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2. Improvement of Itraq-SPROX Protocol Development and Application of Mass Spectrometry-Based Approaches for Thermodynamic Analysis of Protein-Ligand Binding Interactions by Xiaopu (Lorrain) Jin Department of Chemistry Duke University Date:_______________________ Approved: ___________________________ Michael C. Fitzgerald, Supervisor ___________________________ Jie Liu ___________________________ Terrence G. Oas ___________________________ Katherine J. Franz Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry in the Graduate School of Duke University 2017 i v ABSTRACT Development and Application of Mass Spectrometry-Based Approaches for Thermodynamic Analysis of Protein-Ligand Binding Interactions by Xiaopu (Lorrain) Jin Department of Chemistry Duke University Date:_______________________ Approved: ___________________________ Michael C. Fitzgerald, Supervisor ___________________________ Jie Liu ___________________________ Terrence G. Oas ___________________________ Katherine J. Franz An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry in the Graduate School of Duke University 2017 Copyright by Xiaopu (Lorrain) Jin 2017 Abstract The characterization of protein stability changes and protein-ligand interactions on the proteomic scale is important for understanding the biology of cellular processes. The identification and quantification of protein-ligand binding affinities is critical for disease state analyses and drug discovery. A mass spectrometry-based technique, Stability of Proteins from Rates of Oxidation (SPROX), has been established for the thermodynamic analysis of protein stability and protein-ligand interactions. In the first part of this dissertation, a previously published iTRAQ-SPROX protocol is improved by incorporating a filter assisted sample preparation (FASP) protocol to significantly reduce sample loss during the experiment. Also, in order to eliminate methionine as a potential contaminant that can cause signal suppression during LC-MS/MS analysis, TCEP•HCl is used to quench the H2O2 oxidation instead of methionine. This avoids the potential reaction between the free methionine and the iTRAQ reagents. The improved protocol, referred to hereafter as the iTRAQ-FASP-SPROX protocol, is shown to increase the peptide/protein coverages for less concentrated cell lysate samples, and it is applied here to study the protein-ligand interaction networks between human ARPE-19 cells lysis and two different iron chelators (HAPI and Exjade). Information on potential protein targets of these two iron chelators are reported. iv In the second part of this dissertation, a targeted MS-based approach for protein- ligand binding analysis is developed to analyze targeted subsets of proteins in a proteome. The so-called PAB-SPROX protocol is demonstrated to be applicable for the detection and relative quantitation of targeted methionine-containing peptides in +/- ligand samples by using isotopically labeled light and heavy PAB (i.e. 12C6-PAB and 13C6- PAB). Multiple reaction monitoring (MRM) and parallel reaction monitoring (PRM) methods are demonstrated to be amenable to PAB-SPROX analyses. In addition to proof-of-principle studies involving the Cylclophilin A-Cyclosporine A binding interaction, the PAB-SPROX protocol was used to validate the direct interaction between YBX1 protein and tamoxifen using very limited amount of purified YBX1 protein. Applications of PAB-SPROX protocol have also included the validation of potential binding targets of Staurosporine, Manassatin A and Tamoxifen. The PAB-SPROX studies with these latter ligands facilitated the identification of false positives in previous proteome-wide SRPOX studies. v Dedication I would like to dedicate this dissertation to my parents and those who have been supporting me through my life with their love and care, no matter how far we are. vi Contents Abstract ......................................................................................................................................... iv List of Tables ................................................................................................................................ xii List of Figures ............................................................................................................................. xiii Acknowledgements ................................................................................................................... xvi 1. Introduction ................................................................................................................................ 1 1.1 Significance of Protein Folding and Stability Measurements ..................................... 1 1.2 Conventional Techniques for Protein Thermodynamic Stability Measurements ... 2 1.3 Mass Spectrometry-based Proteome-wide Approaches ............................................. 5 1.3.1 Basic Methodologies .................................................................................................... 5 1.3.2 General Workflow of SPROX ..................................................................................... 7 1.3.3 Quantitation Strategies Used in SPROX Analysis ................................................... 8 1.3.3.1 SPROX using Isobaric Mass Tagging Strategy ................................................. 8 1.3.3.2 SPROX using Stable Isotope Labeling with Amino Acids (SILAC) in Cell Culture.............................................................................................................................. 10 1.4 Shotgun vs. Targeted Proteomics ................................................................................. 11 1.5 Focus of Dissertation ...................................................................................................... 15 2. Improvement of iTRAQ-SPROX Protocol ............................................................................ 17 2.1 Introduction ..................................................................................................................... 17 2.2 Experiment ....................................................................................................................... 21 2.2.1 Materials ...................................................................................................................... 21 2.2.2 Cell Culture and Cell Lysate Preparations ............................................................. 22 vii 2.2.3 Evaluation of TCEP·HCl and Methionine Reducing H2O2 .................................. 23 2.2.4 Improved iTRAQ-SPROX Protocol Using TCEP∙HCl and a Filter Aided Sample Preparation (FASP) Strategy ............................................................................... 24 2.2.5 Proteomic Coverage of the Original iTRAQ-SPROX Protocol ............................ 25 2.2.6 LC-MS/MS Analyses .................................................................................................. 28 2.3 Results ............................................................................................................................... 29 2.3.1 Evaluation H2O2 Quenching Reaction using Substance P .................................... 29 2.3.2 iTRAQ-FASP-SPROX Protocol for Protein-ligand Binding Analyses ................ 32 2.3.3 Proteomic Coverage Comparison ............................................................................ 35 2.4 Discussion......................................................................................................................... 35 2.4.1 Protocol Improvement............................................................................................... 35 2.4.2 Starting Protein Amount and Proteomic Coverage .............................................. 37 2.5 Conclusions ...................................................................................................................... 38 3. Identification of Potential Protein Targets for Cellular Iron Chelators Using Improve SPROX protocol ........................................................................................................................... 39 3.1 Introduction ..................................................................................................................... 39 3.1.1 The Differential Biological Profiles of Iron Chelators HAPI and Deferasirox .. 39 3.1.2 Protein Targets Identification using SPROX .......................................................... 42 3.2 Experiment ....................................................................................................................... 44 3.2.1 Materials ...................................................................................................................... 44 3.2.2 ARPE-19 Cell Lysate Preparation ............................................................................ 44 3.2.3 The TMT-SPROX Protocol ........................................................................................ 45 3.2.4 LC-MS/MS Analyses .................................................................................................. 47 viii 3.2.5 TMT-SPROX Data Analysis ...................................................................................... 48 3.3 Results and Discussion ................................................................................................... 52 3.3.1 TMT-SPROX Proteomic Coverage ..........................................................................
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