The development of structural mass spectrometry based techniques for the study of aggregation-prone proteins. Owen Cornwell School of Molecular and Cellular Biology Astbury Centre for Structural Molecular Biology University of Leeds Submitted in accordance with the requirements for the degree of Doctor of Philosophy December 2019 Declaration The candidate confirms that the submitted work is his own and that appropriate credit has been given within the thesis where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from this thesis may be published without proper acknowledgement. © 2019 The University of Leeds and Owen Cornwell i ii Jointly Authored Publications Chapters 4 and 5 contain work from the following published manuscript: Cornwell, O., et al., Comparing Hydrogen Deuterium Exchange and Fast Photochemical Oxidation of Proteins: a Structural Characterisation of Wild-Type and ΔN6 β2-Microglobulin. Journal of The American Society for Mass Spectrometry, 2018. 29(12): p. 2413-2426. In this work, all experimental work and data analysis was performed by OC. Optimisation of the HDX workflow and associated method development was performed by OC and JRA. Initial development and coding of the novel processing algorithm was performed by OC. Final development and coding of the PAVED software available for download was performed by JRA. Chapter 6 contains work from the following published manuscript: Cornwell, O., et al., Long-Range Conformational Changes in Monoclonal Antibodies Revealed Using FPOP-LC-MS/MS. Analytical Chemistry, 2019. 91(23): p. 15163- 15170. In this work, all experimental work and data analysis was performed by OC. Data acquisition was performed at AstraZeneca (Granta Park, Cambridge) by OC under the supervision of NJB. iii iv Acknowledgements Firstly, I’d like to thank my two academic supervisors, Alison and Sheena, not just for taking me on as a student when I had nowhere else to go, but for their continued support and encouragement throughout the four years of my PhD at Leeds. I’d like to recognise James Ault, our MS facility manager, for his significant contribution to the work in this thesis, and for not yelling at me when I text him on Sundays in a panic because the HDX robot has squashed the syringe again. Rachel George, for keeping the lab running smoothly and doing all the jobs that I really should know how to do but don’t, and all past and present members of the lab that helped me out along the way (particularly Anton, Hugh, Tom, Patrick, Emma, Leon and Nasir!). I’d also like to thank my industrial supervisor Nick Bond, as well as Dan Higazi, and Jim Scrivens, my two original supervisors. Similarly, an honourable mention is thoroughly deserved by Sue Slade, for looking after me at several conferences, as well as stocking my formerly deficient kitchen full of a variety of cooking utensils - and a garlic crusher. I’d also like to thank my family, and the doctors and nurses at Royal Papworth Hospital. Their hard work, which makes writing a PhD thesis look unbelievably easy, has given my family and I more time with my dad, for which I will be forever grateful. With recent events I’ve come to appreciate the true significance of my family’s guidance in my life, and just how far away I’d be from getting a PhD, without them. I love you all. Well…most of you. Lastly, I’d like to recognise the essential, if less obvious, contributions made from a number of other people, entities and institutions. I realise I’m running out of space on the page so I’ll be brief. I’d like to thank Mr Clark (my A-level chemistry teacher), Dr Brandon Reeder (my undergraduate project supervisor), the quality control department for KP's dry roasted peanuts, Dr Malcolm Finney and his wife Kathy, Spam, laser pointers, the 'Find and Replace' function in Microsoft Excel, John Cleese, Lemsip Cold & Flu, Chinese takeaway restaurants (especially the one on Town Street), scientific calculators, the complaints department at British Telecom, Robert Palmer, large USB sticks, most small and medium sized USB sticks, the cast and crew of Stargate: SG1, Oxo cubes, VAX essentials vacuum cleaners, pot noodle sandwiches, and last, but by no means least, my dragon tree houseplant - Steve. v vi Oh and my girlfriend Sophie. She was great too… Un-acknowledgements I would also like to not gratefully thank my former landlord, and the staff at the Wakefield branch of Linely & Simpson letting agents, for refusing to fix my leaky bedroom window for two years until I got fed up with it and moved out, somewhere about a third of the way through Chapter 5. vii viii Table of Contents JOINTLY AUTHORED PUBLICATIONS ................................................................... III ACKNOWLEDGEMENTS ........................................................................................... V TABLE OF CONTENTS .............................................................................................. IX LIST OF FIGURES ................................................................................................... XIV LIST OF TABLES ...................................................................................................... XIX LIST OF EQUATIONS .............................................................................................. XX LIST OF ABBREVIATIONS ..................................................................................... XXI ABSTRACT ............................................................................................................ XXV 1 INTRODUCTION I: MASS SPECTROMETRY THEORY ..................................... 2 1.1 Overview and history of mass spectrometry ....................................................... 2 1.2 Ionisation ............................................................................................................... 3 1.2.1 Electrospray ionisation .............................................................................. 4 1.3 Mass analysers ....................................................................................................... 7 1.3.1 Quadrupole analysers ................................................................................. 8 1.3.2 Linear ion traps ........................................................................................ 13 1.3.3 Time-of-flight (ToF) analysers ................................................................ 16 1.3.4 Orbital trapping analysers ....................................................................... 20 1.4 Ion Detectors ....................................................................................................... 25 1.4.1 Electron multipliers ................................................................................. 25 1.4.2 Image current detection .......................................................................... 26 1.5 Analysis of mass spectrometry data ................................................................... 27 1.5.1 Mass error and mass accuracy ................................................................. 29 1.6 Tandem mass spectrometry (MS/MS) ................................................................ 29 ix 1.7 Liquid chromatography – mass spectrometry (LC-MS) .................................... 31 1.8 LC-MS/MS data acquisition methods................................................................. 33 1.8.1 Data dependent acquisition (DDA) ......................................................... 33 1.8.2 Data independent acquisition (DIA) ....................................................... 35 1.9 Ion mobility spectrometry – mass spectrometry (IMS-MS) ............................. 36 1.9.1 Drift tube IMS (DTIMS) .......................................................................... 37 1.9.2 Travelling wave IMS (TWIMS) ............................................................... 38 1.10 Commercial instruments used in this thesis ...................................................... 40 1.10.1 Waters Synapt high definition mass spectrometer ................................ 40 1.10.2 Thermo Q Exactive hybrid quadrupole – orbitrap mass spectrometer . 41 1.10.3 Thermo orbitrap fusion tribrid mass spectrometer ................................ 42 2 INTRODUCTION II: PROTEIN AGGREGATION, STRUCTURE AND DYNAMICS STUDIED BY MASS SPECTROMETRY ................................................ 46 2.1 Protein folding, misfolding and aggregation ..................................................... 49 2.1.1 Protein folding ......................................................................................... 49 2.1.2 Misfolding, aggregation and disease ....................................................... 52 2.1.3 Structure and formation of amyloid ....................................................... 53 2.2 Beta-2 microglobulin (β2m) ................................................................................ 56 2.2.1 Normal function and dialysis related amyloidosis ................................. 56 2.2.2 β2m aggregation in vitro .......................................................................... 59 2.2.3 β2m folding pathway and the IT state ...................................................... 60 2.2.4 ΔN6 β2-microglobulin .............................................................................. 62 2.2.5 D76N β2-microglobulin ............................................................................ 63 2.3 Protein aggregation in biopharmaceuticals