CHARACTERIZATION OF SUPRAMOLECULAR PEPTIDE-POLYMER BIOCONJUGATES USING MULTISTAGE TANDEM MASS SPECTROMETRY A Thesis Presented to The Graduate Faculty of the University of Akron In Partial Fulfillment of the Requirements for the Degree Master of Polymer Science Benqian Wei May 2019 CHARACTERIZATION OF SUPRAMOLECULAR PEPTIDE-POLYMER BIOCONJUGATES USING MULTISTAGE TANDEM MASS SPECTROMETRY Benqian Wei Thesis Approved: Accepted: ___________________________ _____________________________ Advisor Interim Dean of the College Dr. Chrys Wesdemiotis Dr. Ali Dhinojwala ____________________________ _____________________________ Faculty Reader Dean of the Graduate School Dr. Toshikazu Miyoshi Dr. Chand K. Midha _____________________________ _____________________________ Department Chair Date Dr. Tianbo Liu ii ABSTRACT Mass spectrometry (MS) is an essential analytical tool for the characterization of the structure of biological macromolecules, including protein-protein and protein-ligand complexes. One-dimensional MS separates gas-phase analyte ions based on their mass to charge ratio (m/z); however, to obtain more detailed structural information, tandem MS (MS/MS), which involves isolation and subsequent fragmentation of a precursor ion, is required. In this thesis, electrospray ionization multistage tandem mass spectrometry (ESI- MSn) was employed to examine the non-covalent complexes between poly(styrene sulfonate) (PSS) and poly-L-lysine (PLL). During single-stage ion activation, the PLL peptide chain mainly underwent backbone cleavages without disruption of the non- covalent interaction which could only be broken via sequential application of electron transfer dissociation (ETD) and collisionally activated dissociation (CAD), indicating strong binding interactions between the two polyelectrolyte chains. Such binding properties make PSS a potential “non-covalent (supramolecular) label” for determining the surface accessibility of basic residues on a peptide or protein. To probe this premise, non- covalent complexes of substance P and PSS were characterized by ESI-MSn using different ion activation methods. Both MS2 and MS3 experiments on the substance P + PSS complex resulted in the formation of bn (on CAD) or cn (on ETD) fragments attached non-covalently to the intact PSS chain. All peptide fragments containing the intact PSS chain included Arg1, Lys3, and Gln5, pointing out that these residues, which are located near the N- terminus, are most likely involved in the noncovalent interaction with PSS. In contrast, iii Gln6 was excluded from this fragment series, attesting a much weaker interaction with PSS due to lesser accessibility. The strong tendency of PSS to bind peptides noncovalently at sites that can be elucidated by MSn demonstrates a proof-of-concept for the capacity of this approach to unveil higher order structure in proteins. iv ACKNOWLEDGEMENTS I would like to first express my deepest gratitude to my advisor, Dr. Chrys Wesdemiotis for his excellent guidance, valuable suggestions and kindness during the process of my M.S. studies at the University of Akron. His knowledge in chemistry and mass spectrometry directed me towards the right path to achieve my goals during these two years. I would also like to thank him for his help and suggestions regarding my future studies. I would like to thank my committee member Dr. Toshikazu Miyoshi for taking his time to read my thesis and giving me great advice and feedback. He is such a nice person that I do not hesitate to ask for help whenever I have difficulties. I am also grateful for the Department of Polymer Science at The University of Akron. Thanks for holding such an excellent program to support me to do research in such a great environment. The faculty and staff here are all so kind to make me feel at home. I would like to thank all my current and former group members: Dr. Selim Gerişlioğlu, Kevin Endres, Savannah Snyder, Jason O’Neill, Jialin Mao and Chen Du for their friendship, help and suggestions during these two years. Special thanks go to Dr. Selim Gerişlioğlu for his assistance and mentoring throughout my studies. He guided me into the world of mass spectrometry. This thesis could not be completed without him. Lastly, the deepest gratitude goes to my family. I appreciate everything that my parents and family did for me to support my dream to pursue higher level education. I could not finish my M.S. studies without their unconditional love and support. v TABLE OF CONTENTS LIST OF FIGURES................................................................................................... viii LIST OF SCHEMES ................................................................................................... xi CHAPTER I. INTRODUCTION AND PRE-THESIS REVIEW ............................................... 