[giaban]0.000 VNĐ[/giaban] [kythuat]
[/kythuat]
[tomtat]
[tomtat]
Structural analysis of macromolecular complexes using electrospray ionization mass spectrometry based approaches
Table of Contents
Abstract
Acknowledgements
Table of Contents
List of Tables
List of Figures
List of Abbreviations
List of Symbols
1 Overview of the Dissertation
2 Mass Spectrometry: Principles, Instrumentation and Applications
2.1 Principles of MS
2.1.1 The Typical Mass Spectrometer
2.1.2 Ionization Source
2.1.3 Mass Analyzers
2.1.4 Ion Activation Methods
2.1.5 Ion Mobility Mass Spectrometry
2.2 Instrumentation Used in This Dissertation
2.2.1 Synapt HDMS (Waters Corp.)
2.2.2 UltrafleXtreme MALDI-TOF/TOF MS (Bruker Daltonics)
2.3 Application of MS
2.3.1 Protein Identification
2.3.2 Analysis of macromolecular complexes
3 Complete Sequence Determination of Hemoglobin from Endangered Species Using a Combined ESI-MS and X-ray Crystallography Approach
3.1 Introduction
3.2 Materials and Methods
3.2.1 Chemicals and Materials
3.2.2 Preparation of CNmetHb and Its Isolation from pRBCs
3.2.3 Reversed-Phase-HPLC
3.2.4 Proteolytic Digestion
3.2.5 Mass Spectrometry
3.2.6 CNmetHb Nucleation and Crystallization
3.2.7 X-ray Diffraction Data Collection and Refinement
3.3 Results
3.3.1 Hb Sequencing by MS
3.3.2 Hb Sequencing Facilitated by X-ray Diffraction
3.4 Discussion
3.4.1 Hb Sequencing by MS
3.4.2 Hb Sequencing Completed by Electron Density and Sequence Homology Search
3.5 Conclusion
4 Crystallographic Evidence of Naturally Occurring B-form Feline Hemoglobin: What Nature Has Done Differently
4.1 Introduction
4.2 Materials and Methods
4.2.1 Chemicals and Materials
4.2.2 Crystallization of Cyanomethemoglobin
4.2.3 Diffraction Data Collection and Reduction
4.2.4 Structure Determination and Refinement
4.2.5 Structure Analysis.
4.3 Results
4.3.1 CNmetHb X-ray Diffraction Data Collection and Refinement
4.3.2 Structure Comparison of T-state, R-state and Snow Leopard CNmetHb
4.3.3 metHb Quaternary Structure Similarity Comparison
4.3.4 The Role of Feline Hb β Subunit N-terminal Mutation in B-like Quaternary Structure Formation
4.4 Discussion
4.4.1 Crystal Growth, Diffraction Data collection and Refinement
4.4.2 Point Mutation Induced Quaternary Structure Change in Feline Hb
4.4.3 β Subunit N-terminal Acetylation of Feline Hb
4.4.4 Cl- as an Allosteric Effector for Oxygen Affinity
4.5 Conclusions
5 Elucidate Erythrocyte Band 3 N-terminal Interactions with Hemoglobin by ESI-MS
5.1 Introduction
5.2 Materials and Methods
5.2.1 Chemicals and Materials
5.2.2 Preparation of CNmetHbA, CNmetHbS, COHbA and aquometHbA
5.2.3 Preparation of Stock Solutions of Synthetic Band 3 Peptides
5.2.4 ESI-MS Analysis of Hb and Commercial Band 3 N-terminal Peptides
5.3 Result and Discussion
5.3.1 Peptide Design and Human CNmetHbA Analysis under Native ESI-MS Conditions
5.3.2 Binding of CNmetHbA, aquometHbA and COHbA to Full-length Peptide
5.3.3 Binding of CNmetHbA and CNmetHbS with Peptides #1, #2, and #3
5.3.4 Peptide Binding Affects Hb Tetrmer-dimer Equilibrium
5.3.5 Hypothetical Hb-peptide Binding Model
5.5 Conclusions
6 Mass Spectrometric Identification of Silver Nanoparticles: The Case of Ag32(SG)19
6.1 Introduction
6.2 Materials and methods
