Deep time-resolved proteomic and phosphoproteomic profiling of cigarette smoke-induced chronic obstructive pulmonary disease David Skerrett-Byrne BSc Biochem & Mol Bio (Hons)(UCD) MSc Biotech (UU) 25th March 2019 Supervisors: Professor Phil Hansbro, Dr. Matt Dun, Laureate Professor Rodney Scott, Professor Peter Wark, Professor Darryl Knight, Laureate Professor Paul Foster Discipline of Immunology and Microbiology and Priority Research Centre for Healthy Lungs School of Biomedical Science and Pharmacy Faculty of Health and Medicine University of Newcastle and Hunter Medical Research Institute Newcastle, NSW, Australia Submitted in fulfilment of the requirements for the award of a Doctor of Philosophy Declaration Statement of Originality I hereby certify that the work embodied in the thesis is my own work, conducted under normal supervision. The thesis contains no material which has been accepted, or is being examined, for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made. I give consent to the final version of my thesis being made available worldwide when deposited in the University’s Digital Repository, subject to the provisions of the Copyright Act 1968 and any approved embargo. ________________________ David Skerrett-Byrne 25/03/2019 Acknowledgment of Authorship I hereby certify that the work embodied in this thesis contains scholarly work of which I am a joint author. I have included as part of the thesis a written declaration endorsed in writing by my supervisor, attesting to my contribution to the joint scholarly work. By signing below I confirm that David Skerrett-Byrne contributed heavily (90%) to the experimental design, collection and analysis of data, figures generated and the writing of each chapter. Chapter 1 is currently under review with The European Respiratory Journal and Chapters 2, 3, 4 are all in preparation for submission. ____________________ Professor Philip Hansbro 25/03/2019 i Acknowledgements “During the course of any proteomics investigation, you will see interesting results, but you will rarely observe significant ones. To generate interesting and significant results, using a robust hypothesis, you must compare fresh apples.” Dun, M.D. 2016 Firstly my sincerest thanks to my supervisor Prof. Phil Hansbro for taking me on a PhD student and giving me to opportunity to explore and develop my research interests in Australia. I am forever grateful for that. Thank you for your support and patience with our fruitful proteomic inventory. My thanks for co-supervisors Prof. Peter Wark and Laureate Prof. Rodney Scott for their knowledge and experimental support during my studies. Thank you to all co-supervisors, and all past and present Hansbro and Horvat lab members who have been supportive of me during my thesis. Thank you to Dr. Nicole Hansbro for always being there to answer my somewhat frantic questions and ensuring my PhD runs seamlessly. To Matt I owe more than words can express. You have been an incredible mentor to me and you really have shaped the researcher I have become. You have always known when to challenge me and when to provide me with that wonderful unwavering enthusiasm of yours. Even more than that you have been my friend and you will always hold a special place in my heart. I’m excited for what is ahead and delighted we will still be working together. To my friends and colleagues, too many to mention, your friendship has meant the world to me. You have all made me feel so welcome in Australia, created some of my fondest memories, and supported me in many different ways. You are all great craic! Thank you to Abdul, Alex, Amanda, Andrew, Anne Chevalier, Anne Gannon, Brett Hill, Brett Nixon, Cal, Cheryl, David, Diane, Hubert, Jacinta, Jean-Marie, Jon, Mike, Nathan, Nikita, Nikki, Pedro, Phil Dickson, Sam, Severine, Simon, Tabitha, Yanfang, Zac and everyone who has been part of this journey. Heather, you have been the best PhD buddy! Thank you for all our conversations, I have learnt so much for you. You are truly an amazing friend and one the finest researchers I have ever met. Cal, I miss your around lab buddy. You ii have always had a way of brightening my day and I leave every interaction with such joy. Richie, thank you for all the laughter, the craic, and being a true friend mate. To Lizzie, I’m taken aback by your level of support, particularly in these final months of my PhD. I really feel privileged to know you and am a big admirer of your research. Thank you for you mentorship and more importantly your friendship. To Daniel, Steph, Lorcan and Fergie, I can never express how supportive you have all been to me. You are forever my family and I love you all so very much. You welcomed me into your lives without hesitation and allowed me to share in Lorcan growing up, which has been one of the greatest pleasures of my life. I owe so much of this thesis and happiness to you all. To my friends back in Ireland, Daryl, Maria, Zainab, Dave, Mikey, Maykla, Sweeney, Pete, and Katie, thank you all endlessly for your friendship, laughs, support and always thinking of me. To my family, Paddy, Sharon, Roxy and Mam, thank you for supporting me during this PhD and being so far from home. Mam, I’m sure I don’t say enough but I am endlessly appreciative for everything you have done for me in my life, everything I am, have become and achieved is because of you. Thanks for all the Christmas, birthday and St. Patrick’s Day packages, every one of them has brighten my time here. You have been the biggest influence on my life, I love you and I’m incredibly proud to be your son. Lastly and by no means least, thank you to my supportive and beautiful partner Anna. Du bist mein Ein und Alles! While our time apart has been very difficult, I’m so proud of everything we both have achieved and the strength of our love. I know we can achieve anything together and cannot wait to start our lives together and explore all that it will bring. Ich liebe dich für immer und ewig This thesis is dedicated to the two most important people in my life, my Mam und meine Liebste Anna. This thesis was written and researched on Awabakal Lands. Wherever we walk in Australia, we walk on Aboriginal land. iii Table of Contents Declaration .................................................................................................................................. i Acknowledgements .................................................................................................................... ii Synopsis ..................................................................................................................................... v Publications, conference and awards arising from this thesis.................................................. vii Abbreviations ............................................................................................................................ xi CHAPTER 1: Literature review............................................................................................. 1 Use of advanced proteomics and phosphoproteomics to uncover the aetiology of chronic obstructive pulmonary disease CHAPTER 2: .......................................................................................................................... 63 Deep time-resolved proteomic profiling of cigarette smoke-induced chronic obstructive pulmonary disease CHAPTER 3: ........................................................................................................................ 113 Deep time-resolved phosphoproteomic profiling of cigarette smoke-induced chronic obstructive pulmonary disease CHAPTER 4: ........................................................................................................................ 170 Proteomic profiling of OCT-embedded COPD endobronchial biopsies CHAPTER 5: General Discussion & Future Research Directions ................................. 236 APPENDIX: Publications ................................................................................................... 245 iv Synopsis Proteomics has become a mature scientific discipline across many fields and a critical means to understand the dynamic nature of disease states. Strikingly, there are now more than 83,995 citations featuring ‘proteomics’, many of which have arisen in the past 5-10 years. However, in stark contrast to most fields of research, in chronic obstructive pulmonary disease (COPD), proteomics remains an under-utilised research tool with only 202 citations featuring ‘COPD proteomics’ recorded to date (<0.25% of all papers). This is in spite of the severity of the disease with COPD listed as the third leading cause of death worldwide. In this thesis, we have sought to address this fundamental knowledge gap by undertaking a highly comprehensive, comparative and quantitative time resolved analysis of both the proteome and phosphoproteome of lung tissue using our well-characterised mouse model of cigarette smoke-induced COPD. Moreover, we have developed critical platforms for the analysis of endobronchial biopsies from patients through the analysis of samples from clinical cohorts encompassing healthy controls, healthy smokers, mild COPD and severe COPD patients. Key highlights from this body of work include the quantification of 7,324 proteins and 27,857 unique phoshopeptides across a 12-week time course of COPD progression