EVALUATION OF PROTEOMIC AND TRANSCRIPTOMIC BIOMARKER DISCOVERY TECHNOLOGIES IN OVARIAN CANCER. CLARE RITA ELIZABETH COVENEY A thesis submitted in partial fulfilment of the requirements of the Nottingham Trent University for the degree of Doctor of Philosophy October 2016 Copyright Statement “This work is the intellectual property of the author. You may copy up to 5% of this work for private study, or personal, non-commercial research. Any re-use of the information contained within this document should be fully referenced, quoting the author, title, university, degree level and pagination. Queries or requests for any other use, or if a more substantial copy is required, should be directed in the owner(s) of the Intellectual Property Rights.” Acknowledgments This work was funded by The John Lucille van Geest Foundation and undertaken at the John van Geest Cancer Research Centre, at Nottingham Trent University. I would like to extend my foremost gratitude to my supervisory team Professor Graham Ball, Dr David Boocock, Professor Robert Rees for their guidance, knowledge and advice throughout the course of this project. I would also like to show my appreciation of the hard work of Mr Ian Scott, Professor Bob Shaw and Dr Matharoo-Ball, Dr Suman Malhi and later Mr Viren Asher who alongside colleagues at The Nottingham University Medical School and Derby City General Hospital initiated the ovarian serum collection project that lead to this work. I also would like to acknowledge the work of Dr Suha Deen at Queen’s Medical Centre and Professor Andrew Green and Christopher Nolan of the Cancer & Stem Cells Division of the School of Medicine, University of Nottingham for support with the immunohistochemistry. I am grateful for the colleagueship of the numerous and high-quality scientists, both staff and students I have had the opportunity to work alongside during this time. I am indebted for the unconditional and unknowing support of my partner, friends and family, especially my parents. Thank you. Contents CONTENTS FIGURES .................................................................................................................................... i TABLES .................................................................................................................................... iii Abbreviations and Glossary ...................................................................................................... iv HYPOTHESES ........................................................................................................................ vii Thesis Abstract ........................................................................................................................ viii 1. Introduction ............................................................................................................................. 1 1.1. General Concepts ............................................................................................................. 1 1.1.1. Cancer ....................................................................................................................... 1 1.1.2. Ovarian Cancer ......................................................................................................... 4 1.1.3. Biomarkers .............................................................................................................. 10 1.1.3.1. Essential and Desirable biomarker properties .................................................. 11 1.1.4. The Need for Effective Screening Strategies .......................................................... 12 2. Methodological Overview .................................................................................................... 18 2.1. Proteomic Approaches to Biomarker Discovery and Onco-proteomics ........................ 18 2.1.1. Proteomic Techniques for Cancer Biomarker Discovery ....................................... 18 2.1.2. Analysis of the Serum Proteome ............................................................................. 19 2.1.3. Mass Spectrometry and Tandem Mass Spectrometry ............................................. 22 2.1.3.1. An Historic Summary ....................................................................................... 22 2.1.3.2. Soft Ionisation: Matrix Assisted Laser Desorption Ionisation (MALDI) and Electrospray Ionisation (ESI) ......................................................................................... 24 2.1.3.3. Mass Analysers ................................................................................................. 28 2.1.3.4. Tandem Mass Spectrometry ............................................................................. 30 2.1.4. Mass Spectrometry Approaches/Techniques used for Biomarker Discovery ......... 32 2.1.5. Sample Fractionation and Liquid Chromatography ................................................ 33 2.1.5.1. Reducing Sample Complexity .......................................................................... 33 2.1.5.2. The Mechanics of and Sources of Variability in Liquid Chromatography ....... 33 2.1.5.3. Deconvolution of Complex Biological Samples Using Liquid Chromatography ....................................................................................................................................... 36 Contents 2.1.6. Advantages or Disadvantages of Tagging in MALDI-TOF-MS. ............................ 38 2.1.7. Label Free Quantitation Techniques ....................................................................... 38 2.2. Transcriptomic Approaches to Biomarker Discovery and Onco-genomics ................... 39 2.2.1. Gene Expression Profiling ...................................................................................... 40 2.2.1.1. DNA Microarray Experiments .......................................................................... 40 2.2.1.2. Next Generation DNA Sequencing ................................................................... 41 2.2.2. Data Mining ............................................................................................................ 42 2.2.2.1. Statistical Analysis for Omics Data .................................................................. 43 2.2.2.2. Categorical and Continuous Variables in Omics Data ..................................... 44 2.2.2.3. Analysis of Omics Survival Data ...................................................................... 44 2.2.2.4. Machine Learning. ............................................................................................ 48 2.2.2.5. Artificial Neural Networks (ANNs) ................................................................. 49 2.2.3. Curated Data Repositories and Online Tools .......................................................... 54 2.2.3.1. Data Sharing ..................................................................................................... 55 2.2.3.2. Protein interaction databases ............................................................................ 55 2.2.3.3. Ontological Databases ...................................................................................... 59 2.3. Aims of the Project Overall ........................................................................................... 59 3. Proteomic Evaluation: MALDI-MS Profiling Strategy for Biomarker Discovery in Ovarian Cancer ....................................................................................................................................... 60 Chapter Abstract ................................................................................................................... 60 3.1. Introduction .................................................................................................................... 61 3.1.1. MALDI Mass spectrometry and ovarian cancer ..................................................... 61 3.1.2 Aims and Hypotheses of the Chapter. ...................................................................... 67 3.2. Materials and Methods ................................................................................................... 68 3.2.1 Materials .................................................................................................................. 68 3.2.1.1. Equipment used ................................................................................................. 68 3.2.1.2. Reagents used: .................................................................................................. 69 3.2.1.3. Stock Solutions Made and Used ....................................................................... 69 3.2.1.4. Samples ............................................................................................................. 69 3.2.2 Methods ................................................................................................................... 70 Contents 3.2.2.1 Sample Preparation and Data Acquisition ......................................................... 70 3.2.2.2 Biomarker Panel Generation .............................................................................. 71 3.2.2.3 Identification of m/z Values in the Biomarker Panel ......................................... 71 3.2.2.4 ELISA ................................................................................................................ 72 3.2.2.5 Additional Analyses ........................................................................................... 72 3.3. Results ...........................................................................................................................