6.2.1 Chemicals and Materials
6.2.2 Synthesis and Purification of Nanoclusters
6.2.3 UV-Vis Spectrophotometry
6.2.4 Mass Spectrometry
6.3 Results and Discussion
6.4 Conclusion
7 Structure and Stability of Ag32(SG)19 by ESI-MS
7.1 Introduction
7.2 Materials and Methods
7.2.1 Chemicals and Materials
7.2.2 ESI-MS Sample Preparation
7.2.3 ESI-MS Data Collection
7.2.4 Addition of Supercharging Reagent
7.3 Results
7.3.1 Effects of Solvent and Ageing
7.3.2 Collisional Activation of the Au25(SG)18 and Ag32(SG)19 Nanoclusters
7.3.3 IMMS Analysis of Au25(SG)18 and Ag32(SG)19 Clusters
7.3.4 Effect of Adding a Supercharging Agent
7.4 Discussion
7.4.1 The Lost of Pyroglutamate in Au25(SG)18 and Ag Atom in Ag32(SG)19
7.4.2 Solvent Additives and Aging Effects
7.4.3 CID, IMMS and CID-IMMS Analysis of Au25(SG)18 and Ag32(SG)19 Nanoclusters
7.4.4 Supercharging Effect
7.5 Conclusions
8 ESI-MS Structural Analysis of Ag44(pMBA)30 Nanoclusters
8.1 Introduction
8.2 Materials and Methods
8.2.1 Chemicals and Materials
8.2.2 Ag44(pMBA)30 Cluster Synthesis and Purification
8.2.3 ESI-MS Analysis of Ag44(pMBA)30
8.3 Results
8.3.1 MS Detection Optimization
8.3.2 Formula Assignment
8.3.3 CID Fragmentation of Ag44(pMBA)30
8.3.4 IMMS and CID-IMMS
8.4 Discussion
8.4.1 Solution and Instrumental Condition Optimization
8.4.2 Ag43(pMBA)28-3 Is the Fragment Product of Ag44(pMBA)30-4
8.4.3 Fragment as a Result of Secondary Gas-phase Ion Reaction/rearrangement in CID-IMMS
8.5 Conclusions
9 Tandem MS Identification of Periodate-oxidized tRNAala Modification Sites in AlanyltRNA Synthetase
9.1 Introduction
9.2 Materials and Methods
9.2.1 Chemicals and Materials
9.2.2 Preparation and Purification of Oxidized tRNA-modified Alanyl-tRNA Synthetase Peptides
9.2.3 Isolation of otRNAala-modified AlaRS Peptide by RP-HPLC
9.2.4 Tandem MS Analysis of otRNAala-modified AlaRS Peptides
9.3 Results
9.3.1 Preparation and Purification of otRNAala Modified Alanyl-tRNA Synthetase
9.3.2 Identification of otRNAala Modification Site on AlaRS
9.4 Discussion
9.5 Conclusion
10 ESI-MS Structural Analysis of Alanyl-tRNA Synthetase
10.1 Introduction
10.2 Materials and Methods
10.2.1 Chemicals and Materials
10.2.2 Native ESI-MS Analysis of ARS461, ARS875 with Ligands
10.2.3 MS Stability and Binding Studies of ARS461 with Its Cognate/non-cognate Ligands
10.2.4 Global HDX Experiment and Data Analysis
10.3 Results
10.3.1 Native ESI-MS Analysis of ARS461 and ARS875 with Cognate/uncognate Ligands
10.3.2 Effect of pH on Complex Stability
10.3.3 Complex Stability Study by Elevated Collision Energy
10.3.4 Global HDX of ARS461 and Its Complex with ASAd
10.4 Discussion
10.4.1 ARS461-ligand Stability Study
10.4.2 ARS-ligand Analyzed by Global HDX
10.5 Conclusions
11 Future Directions
References
Appendix A Supporting Tables of Chapter 2
Appendix B Supporting Figures and Tables of Chapter 3
Appendix C Supporting Figures and Tables of Chapter 4
Appendix D Supporting Figures of Chapter 5
Appendix E Supporting Figures of Chapter 6
Appendix F Supporting Figures and Tables of Chapter 7
Appendix G Supporting Figures and Tables of Chapter 8
Appendix H Supporting Figures and Tables of Chapter 9
Appendix I Supporting Figures of Chapter 10 Bài viết liên quan
