Washington University in St. Louis Washington University Open Scholarship Arts & Sciences Electronic Theses and Dissertations Arts & Sciences Spring 5-15-2015 Development and Application of Mass Spectrometry-Based Biophysical Approaches Ying Zhang Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Part of the Chemistry Commons Recommended Citation Zhang, Ying, "Development and Application of Mass Spectrometry-Based Biophysical Approaches" (2015). Arts & Sciences Electronic Theses and Dissertations. 357. https://openscholarship.wustl.edu/art_sci_etds/357 This Dissertation is brought to you for free and open access by the Arts & Sciences at Washington University Open Scholarship. It has been accepted for inclusion in Arts & Sciences Electronic Theses and Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Department of Chemistry Dissertation Examination Committee: Michael L. Gross, Chair Liviu M. Mirica, Co-Chair Jan Bieschke Robert E. Blankenship Weikai Li John-Stephen Taylor Development and Application of Mass Spectrometry-Based Biophysical Approaches by Ying Zhang A dissertation presented to the Graduate School of Arts & Sciences of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2015 St. Louis, Missouri © 2015, Ying Zhang Table of Contents List of Figures ................................................................................................................................ vi List of Tables ............................................................................................................................... viii Acknowledgments.......................................................................................................................... ix ABSTRACT OF THE DISSERTATION ....................................................................................... x Chapter 1: Introduction ................................................................................................................... 1 1.1 Hydrogen Deuterium Exchange (HDX) ........................................................................... 2 1.1.1 Types of Hydrogens in Proteins ............................................................................................ 3 1.1.2 pH and Temperature Effect ................................................................................................... 3 1.1.3 HDX-MS ............................................................................................................................... 4 1.1.4 EX1 and EX2 ........................................................................................................................ 6 1.2 Protein-Ligand Interactions .............................................................................................. 6 1.3 Protein-Ligand Affinity Measurements ........................................................................... 8 1.4 Conventional Methods for Ligand Binding Characterization ........................................ 10 1.4.1 Isothermal Titration Calorimetry (ITC) .............................................................................. 10 1.4.2 Fluorescence ....................................................................................................................... 11 1.4.3 Fourier Transform Infrared spectroscopy (FTIR) ............................................................... 11 1.4.4 Surface Plasmon Resonance (SPR) ..................................................................................... 12 1.4.5 Enzyme-Linked Immunosorbent Assay (ELISA) ............................................................... 12 1.4.6 Circular Dichroism (CD) .................................................................................................... 13 1.4.7 Nuclear Magnetic Resonance (NMR) ................................................................................. 13 1.4.8 X-ray Crystallography ......................................................................................................... 14 1.5 Direct Mass Spectrometry Method ................................................................................ 14 1.6 HDX-MS ........................................................................................................................ 15 1.6.1 HDX-MS for Binding Regions ........................................................................................... 15 1.6.2 HDX-MS for Binding Affinity ........................................................................................... 16 1.6.3 HDX-MS for Binding Order ............................................................................................... 34 1.6.3 HDX-MS for Protein-Protein Interactions .......................................................................... 35 1.7 Conclusion ...................................................................................................................... 39 1.8 Acknowledgements ........................................................................................................ 39 ii 1.9 References ...................................................................................................................... 40 Chapter 2: Pulsed Hydrogen/Deuterium Exchange Mass Spectrometry Probes Conformational Changes in Amyloid Beta (A) Aggregation ............................................................................... 46 2.1 Abstract .......................................................................................................................... 47 2.2 Introduction .................................................................................................................... 47 2.3 Materials and Methods ................................................................................................... 49 2.3.1 Chemicals ............................................................................................................................ 49 2.3.2 Protein Preparation .............................................................................................................. 49 2.3.3 Pulsed Hydrogen Deuterium Exchange .............................................................................. 50 2.3.4 A40 Back Exchange ........................................................................................................... 51 2.3.5 Data Analysis and Modeling ............................................................................................... 51 2.4 Results and Discussion ................................................................................................... 53 2.4.1 Comparison of A42 and A Aggregation by Native Gel and Western Blotting ............. 53 2.4.2 Pulsed HDX Analysis of Soluble A Species ..................................................................... 55 2.4.3 Application of Finke-Watzky (F-W) Modeling and Bootstrap Strategy ............................. 63 2.4.4 Pulsed HDX Analysis of A Fibrils ................................................................................. 69 2.4.5 Pulsed HDX Analysis of Factors Affecting A42 Aggregation .......................................... 73 2.5 Conclusion ...................................................................................................................... 77 2.6 Acknowledgments .......................................................................................................... 78 2.7 References ...................................................................................................................... 79 Chapter 3: Structural Analysis of Amyloid Beta Peptides Interacting with Different Ligands by Pulsed Hydrogen Deuterium Exchange Mass Spectrometry ........................................................ 85 3.1 Abstract .......................................................................................................................... 86 3.2 Introduction .................................................................................................................... 86 3.3 Materials and Methods ................................................................................................... 88 3.3.1 Chemicals and Protein Preparation ..................................................................................... 88 3.3.2 Pulsed HDX ........................................................................................................................ 88 3.3.3 LC/MS, Data Analysis, and Finke-Watzky (F-W) modeling .............................................. 89 3.4 Results and Discussion ................................................................................................... 89 3.4.1 Pulsed HDX for L1 ............................................................................................................. 91 3.4.2 Pulsed HDX for L2 ............................................................................................................. 95 3.4.3 Pulsed HDX for AMF ......................................................................................................... 96 iii 3.5 Conclusion ...................................................................................................................... 96 3.6 Acknowledgements .......................................................................................................