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Defining the Mechanism of Small Molecule Inhibition of Amyloid Fibril Formation Using Ion Mobility Spectrometry – Mass Spectrometry

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Defining the Mechanism of Small Molecule Inhibition of Amyloid Fibril Formation Using Ion Mobility Spectrometry – Mass Spectrometry Defining the mechanism of small molecule inhibition of amyloid fibril formation using ion mobility spectrometry – mass spectrometry Lydia Mary Young Submitted in accordance with the requirements for the degree of PhD The University of Leeds Astbury Centre for Structural Molecular Biology September 2015 The candidate confirms that the submitted work is her own, except where work which has formed part of jointly-authored publications has been included. The contribution of the candidate and the other authors to this work has been explicitly indicated overleaf. The candidate confirms the 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. © 2015 The University of Leeds and Lydia Mary Young Jointly Authored Publications Throughout this thesis the work directly attributable to the candidate is as follows: (i) Literature research and compilation of the manuscript stated above. (ii) The candidate performed all the experimental work and data analysis unless otherwise stated. Details of jointly authored publications and the contributions of other authors to these manuscripts: Results Chapter 1 contains work from the following manuscript published in Int J Ion Mobil Spectrom, 2013: Young, L.M., Ndlovu, H., Knapman, T.W., Harris, S. A., Radford, S.E. and Ashcroft, A.E. Monitoring oligomer formation from self-aggregating amylin peptides using ESI- IMS-MS. In this work, TWK performed preliminary experiments and helped in designing the project. I carried out all ESI-IMS-MS experimental work, ThT and TEM analysis and wrote the manuscript along with AEA. HN performed molecular modelling of oligomeric structures. SAH, SER and AEA provided help with scientific discussions, data interpretation and manuscript preparation. Results Chapter 2 contains work from the following manuscript published in J Am Chem Soc, 2014: Young, L.M., Cao, P., Raleigh, D.P., Ashcroft, A.E., and Radford, S.E. Ion Mobility Spectrometry−Mass Spectrometry Defines the Oligomeric Intermediates in Amylin Amyloid Formation and the Mode of Action of Inhibitors. In this work, I designed and carried out all ESI-IMS-MS experimental work, data interpretation, ThT and TEM analysis and wrote the manuscript. PC synthesised i the hIAPP and rIAPP peptides. DPR, SER and AEA provided help with scientific discussions, data interpretation and manuscript preparation. Results Chapter 3 contains work from the following manuscript published in Nat Chem, 2015 and Methods, 2015: Young, L.M.*, Saunders, J.C.*, Mahood, R.A., Revill, C.H., Foster, R.J., Tu, L.-H., Raleigh, D.P., Radford, S.E. and Ashcroft, A.E. Screening and classifying small molecule inhibitors of amyloid formation using ion mobility spectrometry-mass spectrometry. Young, L.M.*, Saunders, J.C.*, Mahood, R.A., Revill, C.H., Foster, R.J., Ashcroft, A.E. and Radford, S.E. ESI-IMS–MS: A method for rapid analysis of protein aggregation and its inhibition by small molecules. *These authors contributed equally to this work. In this work, I designed and carried out all ESI-IMS-MS experimental work, data interpretation and TEM analysis and wrote the manuscript, together with JCS. JCS also provided background information on the small molecules and their published interactions with human islet amyloid polypeptide (hIAPP) and amyloid β peptide residues 1-40 (Aβ40), and provided all the small molecules for in vitro analysis. JCS reviewed and selected the small molecules to be used for seeding the virtual screening library, and calculated LogP values of all 40 small molecules screened. RAM purified the Aβ40 peptide and performed TEM of the sample. CHR and RJF performed the virtual screen. LHT synthesised the hIAPP peptide. RJF, DPR, SER and AEA provided help with scientific discussions, data interpretation and manuscript preparation. ii Acknowledgements These past 4 years have been challenging, rewarding and life changing and I would like to whole-heartedly thank all the people who have carried me through and put up with me. Firstly, thank you to my supervisors, Prof. Alison Ashcroft and Prof. Sheena Radford for their insight, guidance, and encouragement throughout my PhD. Both have experienced first-hand with me the highs and lows of my PhDs – from papers being accepted to sickness bugs at conferences. To my amazing friends and collaborators Janet Saunders and Rachel Mahood, the final 18 months of my PhD were so greatly enriched by your presence and input, both inside and outside of work, thank you! I will miss you like the desert misses rain. To my bestie Anton Calabrese, we have had THE best times and I’m not sure how I’d have coped without you, please stay in Leeds forever!! Big up to all the magnificent members of the Ashcroft and Radford groups. It is a total pleasure to work with people that I consider to be such close friends. LGI Costa trips played a large part in getting me through a good deal of the 4 year PhD, thank you Helen Beeston for fuelling my caffeine addiction. Thanks to the Berry lab beauties Claire Windle and Sasha Derrington (and Alex Moloney) for all the cocktails, fun times and dancing! Sincere thanks to Lucy Woods and James Ault for your patience, training and spoon feeding. You are both stars and I am forever grateful for your valuable input. We had some great fun along the way too! Most importantly my deepest gratitude goes to my crazy but wonderfully amazing family Matthew (my hero), Sarah (my inspiration), Grace (my best mate), Aaron (my lovely bro), Dan and Leah (my babies), never a dull or lonely moment! My family are THE best, fact. Special thanks to Grandma Debs for being by my side through all of my university years, you’ve been amazing!! I would be nowhere without the love and support of you all and I consider myself so deeply blessed that God chose you to be my family. I’m sorry for being a ‘controllious freakious’ and I hope to make you proud. This is for you! iii iv Table of Contents JOINTLY AUTHORED PUBLICATIONS ............................................................................... I TABLE OF CONTENTS ............................................................................................................ V LIST OF FIGURES ................................................................................................................... IX LIST OF TABLES .................................................................................................................. XIII LIST OF ABBREVIATIONS .................................................................................................. XV LIST OF AMINO ACID ABBREVIATIONS ................................................................... XVIII ABSTRACT ............................................................................................................................. XIX 1 INTRODUCTION ............................................................................................................... 1 1.1 History of mass spectrometry ........................................................................................... 1 1.2 Synapt HDMS ............................................................................................................................ 2 1.3 Ionisation of biological molecules .................................................................................... 3 1.4 Ion mobility spectrometry-MS .......................................................................................... 6 1.5 Mass analysers used in a Synapt HDMS ....................................................................... 10 1.6 MS/MS in a Synapt HDMS ................................................................................................. 13 1.7 Mass spectrometry for protein structure elucidation ............................................ 14 1.8 Protein Folding, Misfolding and Aggregation ............................................................ 15 1.9 Amyloid formation and the study of amyloid intermediates .............................. 17 1.10 hIAPP amyloid assembly in vivo ..................................................................................... 21 1.11 Sequence dependence of IAPP amyloid formation.................................................. 23 1.12 Inhibitors of amyloid formation ..................................................................................... 24 1.13 IMS-MS for studying amyloid assembly....................................................................... 29 1.14 Studying oligomeric intermediates using IMS-MS .................................................. 33 1.15 IMS-MS can be used to unravel the mechanism of inhibition of fibril assembly by small molecules ........................................................................................................ 37 2 MATERIALS AND METHODS ..................................................................................... 40 2.1 Materials .................................................................................................................................. 40 2.1.1 Technical equipment ...................................................................................................... 40 2.1.2 Chemicals ............................................................................................................................ 41 2.1.3 Acquisition of IAPP fragment peptides ...................................................................
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