Mass spectrometry has shown its unique potential for studying the structure of proteins. Associated with various specific techniques (H/D exchange, ion mobility, gas-phase spectroscopy, multidimensional mass analysis), it has demonstrated to be an essential tool allowing primary structures to be analyzed and providing a lot of information about high order conformations. This work assesses the capabilities of these emerging mass spectrometry methods, and especially the gas-phase H/D exchange technique, for the structural analysis of nucleic acids. Gas-phase H/D exchange was first used to study single stranded oligonucleotides. The exchange reactions were performed with CD3OD in the collision cell of a 9.4 T FT-ICR MS. In these experimental conditions and in integrating the experimental and theoretical results, gas-phase H/D exchange was shown to be controlled by hydrogen accessibility and not by the chemical nature of the heteroatom bearing the exchangeable hydrogen. This allowed the presence of one structure or several conformers that possess different exchange properties to be detected. Moreover, when several structures were observed, increasing the internal energy of the ions at the entrance of the H/D exchange cell gave access to a qualitative estimation of the relative height of the isomerization barriers compared to the H/D exchange ones. Ion mobility experiments confirmed independently the H/D exchange results. Comparing the ion activation experiments for H/D exchange and for ion mobility revealed that the most compact conformer displays the fastest H/D exchange. This observation showed that H/D exchange and ion mobility provide us with complementary information because accessibility and macromolecule compactness are not univocally associated. Two other methods having independent principles of operations were sequentially combined. The fragmentation of a totally deuterated dinucleotide in exchangeable positions demonstrated the coexistence of several fragmentation channels. The latter were classified according to the involvement of non-labile or labile protons in the fragmentation process. Double resonance experiments were also performed and demonstrated the existence of consecutive fragmentation mechanisms. The involvement of labile, and therefore exchangeable protons in the fragmentation mechanism casts doubt on the use of tandem mass spectrometry to localize incorporated deuteriums in oligonucleotides. Finally, an exploratory work on the gas-phase H/D exchange of non-covalent complexes is presented.

Kong Kam-chuen, Ebenezer. / Thesis submitted in: July 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 131-149). / Abstracts in English and Chinese. / Abstract in English --- p.i / Abstract in Chinese --- p.ii / Table of Content --- p.iii -vii / Acknowledgement --- p.viii / List of Abbreviations --- p.ix -x / List of Tables --- p.xi / List of Figures --- p.xii -xiii / List of Appendices --- p.xiv -xv / Chapter CHAPTER 1 --- Review of Literature / Chapter 1.1 --- Peptidomic Research / Chapter 1.1.1 --- Proteomics and Genomics --- p.1-2 / Chapter 1.1.2 --- Peptidomics and Quantitative Profiling --- p.2-5 / Chapter 1.1.3 --- Proteomics and Peptidomics in Medical Research --- p.5-6 / Chapter 1.1.4 --- Application of Quantitative Peptidomic Profiling