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研究生：	方捷銘 Fang, Chieh-Ming
論文名稱：	利用液相層析串聯式質譜儀鑑定蛋白上雌激素代謝物之特定修飾位點 Identification of Site-Specific Modification of Estrogen Metabolites on Proteins via 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：	2014
畢業學年度：	102
語文別：	英文
論文頁數：	91
中文關鍵詞：	雌激素代謝物、蛋白轉譯後修飾、4-烃雌二醇、胰島素
外文關鍵詞：	estrogen metabolites, proteins post translation modification, 4-hydroxyestradiol, insulin
相關次數：	點閱：142 下載：0
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目前，許多被雌激素以及其活性代謝物以共價鍵修飾的蛋白已被認為與許多疾病的產生及進程有關。然而，因著仍缺乏更細微之分子層面的證據證明此類型活性代謝物所參與的角色以及其特定之修飾位點，阻礙了更深入之研究探討。於此研究中，我們利用奈流液相層析串聯式質譜儀報導了一種在生物體內、體外皆可觀察到修飾於蛋白上的鄰苯二酚雌激素，4-烃雌二醇(4OHE2)。
利用要用胰島素做為模型，搭配兩種二次質譜碎裂技術: 碰撞誘導解離(CID)以及電子轉移解離(ETD),在其A、B兩條蛋白序列上的特定胺基酸，如離胺酸(Lysine)、組胺酸(Histidine)以及胱胺酸(Cysteine)鑑定有4OHE2之共價鍵修飾，此外序列間的雙硫鍵鍵結處亦可發現此修飾。以分子模型之模擬加以佐證發現，4OHE2的修飾使的胰島素在與其受體鍵結之區域有胺基酸位向之改變，此變動可能影響其與受體之鍵結能力進而影響此蛋白之功能性。功能性的測定，如:胰島素刺激之葡萄糖攝取吸收、胰島素誘發其受體之訊號傳導以及細胞周期等，實驗結果發現被4OHE2所修飾之胰島素相較於相同濃度之正常胰島素對於上述功能有降低或是抑制的效果，進而證實分子模型之論述。
進而利用搜尋引擎以及大範圍分析法鑑定在乳癌細胞或是人類血清中被內生性4OHE2所修飾的蛋白。經過多層篩選條件之過濾後的胜肽再利用手動確認其正確性。在鑑定出的蛋白中，由糖尿病患者血清中發現的4OHE2修飾於白蛋白(HSA)之Lys598的位點，此段序列被挑選利用商業化的白蛋白做更進一步的確效。
位點專一性活性代謝物於蛋白上之轉譯後修飾之鑑定有助於因雌激素代謝所帶來的生理紊亂做為機制上或醫療上的目標研究。

Covalent protein modifications by estrogens and their active metabolites have been suspected to play important roles in the development and progression of various diseases. The lack of molecular evidences of the active species involved and sites of modification has however averted more intensive investigation. In this study, we reported the identification of site-specific protein modifications of the catechol estrogen, 4-hydroxyl 17β-estradiol (4-OHE2), at the protein level in-vitro and in-vivo by nano-liquid chromatography-tandem mass spectrometry (LC-MS2).
Using therapeutic insulin as a model, 4OHE2 was identified to spontaneously form single or multiple covalent bonds on lysine and disulfide linkage sites of the A and B chains of insulin by collision-induced dissociation (CID) and electron transfer dissociation (ETD) via bottom-up and semi-top down approach. Molecular modeling showed that 4OHE2 modifications on these identified sites caused significant variations on the exposure of insulin receptor binding domain, which may affect protein interactions. This was evidenced by functional assays which indicated reduced efficacies of insulin-induced glucose uptake and IRS signaling pathway as well as cell cycle progression of MCF-7 cells by 4OHE2-modified insulin.
Endogenous 4OHE2 modified proteins in breast cancer cells (MCF-7 and MDA-MB-231) and human bloods were also identified by large scale analyses via automatic database search using multiple criteria and further validated by manual interpretation. Among the identified proteins, human serum albumin (HSA) in the plasma of female patients who have diabetes mellitus was identified to be modified by 4OHE2 on its lysine site and was further validated by purified HSA protein.
Identification of site-specific protein post translational modifications by active estrogen metabolites reveals new directions for mechanistic or therapeutic target studies of biological disorders associated with estrogen metabolism.

