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研究生： 杜瓊莊
Do, Quynh-Trang
論文名稱： 利用液相層析串聯式質譜搭配蛋白質親和性富集策略和誘導修飾膠體染色法鑑定癌細胞中兒茶酚激素目標蛋白
Identification of Protein Targets of Catechol Estrogens in Cancer Cells by Affinity Enrichment and Modification-induced Gel Staining Coupled with LC-MS/MS
指導教授： 陳淑慧
Chen, Shu-Hui
學位類別： 碩士
Master
系所名稱： 理學院 - 化學系
Department of Chemistry
論文出版年： 2019
畢業學年度： 107
語文別： 英文
論文頁數： 69
外文關鍵詞： Catechol estrogens, affinity purification, click chemistry, LC-MS/MS, redox-cycling
相關次數： 點閱：146  下載：10 
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    • Catechol estrogens (CEs) are toxic metabolites which form stable covalent conjugation with cellular proteins via transformation to catechol quinones and CEs-conjugation has been linked to cancer formation. Identification and characterization of cellular targets of CEs are essential to better understand CEs adduction-induced disease mechanism but challenging goals. We previously used click reaction coupled with dimethyl labeling to identify protein targets of CEs in rat liver microsomes. However, the identification of conjugation sites is still difficult by the previous method. Moreover, preparation and treatment of culture cells related to CEs-related diseases have not yet developed. In this thesis, an improved enrichment method was developed and applied to identify CE targets in MCF-7 breast cancer cells. Moreover, a modification-induced gel staining method was developed to detect the conjugation of high abundant proteins.
    4OHEE2 (4-hydroxyethynylestradiol) was used as a CEs probe to capture protein targets by covalent conjugation. The CEs-conjugated proteins were further enriched by streptavidin beads through a new cleavable biotin linker short of amide functional group. The resulting peptides were found to minimize linker fragmentation and improve protein identification by LC-MSMS-based proteomics. CEs-conjugated peptides were further enriched by borate gel to assist the assignment of multiple conjugation sites on cytosolic, nuclear and histone proteins. We expect to explore functional roles of some identified protein targets and study their usefulness as disease markers or drug targets.

    ABSTRACT I ACKNOWLEDGEMENTS III TABLE OF CONTENT IV LIST OF TABLES VII LIST OF FIGURES VIII ABBREVIATIONS XI CHAPTER 1: ESTROGEN METABOLISM 1 CHAPTER 2: MASS SPECTROMETRY AND PROTEOMICS FOR CATECHOL ESTROGEN MODIFICATION 6 2.1. MASS SPECTROMETRY AND PROTEOMICS 6 2.1.1. Mass spectrometry 6 2.1.2. Introduction of Proteomics 8 2.1.3. LC-MS/MS based bottom-up protein identification 9 2.2. AFFINITY PURIFICATION 12 2.3. CLICK CHEMISTRY COUPLED STREPTAVIDIN BEADS 13 2.4. NBT REDOX-CYCLING METHOD 17 2.5. CATECHOL-BORONATE REVERSIBLE REACTION UTILIZING FOR ENRICHMENT SYSTEM 19 CHAPTER 3: MATERIALS AND METHODS 21 3.1. MATERIALS 21 3.1.1. Reagents 21 3.1.2. Equipment 22 3.2. CATECHOL ESTROGEN TREATED MCF-7 CELLS 23 3.3. SUBCELLULAR FRACTIONATION 24 3.3.1. Cytoplasmic fractionation 24 3.3.2. Nuclear extraction 25 3.3.3. Acid extracted fraction 25 3.3.4. Insolution digestion 26 3.4. PREPARATION OF ESTROGENIZED PROTEIN STANDARDS 27 3.4.1. 4OHEE2- adducted insulin 27 3.4.2. 4OHEE2- adducted human serum albumin 27 3.5. SDS-PAGE AND PROTEIN DETECTION 27 3.5.1. Denaturing polyacryamide gel gel electrophoresis (SDS-PAGE) 27 3.5.2. Coomassie blue staining 28 3.5.3. Electroblotting of proteins 28 3.5.4. Nitro blue tetrazolium redox-cycling staining 28 3.6. AFFINITY PURIFICATION STRATEGIES 29 3.6.1. Enrichment based streptavidin beads 29 3.6.2. Enrichment based boronate gel 31 3.7. LC-MS/MS ANALYSIS 32 3.7.1. Conditions of Liquid Chromatography 32 3.7.2. Conditions of Mass Spectrometry 33 3.8. DATA INTERPRETATION AND BIOINFORMATICS ANALYSIS 33 3.8.1 Mascot 33 3.8.2. Mascot Distiller 34 3.8.3. Peaks 35 3.8.4. Bioinformatics analysis 35 CHAPTER 4: RESULT AND DISCUSSION 36 4.1. SUBCELLULAR FRACTIONATION EFFICIENCY 36 4.2. ENRICHMENT OF CE-MODIFIED PROTEIN STANDARDS 38 4.2.1. Redox-cycling staining method 38 4.2.2. Boronic acid affinity purification 40 4.2.3. Click chemistry coupled streptavidin beads strategy 42 4.2.4. Click chemistry coupled on-streptavidin bead digestion 44 4.3. IDENTIFICATION OF CE TARGET PROTEINS IN SUBCELLULAR FRACTIONS OF MCF-7 CELLS 49 4.3.1. Cytosol fraction 49 4.3.2. Nuclear fraction 53 4.3.3. Acid extracted fraction 55 4.4. FUNCTIONAL ANNOTATIONS OF IDENTIFIED PROTEIN TARGETS IN MCF-7 CELLS 56 CHAPTER 5: CONCLUSIONS 59 CHAPTER 6: REFERENCE 61

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