in Cancer Research --- p.6-8 / Chapter 1.2 --- Technologies for Peptidomic Studies and Limitations --- p.9-11 / Chapter 1.2.1 --- High Performance Liquid Chromatograph (HPLC) --- p.12 / Chapter 1.2.1.1 --- Basic Principle --- p.13-15 / Chapter 1.2.1.2 --- Application in Peptidomic / Proteomic Research --- p.16-17 / Chapter 1.2.2 --- Peptide Gel Electrophoresis --- p.18 -19 / Chapter 1.2.2.1 --- Basic Principle --- p.19-21 / Chapter 1.2.2.2 --- Application in Peptidomic / Proteomic Research --- p.21 -22 / Chapter 1.2.3 --- Capillary Electrophoresis (CE) --- p.23 -24 / Chapter 1.2.3.1 --- Basic Principle --- p.24-29 / Chapter 1.2.4 --- Mass Spectrometry --- p.30 / Chapter 1.2.4.1 --- Electro spray Ionization (ESI) Mass Spectrometry --- p.31 / Chapter 1.2.4.1.1 --- Basic Principle --- p.31-34 / Chapter 1.2.4.2 --- Matrix-assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry --- p.35 / Chapter 1.2.4.2.1 --- Ionization of Sample --- p.35 -38 / Chapter 1.2.4.2.2 --- Time-of-Flight (TOF) Analyzer --- p.39-40 / Chapter 1.2.4.2.3 --- Application in Peptidomic / Proteomic Research --- p.40-42 / Chapter 1.2.4.2.4 --- Isotope-Coded Affinity Tags (ICAT®) --- p.42 -46 / Chapter 1.2.4.2.5 --- Limitations --- p.46 -48 / Chapter 1.2.4.3 --- Surface Enhanced Laser Desorption/Ionization (SELDI) Mass Spectrometry --- p.49 / Chapter 1.2.4.3.1 --- Basic Principle --- p.49 -51 / Chapter 1.2.4.3.2 --- Application in Peptidomic / Proteomic Research --- p.51-52 / Chapter 1.2.4.3.3 --- Retentate Chromatography (RC) --- p.53-54 / Chapter 1.2.4.4. --- Recent Advances in Application of MALDI Technologies --- p.55 -57 / Chapter CHAPTER 2 --- Objectives --- p.58 -59 / Chapter CHAPTER 3 --- Methodologies / Chapter 3.1 --- Method Development / Chapter 3.1.1 --- Nitrocellulose (NC) Preparation --- p.60 / Chapter 3.1.2 --- Matrix Chemicals Preparation --- p.60 / Chapter 3.1.3 --- Spotting Methods --- p.61 / Chapter 3.1.4 --- Standard Preparation --- p.61 -63 / Chapter 3.1.5 --- Data Collection and Analysis --- p.64 / Chapter 3.2 --- Identification of Distinguishing Features for HCC --- p.65 / Chapter 3.2.1 --- Classification Trees --- p.65 -66 / Chapter 3.2.2 --- Statistical Analysis --- p.66 / Chapter CHAPTER 4 --- Results / Chapter 4.1 --- Optimization of Spotting Methods in Protein Quantification --- p.67 -68 / Chapter 4.1.1 --- Detection of Low-Molecular Weight Proteins / Peptides --- p.68 -71 / Chapter 4.1.2 --- Detection of High-Molecular Weight Proteins --- p.71 -74 / Chapter 4.2 --- Assay Linearity of the Nitrocellulose-MALDI-TOF MS for Peptides with Different Masses --- p.75 -84 / Chapter 4.3 --- Accuracy of Mass Measurement --- p.84 -85 / Chapter 4.4 --- Applications in Quantitative Peptidomic Profiling of Serum --- p.86 -88 / Chapter 4.5 --- Application in Tumor Marker Discovery / Chapter 4.5.1 --- Identification of Peptidomic Features For Classification Between the HCC and CLD Patients by Classification Tree Analysis --- p.89 - 92 / Chapter 4.5.2 --- Serum Levels of the Diagnostic Peptides in the HCC and CLD Patient Groups --- p.93 / Chapter 4.5.3 --- Spearman's Rank Correlation Analysis of the Diagnostic Peptides and AFP --- p.93-101 / Chapter 4.5.4 --- Combined Use of the Diagnostic Peptides and AFP in the Diagnosis of HCC --- p.102-105 / Chapter CHAPTER 5 --- Discussion --- p.106-108 / Chapter 5.1 --- Evaluation of Different Matrix Chemical and Sample Spotting Techniques / Chapter 5.1.1 --- Effect of CHCA and SA --- p.109 / Chapter 5.1.2 --- Effect of Nitrocellulose in Peptide Ions Formation --- p.110-112 / Chapter 5.2 --- MS Automation and High-Throughput Sampling --- p.113 / Chapter 5.3 --- Reproducibility and Signal Quantitations --- p.114-115 / Chapter 5.4 --- Peptidomics: The Study of Entire Peptidome --- p.116 / Chapter 5.5 --- Serum Peptides --- p.117-118 / Chapter 5.6 --- Application of Peptidomics to Discover Markers for HCC / Chapter 5.6.1 --- Hepatocellular Carcinoma --- p.119 / Chapter 5.6.2 --- Causes of HCC --- p.119-120 / Chapter 5.6.3 --- HCC Tumor Markers --- p.120-122 / Chapter 5.6.4 --- HCC Tumor Markers Identified in the Current Studies --- p.122-124 / Chapter 5.7 --- "Role of MALDI-TOF MS, SELDI-TOF MS and 2-DE in Peptidomics" --- p.125-128 / Chapter CHAPTER 6 --- Conclusion --- p.129-130 / Chapter CHAPTER 7 --- References --- p.131-140 / Chapter CHAPTER 8 --- Original Data --- p.150 / Chapter CHAPTER 9 --- Appendices --- p.151-167

Law Wai Keung. / Thesis (M.Sc.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 98-108). / Abstracts in English and Chinese. / Abstract --- p.v / 摘要 --- p.viii / Acknowledgement --- p.x / List of Abbreviations --- p.i / Index of tables --- p.xiv / Index of figures --- p.xv / Chapter 1. --- Introduction --- p.1 / Chapter 2. --- Literature review --- p.3 / Chapter 2.1 --- Immunosuppression in Organ Transplantation --- p.3 / Chapter 2.2 --- Mechanism of Graft Rejection --- p.4 / Chapter 2.3 --- Conventional immunosuppressive drugs --- p.4 / Chapter 2.3.1 --- Corticosteriod --- p.6 / Chapter 2.3.2 --- Azathioprine --- p.6 / Chapter 2.3.3 --- Polyclonal antilymphocyte globulin and OKT3 --- p.7 / Chapter 2.4 --- Cyclosporine A (CsA) --- p.8 / Chapter 2.4.1 --- Mechanisms of CsA --- p.8 / Chapter 2.4.2 --- Pharmacokinetics of CsA --- p.10 / Chapter 2.4.2.1 --- Absorption --- p.10 / Chapter 2.4.2.2 --- Distribution --- p.11 / Chapter 2.4.2.3 --- Metabolism and elimination --- p.11 / Chapter 2.4.2.4 --- Toxicity --- p.12 / Chapter 2.4.3 --- Therapeutic drug monitoring of CsA --- p.13 / Chapter 2.4.3.1 --- CsA trough monitoring --- p.13 / Chapter 2.4.3.2 --- Full AUC monitoring --- p.15 / Chapter 2.4.3.3 --- Limited sampling strategy --- p.16 / Chapter 2.4.3.4 --- Two-hour post dose CsA level monitoring --- p.20 / Chapter 2.4.4 --- Conventional techniques of measuring cyclosporine concentration --- p.23 / Chapter 2.4.4.1 --- High performance liquid chromatography --- p.23 / Chapter 2.4.4.2 --- Non-specific immunoassays --- p.25 / Chapter 2.4.4.3 --- Specific radioimmunoassays --- p.26 / Chapter 2.4.4.4 --- Specific fluorescent polarization immunoassay --- p.26 / Chapter 2.4.4.5 --- Enzyme multiplied immunoassay technique --- p.28 / Chapter 2.4.4.6 --- Cloned enzyme donor immunoassay --- p.29 / Chapter 2.4.4.7 --- Summary for conventional techniques --- p.29 / Chapter 2.5 --- Liquid chromatography mass spectrometry for CsA measurement --- p.30 / Chapter 2.5.1 --- Main components of MS --- p.31 / Chapter 2.5.1.1 --- Specific interfaces to LC --- p.31 / Chapter 2.5.1.2 --- Mass analyzer --- p.33 / Chapter 2.5.1.3 --- Electron multiplier --- p.36 / Chapter 2.5.2 --- Sample