Proteomics analysis of brain AVM endothelium post irradiation in pursuit of targets for AVM molecular therapy Margaret Simonian, BSc, MPhil A thesis presented for the degree of Doctor of Philosophy Australian School of Advanced Medicine Faculty of Medicine and Health Sciences Macquarie University Table of Contents LIST OF FIGURES AND TABLES ......................................................................................... 6 DECLARATION......................................................................................................................11 ACKNOWLEDGMENT ......................................................................................................... 12 Summary .................................................................................................................................. 13 Chapter1. General Introduction ............................................................................................... 14 1.1. Arteriovenous malformations and goals of project ........................................................... 15 1.1.1. Treatment options .......................................................................................................... 16 1.1.2. Development of new treatments for brain AVMs ........................................................... 20 1.2. Vascular endothelium ....................................................................................................... 24 1.2.1. Function of vascular endothelium ................................................................................. 25 1.2.2. Thrombogenesis ............................................................................................................. 25 1.2.3. Thromboresistant properties of vascular endothelium .................................................. 25 - Plasminogen activators……………………………………………………………….26 - Heparin-like molecules………………………………………………………………..26 - Antithrombin III……………………………………………………………………….26 - Annexin V……………………………………………………………………………...26 1.2.4. Endothelial response to irradiation ............................................................................... 27 1.2.5. Selected endothelial vascular molecules and their response to irradiation .................. 28 - PECAM-1……………………………………………………………………………….28 - Cadherin-5…………………………………………………………………………….29 - Integrins……………………………………………………………………………….30 1.3. Membrane proteins ........................................................................................................... 31 1.4.1. Biotinylation ................................................................................................................... 35 1.5. Proteomics .................................................................................................................... 36 1.5.1. Proteomics in medicine .................................................................................................. 38 1.5.2. Quantitative proteomic techniques ................................................................................ 38 - Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) ..................................... 39 - Mass spectrometry expression (MSE) ............................................................................ 40 Chapter 2: In vitro biotinylation protocol optimizations of murine endothelial cell cultures (bEnd3) .................................................................................................................................... 41 2.1 Introduction ........................................................................................................................ 42 2.2. In vitro biotinylation of bEnd.3 cultures - first trial ......................................................... 42 1 2.2.1. Materials and methods .................................................................................................. 42 2.2.1.1. Mouse endothelial cell cultures (bEnd.3) ................................................................... 42 2.2.1.2. Cell density and total protein concentrations ............................................................. 42 2.2.1.3. In vitro biotinylation ................................................................................................... 43 2.2.1.4. Digestion of eluted proteins and LC-MS/MS analysis ................................................ 44 2.2.1.5. Data processing .......................................................................................................... 44 2.3.1. Materials and methods .................................................................................................. 46 2.3.1.1. In vitro biotinylation ................................................................................................... 46 2.3.1.2. One dimensional electrophoreses ............................................................................... 46 2.3.1.3. MALDI-TOF mass spectrometry ................................................................................ 46 2.3.1.4. Data processing .......................................................................................................... 47 2.3.2. Results............................................................................................................................ 47 2.4. In vitro biotinylation of BSA and E.coli - First trial ......................................................... 51 2.4.1.1. Total protein concentration of E.coli.......................................................................... 51 2.4.1.2. In vitro biotinylation of BSA ....................................................................................... 52 2.4.1.3. Capture of biotinylated BSA ....................................................................................... 52 2.4.1.4. Protein digestion, purification and LC-MS/MS analysis ............................................ 52 2.4.2. Results............................................................................................................................ 53 2.5. In vitro biotinylation of BSA and E.coli - Second trial .................................................... 58 2.5.1. Materials and methods .................................................................................................. 58 2.5.1.1. In vitro biotinylation of BSA ....................................................................................... 58 2.5.1.2. Capture of biotinylated BSA ....................................................................................... 58 2.5.1.3. Protein digestion, purification and LC-MS/MS analysis ............................................ 59 2.5.1. Results............................................................................................................................ 59 2.6. In vitro biotinylation of BSA with different biotin concentrations - Third trial ............... 66 2.6.1. Materials and methods .................................................................................................. 66 2.6.1.1. In vitro biotinylation ................................................................................................... 66 2.6.2. Results............................................................................................................................ 67 2.7. In vitro biotinylation of BSA using different biotin concentrations with E.coli .............. 77 2.7.1. Materials and methods .................................................................................................. 77 2.7.1.1. In vitro biotinylation ................................................................................................... 77 2.7.1.2 Results .......................................................................................................................... 78 2.8. In vitro biotinylation of bEnd3 cell cultures - new trials .................................................. 83 2.8.1. Materials and methods .................................................................................................. 83 2.8.1.1. In vitro biotinylation ................................................................................................... 83 2 2.8.1.2. Tryptic digestion of biotinylated proteins and LC-MS/MS analysis ........................... 84 2.8.1.3. Data processing .......................................................................................................... 85 2.8.2. Results ............................................................................................................................ 85 2.9 Western blot analysis of bEnd.3 cells ................................................................................ 88 Chapter3. In vitro biotinylation and iTRAQ-mass spectrometry of bEnd.3 cells ................. 90 3.1 Introduction ........................................................................................................................ 91 3.2 Materials and Methods ....................................................................................................... 91 3.2.1 Mouse endothelial cell cultures (bEnd.3) ....................................................................... 91 3.2.2 Cell density and total protein concentration ................................................................... 91 3.2.3 Irradiation of bEnd.3 cells .............................................................................................. 92 3.2.4 In vitro biotinylation of bEnd.3 cells .............................................................................