Organellar Proteomics of the Golgi Apparatus and Golgi Derived COPI Vesicles. Catherine Elaine Au Department of Anatomy and Cell Biology McGill University, Montreal January 2008 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy. ©Catherine Elaine Au 2008 Table of Contents Table of Contents .................................................................................................1 List of Figures .......................................................................................................7 List of Tables ........................................................................................................9 List of Abbreviations ...........................................................................................10 Abstract...............................................................................................................13 Resumé ..............................................................................................................14 Original Contributions .........................................................................................18 Acknowledgements.............................................................................................19 Chapter 1 -- Introduction.....................................................................................21 Chapter 2 -- Literature Review............................................................................24 2.1 The early secretory pathway. ........................................................ 24 2.1.1 The endoplasmic reticulum. ........................................................... 24 2.1.1.1 The rough endoplasmic reticulum. ................................... 24 2.1.1.2 The smooth endoplasmic reticulum.................................. 25 2.1.1.3 The transitional endoplasmic reticulum. ........................... 26 2.1.2 The ER Golgi intermediate compartment....................................... 26 2.1.3 The Golgi apparatus. ..................................................................... 26 2.1.3.1 Golgi ultrastructure........................................................... 27 2.1.3.2 Post-translational modification and the Golgi. .................. 27 2.1.3.3 Other Golgi components. ................................................. 28 2.1.4 COPI vesicles. ............................................................................... 28 2.2 Cisternal maturation and vesicular transport. ................................ 31 2.3 Mass Spectrometry Based Proteomics. ........................................ 34 2.3.1 Mass spectrometric techniques used in proteomics....................... 34 2.3.1.1 Time of Flight (ToF).......................................................... 34 2.3.1.2 Fourier transform ion cyclotron resonance (FTICR). ........ 34 2.3.1.3 Tandem MS (MS/MS)....................................................... 35 2.3.1.4 Gas chromatography/MS (GC MS). ................................. 35 2.3.1.5 Liquid chromatography/MS (LC MS). ............................... 35 2.3.1.6 Electrospray ionization (ESI)............................................ 35 2.3.1.7 Matrix-assisted laser desorption/ionization (MALDI). ....... 36 2.3.2 Interpretation of mass spectra. ...................................................... 36 2.3.3 Quantitation and proteomics. ......................................................... 37 2.3.4 Sample preparation for proteomics................................................ 38 2.4 Proteomics and Cell Biology. ........................................................ 40 2.4.1 Organellar proteomics.................................................................... 40 2.4.1.1 Synaptic vesicles and clathrin coated vesicles from rat brain. ............................................................................................. 40 2.4.1.2 ER-phagosome controversy............................................. 41 2.4.1.3 Endosomes and p14. ....................................................... 42 2.4.1.4 The exosome. .................................................................. 42 2.4.1.5 The mitochondria. ............................................................ 43 2.4.1.6 The peroxisome. .............................................................. 44 1 2.4.1.7 The endoplasmic reticulum. ............................................. 44 2.4.1.8 The ERGIC....................................................................... 45 2.4.1.9 The Golgi apparatus......................................................... 45 2.4.1.10 The COPI vesicles. ........................................................ 46 2.4.1.11 The lysosome................................................................. 46 2.4.1.11 The nuclear envelope..................................................... 46 2.4.2. Organelle contamination. .............................................................. 47 Chapter 3 -- Validity of a quantitative proteomics method for organellar proteomics. .........................................................................................................50 3.1 Abstract ......................................................................................... 50 3.2 Introduction ................................................................................... 52 3.3 Methods ........................................................................................ 54 3.3.1 Organelle isolation. ........................................................................ 54 3.3.2 Enzyme assays.............................................................................. 54 3.3.3 SDS-PAGE and immunoblots. ....................................................... 54 3.3.4 Cryoimmune electron microscopy.................................................. 54 3.3.5 Peptide Counting. .......................................................................... 55 3.3.5.1 The principle of peptide counting. .................................... 55 3.3.5.2 Comparison between samples. ........................................ 56 3.4 Results .......................................................................................... 57 3.4.1 Sensitivity of the approach............................................................. 57 3.4.2 Complex protein mixtures. ............................................................. 57 3.4.3 In situ distribution of albumin in rat liver parenchyma. ................... 57 3.4.4 Comparison of marker enzyme enrichment and marker protein peptides detected by tandem MS. ........................................................... 58 3.5 Discussion..................................................................................... 60 Figures ................................................................................................. 63 Figure 3.1. Sensitivity of the in-gel trypsin digested 1D SDS-PAGE tandem MS method. ................................................................................ 63 Figure 3.2. Characterization of albumin content in RM, SM and Golgi fractions. .................................................................................................. 65 Figure 3.3. Albumin is concentrated in the Golgi apparatus from the CGN. ................................................................................................................. 67 Figure 3.4. Distribution of albumin and β-COP in isolated rat liver Golgi fractions. .................................................................................................. 70 Figure 3.5. Comparison of marker enzyme activities and peptide counts for cognate marker proteins. .................................................................... 72 Figure 3.6. Marker proteins as detected by immunoblotting and peptide counting. .................................................................................................. 74 Chapter 4 -- The Pipeline....................................................................................76 4.1 Abstract ......................................................................................... 76 4.2 Introduction ................................................................................... 77 4.3 Methods ........................................................................................ 78 4.3.1 Sample preparation........................................................................ 78 4.3.1.1 Rough and smooth microsomes....................................... 78 4.3.1.2 Golgi apparatus................................................................ 78 2 4.3.1.3 COPI vesicles................................................................... 79 4.3.2 Sample characterization. ............................................................... 81 4.3.2.1 Preparation of samples for electron microscopy. ............. 81 4.3.2.2 Quantification of vesicle profiles on electron micrographs. ...................................................................................................... 82 4.3.2.3 Enzyme assays. ............................................................... 82 4.3.2.4 SDS-PAGE and immunoblots. ......................................... 83 4.3.3 Tandem MS (MS/MS) analysis and data processing. .................... 83 4.3.4 Data processing............................................................................. 86 4.3.5 Data analysis. ................................................................................ 87 4.3.5.1 Principal coordinates