental design ............................................................................................ 21 2.3.1 Materials. .............................................................................................................. 21 2.3.2 Protein removal and metabolite extraction. .......................................................... 22 2.3.3 TSP as an internal standard for serum extraction. ............................................... 23 2.3.4 Metabolite spike-in experiment. ............................................................................ 24 2.3.5 NMR experiments and data processing. .............................................................. 24 2.3.6 Statistical analysis. ............................................................................................... 26 2.3.7 Quantitation of serum metabolites. ....................................................................... 26 2.4 Results and Discussion ........................................................................................ 27 2.4.1 TSP as an internal quantitative reference for serum preparation and analysis by NMR. ............................................................................................................................. 27 2.4.2 Comparison of serum preparation protocols. ....................................................... 29 2.4.3 PCA analysis of serum sample preparation methods. .......................................... 30 2.4.4 Spike-in experiment proved the accuracy of quantitative analysis. ...................... 31 2.5 Conclusion ........................................................................................................... 33 2.6 References ........................................................................................................... 36 CHAPTER 3 Optimized urinary metabolite separation into two-phase solvent system for improved signal assignment in NMR-based metabolomics ........................................... 42 3.1 Abstract ................................................................................................................ 42 3.2 Introduction .......................................................................................................... 43 iv 3.3 Experimental design ............................................................................................ 45 3.3.1 Materials and urine sample. ................................................................................. 45 3.3.2 Two-phase urinary metabolite separation. ........................................................... 45 3.3.3 NMR spectroscopy. .............................................................................................. 47 3.3.4 Statistical analysis. ............................................................................................... 49 3.4 Results and Discussion ........................................................................................ 49 3.4.1 Complexity of NMR spectra of urine. .................................................................... 49 3.4.2 Sample pre-processing and optimization of the two-phase solvent systems. ....... 50 3.4.3 NMR analysis of non-polar and polar solvent phases. ......................................... 51 3.4.4 PCA analysis. ....................................................................................................... 57 3.5 Conclusions ......................................................................................................... 59 3.6 References ........................................................................................................... 61 CHAPTER 4 Prostate cancer diagnosis using NMR-based metabolic profiling of serum ...................................................................................................................................... 68 4.1 Abstract ................................................................................................................ 68 4.2 Introduction .......................................................................................................... 69 4.3 Materials and Methods ......................................................................................... 71 4.3.1 Prostate serum sample collection ......................................................................... 71 4.3.2 Sample preparation for NMR metabolic profiling .................................................. 71 4.3.3 1H MAS NMR spectroscopy ................................................................................. 72 4.3.4 Multivariate modeling ........................................................................................... 73 4.3.5 Univariate analysis ............................................................................................... 73 v 4.4 Results and discussion ........................................................................................ 74 4.4.1 1H NMR metabolic profiles ................................................................................... 75 4.4.2 Statistical analysis ................................................................................................ 77 4.4.3 Metabolic alterations and signal transduction pathways ....................................... 80 4.5 Conclusion ........................................................................................................... 81 4.6 References ........................................................................................................... 81 CHAPTER 5 NMR-based metabolomic profiling of urine nominates a signal biomarker panel for prostate cancer detection ..............................................................................
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