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Cloning, expression and purification of the different human haptoglobin chains and initial characterization by mass spectrometry
Table of Contents
Abstract
Acknowledgements
Table of Contents
List of Tables
List of Figures
List of Abbreviations
1 Introduction
1.1 Haptoglobin
1.1 DnaT
1.2 Project Goals
1.2.1 Haptoglobin
1.2.2 DnaT
1.3 Organization of this Thesis
2 Mass Spectrometry: Principles and Instruments Design
2.1 Principles of Mass Spectrometry
2.1.1 Typical Instrument Construction
2.1.2 Definition and Principle
2.1.3 The Typical Mass Spectrum
2.2 Ionization Methods
2.2.1 Electrospray Ionization (ESI)
2.2.2 Matrix-Assisted Laser Desorption/Ionization (MALDI)
2.3 Mass Analyzers
2.3.1 Time of Flight Mass Analyzer
2.3.2 Hybrid Quadrupole Time-of-Flight Mass Analyzer
2.3.3 TOF-TOF Mass Analyzer
2.4 Ion Activation Methods
2.4.1 Overview of Ion Activation Methods
2.4.2 Collision Induced Dissociation
2.4.3 Peptide and Protein Fragmentation and Fragment Nomenclature
2.5 Ion Mobility Mass Spectrometry
2.6 Instrumentation Used in this Research
2.6.1 Synapt HDMS (Waters Corp.)
2.6.2 UltrafleXtreme MALDI-TOF/TOF Mass Spectrometer
3 Applications of Mass Spectrometry to Protein Identification, Protein Structure and Protein Dynamics
3.1 Proteomics and protein identification
3.1.1 The top-down approach
3.1.2 Bottom-up approach
3.2 Protein structural analyses
3.2.1 Charge state distributions
3.2.2 IMMS
3.2.3 Mapping of Disulfide Bridging Patterns and their Verification for Recombinant Proteins
3.2.4 Noncovalent Protein Complexes
3.2.5 MS as a Tool for Protein Crystallography
4 Cloning, Expression and Purification of the Individual Subunits of Human Haptoglobin Chains
4.1 Material and Methods
4.1.1 List of Materials and Reagents
4.1.2 Polymerase Chain Reaction (PCR)
4.1.3 Insertion of the GOI into the entry vector
4.1.4 Insertion of the GOI into the expression vector
4.1.5 Protein Expression
4.1.6 Cell Lysis
4.1.7 Purification of Hp Chains
4.1.8 Electrophoresis
4.1.9 Trypsin Digestion
4.1.10 In Silico Digest
4.1.11 MALDI-TOF/TOF MS Experiments
4.2 Results and Discussion
4.2.1 Protein Cloning, Expression and Purification of the Light Chains (L1 and L2) of Human Haptoglobin
4.2.2 Protein Cloning of the H-chain of Human Haptoglobin
4.3 Conclusion
5 Initial Characterization of the Haptoglobin Chains by Mass Spectrometry
5.1 Materials and Methods
5.1.1 Sample Preparation for Mass Spectrometry Analysis
5.1.2 Reaction for Mapping of the Disulfide Bridges
5.1.3 In-solution Trypsin Digestion
5.1.4 Mass Spectrometry Analysis
5.2 Results and Discussion
5.2.1 Analysis of the Intact Light Chains
5.2.2 Establishing the Disulfide Pattern of the Light Chains
5.2.3 Molecular Modeling
5.3 Conclusion
6 Protein Disorder Determination of DnaT by Mass Spectrometry
6.1 Material and Methods
6.1.1 Protein Expression
6.1.2 Lysis and Ammonium Sulfate Precipitation
6.1.3 Purification of DnaT
6.1.4 Sample Preparation for Mass Spectrometry Experiments
6.1.5 Mass Spectrometry Experiments
6.2 Results and Discussion
6.2.1 Expression and Purification of DnaT
6.2.2 DnaT: Degradation Products
6.2.3 Identification of the Flexible Domain of DnaT
6.2.4 Study and Identification of DnaT Conformers in Solution
6.3 Conclusion
7 Summary and Future Directions
References
Appendix A
Appendix B
Appendix C Bài viết liên quan