1 II. INSTRUMENTAL METHODS AND BACKGROUND ...................................... 7 2.1. Mass Spectrometry ............................................................................................... 7 2.1.1. Ionization Techniques .......................................................................... 8 2.1.1.1. Electrospray Ionization .................................................................. 8 2.1.1.2. Matrix Assisted Laser Desorption/Ionization .............................. 11 2.1.2.1. Quadrupole Mass Analyzer ......................................................... 13 2.1.2.2. Quadrupole Ion Trap Mass Analyzer .......................................... 14 2.2. Tandem Mass Spectrometry ............................................................................... 16 2.2.1. Collisionally Activated Dissociation (CAD) ..................................... 17 2.2.2. Electron Transfer Dissociation (ETD) ............................................... 17 III. MATERIALS AND INSTRUMENTATION ...................................................... 20 3.1. Materials ............................................................................................................. 20 3.2. Instrumentation ................................................................................................... 21 3.2.1. Bruker© HCTultraTM II Quadrupole Ion Trap Mass Spectrometer.... 21 vi IV CHARACTERIZATION OF POLYMER-PEPTIDE SUPRAMOLECULAR BIOCONJUGATES USING MASS SPECTROMETRY ......................................... 24 4.1. Background ......................................................................................................... 24 4.2. Experimental ....................................................................................................... 26 4.2.1. Sample Preparation ............................................................................ 26 4.2.2. Instrumental Conditions .................................................................... 26 4.3. Results and Discussion ....................................................................................... 27 4.3.1. PLL-PSS Complexes ......................................................................... 27 4.3.2. Substance P-PSS Complexes ............................................................. 35 V. CONCLUSIONS ................................................................................................... 41 REFERENCES ........................................................................................................... 43 APPENDIX ............................................................................................................... 55 vii LIST OF FIGURES Page Figure. 2. 1. Components of a mass spectrometer. ............................................................. 7 Figure. 2. 2. Diagram of electrospray ionization source (Reproduced from reference 1 with permission). ......................................................................................................................... 9 Figure. 2. 3. Electrospray ionization mechanisms (Reproduced from reference 83 with permission). ....................................................................................................................... 10 Figure. 2. 4. Diagram of the principle of MALDI ((Reproduced from reference 1 with permission). ....................................................................................................................... 12 Figure. 2. 5. Diagram of the principle of a quadrupole mass analyzer (Reproduced from reference 1 with permission). ............................................................................................ 13 Figure. 2. 6. Schematic view of a 3D ion trap, and direction of the x, y and z coordinates. (Reproduced from reference 1 with permission). ............................................................. 16 Figure. 3. 1. Schematic of Bruker© HCTultraTM II quadrupole ion trap mass spectrometer. (Reproduced from reference 91 with permission). ........................................................... 22 Figure. 4. 1. (a) ESI-MS spectrum of the PLL + PSS mixture and (b) zoom-in version of the m/z 500-1000 region. The doubly and triply charged distributions of the PLL-PSS complexes observed are marked on top of the corresponding peaks in red and blue color, respectively. L and S represent the PLL and PSS repeat units, respectively. The peaks at m/z 403.4, 531.4, and 659.6 represent [Ln + H]+ (n = 3-5) ions. (c) A possible structure of 2+ [L6S4 + 2H] (m/z 791.4). ................................................................................................ 27 Figure. 4. 2. Supramolecular PLL-PSS complex between the PLL 8-mer (L8) and the PSS 2+ 4-mer