preparation for LC-MS/MS for CsA measurement --- p.36 / Chapter 2.5.2.1 --- Liquid-liquid extraction --- p.37 / Chapter 2.5.2.2 --- Solid phase extraction --- p.38 / Chapter 2.5.2.3 --- Column switching --- p.39 / Chapter 2.5.2.4 --- Dilute and shoot --- p.40 / Chapter 2.5.3 --- LC-MS/MS for CsA measurement --- p.40 / Chapter 2.6 --- Summary --- p.42 / Chapter 3. --- Aim of study --- p.43 / Chapter 4. --- Materials and methods --- p.44 / Chapter 4.1 --- Materials --- p.44 / Chapter 4.1.1 --- Chemicals --- p.44 / Chapter 4.1.2 --- Equipment --- p.44 / Chapter 4.1.3 --- Reagent preparation for CsA analysis --- p.45 / Chapter 4.2 --- Methods --- p.48 / Chapter 4.2.1 --- Immunoassay --- p.48 / Chapter 4.2.2 --- Operation of tandem mass spectrometer --- p.48 / Chapter 4.2.2.1 --- Optimization of cone voltage --- p.50 / Chapter 4.2.2.2 --- Optimization of collision energy --- p.50 / Chapter 4.2.3 --- Optimization of LC-MS/MS --- p.51 / Chapter 4.2.3.1 --- Deproteinization procedures of whole blood --- p.52 / Chapter 4.2.3.2 --- Optimization of mobile phase flow rate --- p.52 / Chapter 4.2.3.3 --- Optimization of source temperature --- p.53 / Chapter 4.2.3.4 --- Optimization of the drying gas flow rate --- p.53 / Chapter 4.2.4 --- Matrix interference on MS/MS response --- p.53 / Chapter 4.2.5 --- Analytical performance of CsA on LC-MS/MS --- p.54 / Chapter 4.2.5.1 --- Linearity study --- p.54 / Chapter 4.2.5.2 --- Precision performance --- p.54 / Chapter 4.2.5.3 --- Accuracy performance --- p.54 / Chapter 4.2.5.4 --- The lowest detection limit of the CsA analysis --- p.55 / Chapter 4.2.5.5 --- Correlation study of the CsA analysis --- p.55 / Chapter 4.3 --- CsA pharmacokinetic studies in Chinese patients --- p.56 / Chapter 4.3.1 --- Determining the time point of CsA correlating better with AUC --- p.56 / Chapter 4.3.1.1 --- Patient and method --- p.56 / Chapter 4.3.1.2 --- Statistical analysis --- p.57 / Chapter 4.3.2 --- "Intra-individual variability of CO, C1 and C2" --- p.57 / Chapter 4.3.2.1 --- Patient and method --- p.57 / Chapter 4.3.2.2 --- Statistical analysis --- p.57 / Chapter 5. --- Results and discussion --- p.59 / Chapter 5.1 --- Optimization of MS parameters --- p.5 9 / Chapter 5.1.1 --- Optimization of cone voltage --- p.61 / Chapter 5.1.2 --- Optimization of collision energy --- p.63 / Chapter 5.2 --- Optimization of LC-MS/MS --- p.63 / Chapter 5.2.1 --- Optimization of mobile phase flow rate --- p.63 / Chapter 5.2.2 --- Optimization of ion source temperature and drying gas flow rate --- p.67 / Chapter 5.3 --- Matrix interference on MS/MS response --- p.69 / Chapter 5.4 --- Analytical performances of CsA on LC-MS/MS method --- p.71 / Chapter 5.4.1 --- Linearity --- p.71 / Chapter 5.4.2 --- Precision performance --- p.71 / Chapter 5.4.3 --- Accuracy performance --- p.72 / Chapter 5.4.4 --- The lowest limit of detection --- p.73 / Chapter 5.4.5 --- Correlation study of the CsA analysis --- p.80 / Chapter 5.5 --- The correlation between CsA at different point and AUCo-6 --- p.84 / Chapter 5.6 --- "Intra-individual variability of CO, C1 and C2" --- p.88 / Chapter 5.7 --- Therapeutic ranges of C2 --- p.90 / Chapter 5.8 --- Practical consideration for C2 measurement by LC-MS/MS method --- p.94 / Chapter 6. --- Conclusions --- p.97 / References --- p.98