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系統識別號 U0026-2401201116545200
論文名稱(中文) 以定量磷酸蛋白質體學分析N端的磷酸化位置及雌激素所引起之轉錄調節
論文名稱(英文) Quantitative Phosphoproteomics for the Analysis of N-terminal Phosphorylation Sites and Estrogen-Induced Transcriptional Regulation
校院名稱 成功大學
系所名稱(中) 化學系碩博士班
系所名稱(英) Department of Chemistry
學年度 99
學期 1
出版年 100
研究生(中文) 吳晉任
研究生(英文) Chin-Jen Wu
學號 L3893104
學位類別 博士
語文別 英文
論文頁數 121頁
口試委員 指導教授-陳淑慧
口試委員-戴榮湘
口試委員-陳彥文
口試委員-吳慧芬
口試委員-洪建中
中文關鍵字 蛋白質磷酸化  定量分析  磷酸化位置  穩定同位素二甲基標定  磷酸化蛋白質體  親水性層析  固定化金屬親和性層析  質譜  乳癌細胞 
英文關鍵字 Protein phosphorylation  quantitative ananlysis  phosphorylation site  stable isotope dimethyl labeling  phosphoproteomics  HILIC  IMAC  mass spectrometry  MCF-7 cell 
學科別分類
中文摘要 蛋白質的磷酸化及去磷酸化扮演了一個很關鍵的角色,它可以調節生物體中的許多過程,包含了訊號的傳遞、細胞的增生及基因的表現等。因此,全面性的分析蛋白質的磷酸化及其磷酸化量的改變進而得知訊號傳遞的路逕並了解疾病的產生機制就變得相當重要。最近幾年,對於蛋白質磷酸化的分析及相對定量而言,質譜已經成為一項很有價值的分析工具。不過利用質譜來分析蛋白質的磷酸化常常會遭遇失敗,這是因為具有調節功能的磷酸化蛋白質僅存在非常少的量。此外,相較於其他非磷酸化的胜肽,磷酸化胜肽在質譜的正電偵測模式下,通常會有較差的離子化效率,這會使磷酸化胜肽的訊號受到抑制。為了解決這些問題,固定化金屬親和性層析法(Immobilized metal affinity chromatography, IMAC)已經廣泛地被利用,它可以在質譜偵測前,先將磷酸化胜肽從複雜的胜肽混合物中給純化出來。但是一些含有酸性胺基酸(如:麩氨酸、天門冬氨酸、組氨酸及半胱氨酸)的非磷酸化胜肽經常會跟固定化金屬親和性層析中的金屬離子產生非專一性的吸附,因此降低了純化磷酸化胜肽的效率。除此之外,固定化金屬親和性層析法所純化出的磷酸化胜肽通常很複雜,這些磷酸化胜肽無法在一次的質譜偵測中,完全地被分析。所以在此研究中,一個改良過的固定化金屬親和性層析法被發展出來解決上述所談到的缺點,它可以結合親水性的層析管柱及二甲基標定,有效地對乳癌細胞中的磷酸化蛋白質做定性及定量的分析。
