NANO-SCALE LIQUID CHROMATOGRAPHY ELECTROSPRAY TANDEM MASS SPECTROMETRIC IDENTIFICATION OF MULTIPLE CYTOCHROME P450 ISOFORMS IN TISSUES Sonia Nisar W A Thesis Submitted for the degree of Doctor of Philosophy of the University of London Department of Pharmaceutical and Biological Chemistry The School of Pharmacy University of London 29-39 Brunswick Square London WCIN lAX February 2005 ProQuest Number: 10104297 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10104297 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract The cytochromes P450 (CYPs) are membrane bound proteins that collectively carry out a wide range of biological oxidations important in the metabolism of steroids, ecosanoids and xenobiotics including anticancer agents. A method of CYP separation and analysis has been developed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and nano-scale liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS) for the direct identification of multiple CYPs found in rat liver, human tissues and tumours. Endoplasmic reticulum from selected tissues was prepared as microsomes using ultracentrifugation and subjected to SDS-PAGE. The proteins present in the 48-62 kDa gel band region were excised, cut into small pieces and digested with trypsin. The resultant peptides were solvent extracted, separated and analysed by LC-ESI-MS/MS. CYP identifications were made by searching the MS/MS data using Sequest software against a protein database (obtained from National Centre for Biotechnology Information). Twenty-four CYP isoforms from the sub-families lA, 2A, 2B, 2C, 2D, 2E, 3A, 4A, 4F, CYP 17 and CYP 19 were positively identified in rat liver, fourteen isoforms of which were shown to be gender biased consistent with previous studies on sexual dimorphism or gender predominance. Fifteen CYP isoforms namely, 1A2, 2A6, 2B6, 2C8, 2C9, 2C17, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, 4A11, 4F1 and 4F3 were positively identified to be present in normal human liver. CYPs were also identified from paired samples of human liver: 1A2, 2A6, 2B6, 2C8, 2C9, 2C17, 2C19, 2D6, 2E1, 3A4, 4A11, 4F2 and 4F8 and colon metastasis in the liver: 1A2, 2A6, 2C8, 2C9, 2D6, 2E1, 3A4, 4A11, 4F2 and 4F8. In normal colon and colon primary tumour only the CYP3A subfamily was identified. These results confirm the use of an MS based proteomic approach to the identification of multiple CYPs that obviates the need to predetermine which CYPs are present. They also suggest that the liver tissue environment is significantly increasing the expression profile of CYPs in colon tumours that metastasise to the liver. Contents Sections pp. Abstract..................................................................................................................... 2 Table of Contents .................................................................................................... 3-6 List of Figures ........................................................................................................... 7-11 List of Tables .......................................................................................................... 12-13 List of Abbreviations ............................................................................................... 14-17 Acknowledgments .................................................................................................... 18 Chapter 1: Introduction 1.1 Cytochromes P450 ....................................................................................... 21-25 1.2 Catalytic cycle and structure of CYP450 ................................................. 25-29 1.3 Expression of CYPs in tissues .................................................................... 30-32 1.4 Development of an anti tumour Prodrug ................................................... 32-33 1.5 Polymorphism ............................................................................................. 33-34 1.6 Why proteomics to study CYPs? ............................................................... 36-37 1.6.1. Gel electrophoresis ........................................................................ 38-41 1.6.2. High performance liquid chromatography .................................. 41-43 1.6.3. Mass spectrometry ........................................................................ 43-46 1.6.3a. Matrix assisted laser desorption ionisation ...................... 46-50 1.6.3b. Electrospray ionisation tandem mass spectrometry 51-58 3 1.7. Peptide sequencing via database searching of tandem mass spectra 58-59 1.8 Quantitative proteome analysis .................................................................... 59-63 1.9 Aims.............................................................................................................. 64 Chapter 2: Materials and methods Materials................................................................................................................. 66 2.1. Cell culture....................................................................................................... 67 2.2. Preparation of microsomes ............................................................................. 68-72 2.2.1. Preparation of microsomes from MCF-7 cell lines ...................... 68 2.2.2. Preparation of rat liver microsomes ............................................... 68-69 2.2.3. Preparation of human liver microsomes ....................................... 69-70 2.2.4. Preparation of microsomes from human colorectal metastasis and paired normal liver ................................................................... 70-71 2.2.5. Preparation of microsomes from xenograft samples .................. 72 2.3. Bradford Assay ................................................................................................ 72-73 2.4. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis ................ 73-75 2.5. Western blotting ................................................................................................ 76-77 2.6. Trypsin digestion and extracting peptides from polyacrylamide gel 77-79 2.6.1. Preparation of gel slices for tryptic digest ...................................... 77-78 2.6.2. Preparation of trypsin stock solution ............................................... 78 2.6.3. Digestion of gel slices with trypsin ................................................. 78 4 2.6.4. Extracting digested peptides from trypsinised gel pieces 78 2.6.5. Desalting the digest samples .......................................................... 79 2.7. Nano-HPLC .......................................................................................................... 80-81 2.8. LCQ ....................................................................................................................... 81-83 2.8.1. Static nano spray .................................................................................... 82-83 2.8.2. LC-ES-MS/MS ................................................................................ 83 2.9. Protein identification ............................................................................................ 83-87 2.10. Optimisation of protein identification .............................................................. 87-88 2.11. Absolute Quantification (AQUA) of proteins ................................................. 88-89 Chapter 3: Results A proteomic approach to the identification of cytochrome P450 isoforms in tissues by nano-scale liquid chromatography electrospray ionisation tandem mass spectrometry 3.1. Rat liver.......................................................................................................... 91-100 3.2. Human liver......................................................................................................... 101-108 3.3. CYP expression in normal human liver, colorectal métastasés, normal colon and primary colon tumours .................................................... 109-118 3.4. Experimental tumours ..................................................................................... 119-123 3.5. Improvement of identification methods ....................................................... 124-128 3.6. Validation of absolute quantification of proteins and applications to CYPs 129-133 Chapter 4: Discussion 4.1. CYP expression in male and female rat liver............................................... 135-141 4.2. Human liver..................................................................................................... 141-145 4.3. Comparison of CYPs in normal liver, colorectal métastasés, normal colon and primary colon tumours .......................................................................................... 146-149 4.4. Experimental tumours and MCF-7 cell lines............................................... 150-151 4.5. Optimisation of protein identification .......................................................... 152-153 4.6. Absolute