Yang Yu PhD Thesis Lipidomics Investigations in Cell Biology International Doctoral School in Biomolecular Sciences 26th cycle Lipidomics Investigations in Cell Biology PhD thesis of: Yang Yu Centre for Integrative Biology (CIBIO) Academic Year 2012 – 2013 Tutor: Prof. Graziano Guella Bioorganic Chemistry Laboratory (Dept. Physics) Advisors: Prof. Paolo Macchi (CIBIO), Dr. Urska Vrhovsek (FEM) 1 Yang Yu PhD Thesis Lipidomics Investigations in Cell Biology 'Declaration of Authorship: I (Yang Yu) confirm that this is my own work and the use of all material from other sources has been properly and fully acknowledged.' Tutor: Prof. Graziano Guella PhD Candidate: Yang Yu 2 Yang Yu PhD Thesis Lipidomics Investigations in Cell Biology Abbreviations ADMEM Advanced-Dulbecco’s Modified Essential Medium CAD Collision gas CE Collision Energy Chol Cholesterol CID Collision-Induced Dissociation CUR CURtain gas CXP Collision Cell Exit Potential DAG Diglycerides DESI Desorption Electrospray Ionization DHAP DiHydroxyAcetonePhosphate DHAPAT DiHydroxyAcetonePhosphate AcylTransferase DLPC 1,2-Dilauroyl-sn-glycero-3-PhosphoCholine DMEM Dulbecco’s Modified Essential Medium DOPA 1,2-Dioleoyl-sn-glycero-3-Phosphate DOPC 1,2-Dioleoyl-sn-glycero-3-PhosphoCholine DOPS 1,2-Dioleoyl-sn-glycero-3-Phospho-L-Serine DP Declustering Potential DSPG 1,2-Dioctadecanoyl-sn-glycero-3-Phospho-(1'-rac-Glycerol) EI Electron Ionization EIC Extracted Ion Chromatogram EP Entrance Potential ePC plasmanyl (or ether) PC ER Endoplasmic Reticulum ESI Electrospray Ionization FBS Fetal Bovine Serum FP Focusing Potential GC-MS Gas Chromatography–Mass Spectrometry GL GlycoLipids GlcCer GlucosylCeramide GPL GlyceroPhosphoLipids GSL GlycoSphingoLipids 1H DQCOSY Double Quantum COherence SpectroscopY 1H-13C HMBC proton-carbon Multiple-Bond Correlation 1H-13C HSQC proton-carbon Single Quantum Correlation HIF 1-a Hypoxia-Inducible Factor 1-alpha HIF Hypoxia-Inducible Factor HPLC High Performance Liquid Chromatography IS IonSpray 3 Yang Yu PhD Thesis Lipidomics Investigations in Cell Biology LPC Lyso PhosphoCholines LPE Lyso PhosphoEthanolamines LPL Lyso PhosphoLipids MALDI TOF MS Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass spectrometer MS Mass Spectrometry MUFA Mono-Unsaturated Fatty Acid MVDA MultiVariate Data Analysis NEB NEBulizer gas NLS Neutral Loss Scanning NMR Nuclear Magnetic Resonance NPLC Normal Phase Liquid Chromatography OPLS-DA Orthogonal Projections to Latent Structures-Discriminant Analysis PA Phosphatidic Acids PAF Platelet-Activating Factor PC PhosphoCholines PC1 Principal Component 1 PC2 Principal Component 2 PCA Principal Component Analysis PE PhosphoEthanolamines PG PhosphoGlycerols PI PhosphoInositols PIS Precursor Ion Scanning PL PhosphoLipids PLS-DA Partial Least Squares Discriminant Analysis pPC plasmenyl (or vinyl-ether) PC PS PhosphoSerines PUFA Poly-Unsaturated Fatty Acid QQQ MS Triple Quadruple Mass Spectrometer REIMS Rapid Evaporative Ionization Mass Spectrometry RP-HPLC Reversed Phase High Performance Liquid Chromatography RTK Receptor Tyrosine Kinases S1P Sphingosine-1-Phophate SL SphingoLipids TAG Triglycerides TEM TEMperature TIC Total Ion Chromatogram TOCSY TOtally Correlated SpectroscopY UI Unsaturation Index UVDA UniVariate Data Analysis VIP Variable Influence on Projection 4 Yang Yu PhD Thesis Lipidomics Investigations in Cell Biology Table of Contents Chapter 1. Abstract ........................................................................................................................... 7 Chapter 2. Introduction .................................................................................................................. 9 2.1 Lipids and lipidomics .................................................................................................................... 9 2.2 NMR and Mass spectrometry .................................................................................................. 15 2.3 Sample preparation for lipidomics analysis......................................................................... 20 2.4 Lipidomics data processing ...................................................................................................... 21 2.5 Aim of the thesis ......................................................................................................................... 23 2.6 References .................................................................................................................................... 24 Chapter 3. A lipidomics investigation of the induced hypoxia stress on HeLa cells by using MS and NMR techniques .............................................................................. 28 3.1 Abstract ......................................................................................................................................... 28 3.2 Introduction ................................................................................................................................. 29 3.3 Materials and Methods ............................................................................................................. 33 Chemicals ............................................................................................................................................ 33 Cell culture and Hypoxia treatment .................................................................................................... 33 Immunocytochemistry and fluorescence microscopy ........................................................................ 33 Phospholipids extraction and sample preparation ............................................................................. 34 NMR measurements ........................................................................................................................... 35 Optimization of precursor ion scanning and neutral loss scanning for ESI MS/MS analysis .............. 35 HPLC-ESI MS/MS measurements ........................................................................................................ 36 Data processing ................................................................................................................................... 37 3.4 Results and discussion ............................................................................................................... 38 Cobalt (II) chloride induces hypoxia .................................................................................................... 38 NMR analysis of phospholipid classes................................................................................................. 39 Lipid class-specific (head groups) survey scans through MS/MS measurements .............................. 42 5 Yang Yu PhD Thesis Lipidomics Investigations in Cell Biology HeLa cellular phospholipids profiling and hypoxia-induced lipid alterations ..................................... 45 Multivariate analysis and individual potential marker analysis .......................................................... 49 Unsaturation Index analysis of each phospholipid class..................................................................... 54 3.5 Conclusions ................................................................................................................................... 56 3.6 Acknowledgments ...................................................................................................................... 58 3.7 Notes and References ............................................................................................................... 58 Chapter 4. Comparative Lipidomic studies of in vitro urinary bladder cancer models RT4 and T24: an investigation on urothelial metastasis ......................... 61 4.1 Abstract ......................................................................................................................................... 61 4.2 Introduction ................................................................................................................................. 61 4.3 Material and methods ............................................................................................................... 64 Chemicals ............................................................................................................................................ 64 Urothelial cell cultures of RT4 and T24 ............................................................................................... 64 Lipid extraction and sample preparation ............................................................................................ 65 NMR analysis and MS measurements ................................................................................................ 66 Data processing ................................................................................................................................... 66 4.4 Results and discussions ............................................................................................................. 67 Morphological comparison between RT4 and T24 ............................................................................. 67 1H-NMR and 31P-NMR analysis of RT4 and T24 lipid extracts ............................................................. 68 Cellular lipids comparison between RT4 and T24 via HPLC-ESI MS/MS measurements .................... 72 Multivariate analysis and individual potential marker analysis .......................................................... 78 Fatty acyl