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Novel Lipidomic Approaches to Analyse NOVEL LIPIDOMIC APPROACHES TO ANALYSE GLYCEROPHOSPHOLIPIDS AND SPHINGOLIPIDS IN COMPLEX MIXTURES USING MASS SPECTROMETRY GUAN XUE LI NATIONAL UNIVERSITY OF SINGAPORE 2008 NOVEL LIPIDOMIC APPROACHES TO ANALYSE GLYCEROPHOSPHOLIPIDS AND SPHINGOLIPIDS IN COMPLEX MIXTURES USING MASS SPECTROMETRY GUAN XUE LI (B.Sc. (Hons.), National University of Singapore) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2008 Acknowledgements Very sincerely, I thank my supervisor, Markus R. Wenk, for being a mentor with a very unique style and for paving the way and filling it with immense support, unceasing patience, many deep insights and stimulating ideas. And with utmost appreciation, thank you very much for firmly believing in me. I thank Howard Riezman, not only for his contribution to a major focus in my thesis, and the opportunity to work in his laboratory, but his unceasing enthusiasm and engagement in science is inspirational. And for making a difference during this journey, to both Howard and Isabelle Riezman, I express my heartfelt gratitude. To Shui Guanghou, Anne K. Bendt, Chua Gek Huey and Aaron Fernandis, thank you for the encouragement, the stimulation to find a better person in me, the knowledge shared, the support during those dark moments, for everything. To Sashi Kesavapany and Maxey Chung, thank you for being in my thesis committee and providing all the constructive feedback. To Lim Tit Meng, thank you for all the support through these years. To Gisou van der Goot, thank you for the helpful discussions, and the enthusiasm and immense support, particularly for the Swiss exchange which had been an invaluable experience. To Ernst Hafen and his group members, Katja Kohler and Irena Jevtov, thank you for collaborating and the helpful discussions on fly biology. To Marcos Gonzalez, thank you for being an enthusiastic partner for fly lipidomics. To Ong Wei Yi and his then PhD student, He Xin, thank you for collaborating and the expertise in animal work. To all my other collaborators, thank you for the interest, the enthusiasm, and the opportunities to learn about many amazing things beyond the scope of my thesis work. To all past and present members of the Wenk and neighbouring laboratories, thank you for providing a pleasant scientific as well as non-scientific environment. And also to members of the Riezman laboratory, thank you for the hospitality during the exchange. Cleiton Martins de Souza, then Howard Riezman’s PhD student, is acknowledged for his enthusiasm and help throughout the collaboration. i To my friends outside the laboratory, Heiny, Petrina Fan, Goh Shu Shang and Tan Yong Wah, thank you for always being there and for whom I can turn to especially when I need a break from those greasy works. And to my family, thank you very much for the unconditional and silent support. And for providing a place to fall back on when all else fail, thank you. I would also like to acknowledge the European Molecular Biology Organization (EMBO) for its generous funding of a short term fellowship (ATSF 07-2008) for a two-month exchange to Howard Riezman’s laboratory in University of Geneva in 2008, the Yong Loo Lin School of Medicine for the research scholarship during my PhD candidature, the Pediatric Dengue Vaccine Initiative (PDVI) for a travel award for the 3rd Asian Regional Dengue Research Network Meeting in 2007 and the National University of Singapore for the prestigious President’s Graduate Fellowship in 2006/2007. ii Table of Contents Acknowledgements .........................................................................................................................i Table of Contents ..........................................................................................................................iii Summary........................................................................................................................................vi List of Tables ...............................................................................................................................viii List of Figures................................................................................................................................ix List of Abbreviations ....................................................................................................................xi List of Publications .....................................................................................................................xiv Chapter 1. Introduction.................................................................................................................1 1.1 Membrane Lipids................................................................................................................3 1.1.1 Structural diversity .........................................................................................................3 1.1.2 Biological functions of lipids .........................................................................................7 1.2 Biochemical analysis of lipids ..........................................................................................13 1.2.1 Isolation and purification of membrane lipids..............................................................13 1.2.2 Mass spectrometry........................................................................................................15 1.3 Lipidomics as a pathway discovery tool...........................................................................24 1.3.1 Unbiased discovery lipidomics.....................................................................................25 1.3.2 Targeted lipidomic analysis..........................................................................................28 1.4 Motivations and aims........................................................................................................30 Chapter 2. Novel Analytical Approach to Study Mammalian Glycerophospholipids and Sphingolipids ................................................................................................................................37 2.1 Introduction.......................................................................................................................38 2.2 Materials and Methods......................................................................................................38 2.2.1 Chemicals and reagents ................................................................................................38 2.2.2 Animal handling and collection of brain tissue ............................................................39 2.2.3 Sample preparation and collection of brain tissue........................................................39 2.2.4 Internal standards..........................................................................................................39 2.2.5 Lipid extraction ............................................................................................................40 2.2.6 Lipid analysis by electrospray ionisation mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS).....................................................................................................40 2.2.7 Data processing ............................................................................................................42 2.3 Results...............................................................................................................................43 2.3.1 Profiling of mammalian brain lipids by negative ion ESI-MS .....................................43 2.3.2 Non-targeted differential profiling based on ESI-MS and chemometry.......................46 2.4 Discussion.........................................................................................................................50 Chapter 3. High Resolution and Targeted Profiling of Glycerophospholipids and Sphingolipids in Extracts from Saccharomyces cerevisiae .......................................................53 3.1 Introduction.......................................................................................................................54 3.2 Materials and Methods......................................................................................................57 3.2.1 Strains, media and culture condition ............................................................................57 3.2.2 Lipid standards .............................................................................................................57 3.2.3 Lipid extraction ............................................................................................................58 3.2.4 Lipid analysis by ESI-MS, MS/MS and MS3 ...............................................................59 iii 3.2.5 Data analysis.................................................................................................................60 3.2.6 Statistical analysis ........................................................................................................61 3.3 Results...............................................................................................................................61 3.3.1 Theoretical calculation of the masses of yeast glycerophospholipids and sphingolipid molecular species.......................................................................................................................62 3.3.2 Rapid isolation and profiling of polar lipids from Saccharomyces
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