Membrane Proteomics Characterization of Brush Border membrane proteins of mice intestinal mucosa. Case study: cholesterol absorption Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften vorgelegt beim Fachbereich Biochemie, Chemie und Pharmazie der Johann Wolfgang Goethe-Universität in Frankfurt am Main von Eirini Tsirogianni aus Athen (Griechenland) Frankfurt (2008) 2 Vom Fachbereich Biochemie, Chemie und Pharmazie der Johann Wolfgang Goethe-Universität als Dissertation angenommen. Dekan: Prof. Dr. D. Steinhilber Gutachter: Prof. Dr. M. Karas PD Dr. H. Langen Datum der Disputation: 18.05.2009 3 4 To Axel and Nephelie (Where there is a will, there is a way) 5 6 Table of content SUMMARY........................................................................................................11 ZUSAMMENFASSUNG ..................................................................................17 1.1 Brush Border Membrane BBM .................................................................23 1.1.1 Physiology of the small intestine .............................................................................. 23 1.1.2 Brush Border Membrane: Location and Function ................................................ 24 1.1.3 Brush Border Membrane and Lipid Rafts microdomains.................................... 25 1.2 Cholesterol homeostasis..............................................................................26 1.2.1 Cholesterol absorption in the Small Intestine......................................................... 27 1.3 Membrane proteomics ................................................................................29 1.3.1 Proteomics – Definition and workflow.................................................................... 29 1.3.2 Membrane Proteins - Importance and Characteristics ......................................... 31 1.3.3 Proteomic approaches for Membrane Proteins...................................................... 32 1.4 Mass spectrometry ......................................................................................34 1.4.1 A brief history of MS in biology............................................................................... 34 1.4.2 Ionization technique.................................................................................................. 35 1.4.2.1 Matrix-assisted laser desorption/ionization (MALDI) ............................................................... 35 1.4.2.2 Electrospray ionization (ESI) ....................................................................................................... 37 1.4.3 The Mass Analyzer.................................................................................................... 38 1.4.3.1 The Linear Ion Trap-Orbitrap Mass Spectrometer ................................................................... 41 1.5 Data analysis and Bioinformatics tools .....................................................44 1.5.1 Protein Identification ................................................................................................ 44 1.5.1.1 Peptide Mass Fingerprint (PMF method).................................................................................... 45 1.5.1.2 Fragmentation Mass Fingerprinting (FMF) ............................................................................... 46 1.5.2 Validation of peptide and protein identification .................................................... 49 1.6 Quantification in Proteomics .....................................................................50 1.6.1 Semi-quantitative analysis based on Spectral Counting........................................ 51 1.6.2 Relative Quantification based on Differential Stable Isotope labeling................. 52 1.6.3 Label free quantitation of LC-MS data................................................................... 56 2. OBJECTIVES................................................................................................59 3. MATERIALS AND METHODS .................................................................61 3.1 Materials / Chemicals..................................................................................61 3.2 Methods ........................................................................................................62 3.2.1 BBM Preparation ...................................................................................................... 62 3.2.2 Protein concentration estimation by the BCA method .......................................... 62 3.2.3 Protein Deglycosylation ............................................................................................ 63 3.2.4 1D SDS-PAGE electrophoresis ................................................................................ 64 7 3.2.4.1 Sample preparation and electrophoresis ..................................................................................... 64 3.2.4.2 Protein staining.............................................................................................................................. 65 3.2.5 Western Blotting........................................................................................................ 65 3.2.6 In-gel protein digestion ............................................................................................. 66 3.2.7 Mass spectrometry .................................................................................................... 67 3.2.7.1 Packing of NanoLC columns ........................................................................................................ 67 3.2.7.2 Method development for NanoLC ESI-MS/MS.......................................................................... 67 3.2.7.3 Data Processing Method and Protein Identification................................................................... 68 3.2.7.4 Sequence and topology analysis.................................................................................................... 69 3.2.8 RNA extraction from the small Intestine ................................................................ 70 3.2.9 RNA Electrophoresis................................................................................................. 71 4. RESULTS AND DISCUSSION ...................................................................73 4.1 An improved protocol for the specific isolation of BBM from small intenstine ............................................................................................................73 4.1.1 1D-SDS-PAGE analysis of BBM fractions.............................................................. 79 4.1.2 BBM preparation and protein degradation............................................................ 81 4.1.2.1 Protein deglycosylation ................................................................................................................. 81 4.1.2.2 Western Blot analysis of Aminopeptidase N ............................................................................... 82 4.1.2.3 Inhibition of protein degradation................................................................................................. 83 4.2 Protein identification of BBM mice intestinal mucosa ............................87 4.3 Examples of protein localization................................................................95 4.4 Cholesterol absorption................................................................................97 4.4.1 Identified proteins related to Cholesterol absorption............................................ 97 4.4.2 Comparison of protein expression in the BBM of wild type mice and ApoE knockout mice................................................................................................................... 100 4.5 Assessing the reproducibility of the improved BBM preparation .......104 4.5.1 BBM preparation procedure.................................................................................. 105 4.5.2 Comparing the variability of the technical steps: design of experiment............ 106 4.5.3 Estimation of experimental reproducibility.......................................................... 108 4.5.3.1 Estimation of experimental reproducibility based on Protein identification ......................... 108 4.5.3.1.1 Venn diagrams representation............................................................................................... 108 4.5.3.2 Estimation of experimental reproducibility based on LC-MS signals .................................... 115 4.5.3.2.1 Comparative analysis of a standard peptide mixture............................................................. 115 4.5.3.2.2 Comparative analysis of the BBM SDS gel bands 2, 9 and 11. ............................................. 121 4.5.3.2.3 LC-MS reproducibility........................................................................................................... 129 4.5.3.3 Findings and discussion............................................................................................................... 131 4.6 Peptide identification by LC-MS/MS......................................................134 4.6.1 Characteristics of identified peptides .................................................................... 135 4.6.2 Comparison of identified peptides with predicted tryptic transmembrane peptides.............................................................................................................................. 137 4.6.3 Discussion................................................................................................................