中文摘要	 I-II
Abstract 	 III-IV
誌謝	 V
Table of Contents 	 VI-VIII
List of Figures and Tables 	 IX-X
Abbreviations 	 XI
	
Chapter1: Introductions
1.1 4-Hydroxy 17-ß Estradiol and Its Protein Adduct	1
1.2 Bottom-Up Proteomics by LC-MS/MS	4
1.2.1 Interpretation of MS2 Spectrum of CID and ETD Fragment Ions 	4
1.3automatic Database Search 	8
1.3.1 Scoring Scheme 	10
1.3.2 Automatic Error Tolerant Search 	12
1.3.3 Decoy Database Search 	14
1.4 Introduction of Insulin 	15
1.4.1 Signaling Transduction of Insulin 	16
1.4.2 Relationship between Insulin and Cell Proliferation 	18


Chapter 2: Experiment Procedure
2.1 Materials 	20
2.2 4OHE2 covalent-bound amino acid and protein 	21
2.3 Sample Preparation 	22
2.3.1 Human Blood Collection 	22
2.3.2 Cell Culture 	22
2.3.3 Protein Digestion 	23
2.3.4 Off-Line HILIC Fractionations 	24
2.4 LC-MS/MS Analysis 	25
2.5 Database Search 	26
2.6 Functional Assay 	27
2.6.1 Cell Culture 	27
2.6.2 2-NBDG Glucose Uptake Measurements 	27
2.6.3 Cell Signaling 	28
2.7 Molecular Modeling 	29

Chapter 3: Result
3.1 The mass shift resulted from 4OHE2 adduction 	30
3.2 Identification of specific binding sites of 4OHE2 on insulin 	32
3.3 Molecular modeling of 4OHE2-adducted insulin. 	38
3.4 Functional effect of 4OHE2-adducted insulin 	40
3.4.1 Glucose uptake 	40
3.4.2 Insulin triggering signaling transduction pathway 	42
3.4.3 Cell cycle 	45
3.5 Endogenous 4OHE2 modified proteins 	47
3.5.1 Initial search result by Mascot 	49
3.5.2 Further validation of human serum albumin 	51
Chapter 4: Conclusion	59
Reference	61
Appendix	67

List of Figures and Tables
Fig.1 Formation of estrogen metabolites and its DNA adducts	3
Fig.2 (A) b, y types of ions that generate from CID fragmentation. (B) c, z type of ions that generate from ETD fragmentation.	6
Fig.3 Neutral loss 	7
Fig.4 Workflow of automatic database search in large-scale proteomics 	9
Fig.5 The histogram of the score distribution with threshold p<0.05 and mass tolerance (A) ±0.1 Da (B) ± 1.0 Da (C) ± 2.5 Da 	11
Fig.6 The result format in error tolerance search	13
Fig.7 The insulin signaling network 	17
Fig.8 Insulin receptor binding sites on insulin 	17
Fig.9 Phases of the cell cycle 	19
Fig.10 Formation of 4OHE2-conjugated amino acids and disulfide linkage compound 	31
Fig.11 XIC for 4OHE2 adducted and normal insulin 	34
Fig.12 Time course study for 4OHE2 adducted insulin 	35
Fig.13 (A) Fragmentation in ETD, which preferentially cleavages on the disulfide linkage site. (B) Fragmentation in CID 	36
Fig.14  Molecular modeling of 4OHE2 modified on Cys7 of insulin ß-chain. 	39
Fig.15  Molecular modeling of 4OHE2 modified on Cys7 and His10 of insulin ß-chain 	39
Fig.16 Glucose uptake of normal insulin and the mixture 	41
Fig.17 Cell signaling via phosphorylation of IRS in (A) L6 cells (B) MCF-7 cells 	43
Fig.18 G0/G1 phase of MCF-7 cell, treating with 4OHE2-adduted insulin 	46
Fig.19 S+G2/M phase of MCF-7 cell, treating with 4OHE2-adduted insulin 	46
Fig.20 Workflow and filtering criteria for Mascot search 	48
Fig.21 XIC for unmodified and 4OHE2-modified peptide in (A) pure HSA (B) patient’s blood (C) pure HSA incubated with 4OHE2, (D) spiked pure HSA+4OHE2 into patient’s blood  	53
Fig.22 MS2 for (A) In-vivo 4OHE2-modified peptide; (B) In-vitro 4OHE2-modified peptide; (C) a synthetic peptide (D) In-vivo unmodified peptide  	54
Fig.23 Validation of 4OHE2-modified peptide 	58

Table. 1 Common database search engine and websites 	9
Table.2 Identification of in-vitro 4OHE2 modification sites on insulin 	37
Table.3 Possible in-vivo 4OHE2-modified peptides from the diabetes patient’s blood 	50


                                    

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