第一個研究主題 (第二章): 在碰撞誘導解離作用後,二甲基標定的胜肽會產生訊號很強的a1離子,這個獨特的a1離子可以用來幫忙確認胜肽N端的胺基酸。在此研究中,我們證明使用穩定的二甲基同位素標定能更進一步的利用a1離子來幫忙鑑定靠近胜肽N端的磷酸化位置。我們發現二甲基標定的胜肽如果在N端有絲氨酸或蘇氨酸被磷酸化的話,a1離子的訊號將會被抑制。但是如果是在胜肽中端的絲氨酸和蘇氨酸或是N端的酪氨酸被磷酸化的話,增強的a1離子訊號仍會產生。除此之外,N端有絲氨酸或蘇氨酸被磷酸化的胜肽,在二甲基標定及碰撞誘導解離後,於二次質譜圖中也會產生許多去掉磷酸根的b離子和一根很強且去掉磷酸根的分子離子。由上述的現象我們可以推斷,二甲基同位素標定會增加胜肽N端的鹼度,而使N端有絲氨酸或蘇氨酸被磷酸化的胜肽加速去掉磷酸根,去掉磷酸根後將會在N端胺基酸的α碳上形成不飽和的共價鍵,進而抑制a1離子的產生。藉由這個方法的使用,我們找出A431細胞中3個被質譜及資料庫所錯判的磷酸化位置,這3個磷酸化位置的其中2個,在以往也被其他期刊所報導過。在此,除了利用二甲基標定做磷酸化位置的確認外,我們也用它來定量這些磷酸化位置,於EGF刺激後所產生的變化。最後,我們也利用這個方法於懷孕的老鼠子宮中找到一個新的磷酸化位置,這個磷酸化位置位於SVH這個蛋白質上,為43號的磷酸化絲氨酸。這個磷酸化位置在經過cAMP刺激下會產生輕微的下降。
第二個研究主題 (第三章):雌二醇會引起蛋白激酶訊息放大效應,導致雌激素接受器及其他功能性蛋白質產生磷酸化,進而調節轉錄活性。為了解哪些磷酸化蛋白質是受雌二醇所調控,我們運用定量磷酸化蛋白質體學去研究乳癌細胞在雌二醇刺激下,所有磷酸化蛋白質其磷酸化量的改變並鑑定磷酸化蛋白質的身份。在蛋白質體分析的部份,我們使用二甲基同位素標定結合固定化金屬親和性─親水性層析的分析方法,針對含有1毫克蛋白質混合物的乳癌細胞萃取液,在酵素消化、二甲基標定及磷酸化胜肽純化後做分段分離,最後再將不同段的分離樣品以質譜做個別的偵測。在此我們鑑定到1338個蛋白質當中包含了2857個磷酸化位置,而且大部份被鑑定到的磷酸化胜肽僅出現在親水性層析的特定一段分離樣品中。除了雌激素接受器118號的磷酸化絲氨酸在雌二醇刺激30分後有明顯量的改變外,我們也另外找到428個磷酸化位置,在此狀態下也產生有意義地變化,而這些磷酸化位置中有112個磷酸化位置是新的且未被報導過的。有趣的是,這些有變化的磷酸化位置,有絕大部份都是絲裂原激活蛋白激酶(ERK1/2)的受質。這些受質在雌二醇刺激下,磷酸化不僅產生了上調節,有些也出現了下調節的情況。除此之外,在我們蛋白質體的數據中也發現,在雌二醇刺激30分後,絲裂原激活蛋白激酶(ERK1)本身只有202號的磷酸化蘇氨酸或是204號的磷酸化酪氨酸產生上調節,並沒有看到202號的磷酸化蘇氨酸和204號的磷酸化酪氨酸同時存在且形成上調節。由這些現象可以看出在這30分的刺激時間下,絲裂原激活蛋白激酶的訊號傳遞將會產生一個去活化的回饋機制,以用來平衡刺激早期絲裂原激活蛋白激酶產生磷酸化而形成的訊號傳遞。有變化的磷酸化位置除了大部份是絲裂原激活蛋白激酶的受質外,有些也是環核苷酸活化激酶 (PKA)及酪蛋白激酶 (CKII)的受質,但是這些受質在雌二醇刺激30分鐘下,大部份只產生上調節的情況。其中一個例子就是HSP90β其226號的磷酸化絲氨酸,它為酪蛋白激酶的受質,經由西方墨點轉漬法的再次確認,顯現出透過雌二醇的刺激,可以在不改變蛋白質表現量的情況下,增加磷酸化的表現達2至3倍。除了226號的磷酸化絲氨酸,雌二醇也會影響255號的磷酸化絲氨酸,使它產生些微的上調節。和HSP90β同樣的情況,HSP90α在受雌二醇的刺激下,其353號的磷酸化絲氨酸也有上調節的狀態產生,這個磷酸化位置也是一個酪蛋白激酶的受質。綜合上述所觀察到的現象,顯示出雌二醇會藉由調控某些調節蛋白質上的磷酸化位置,來影響這些蛋白質的穩定性、功能及其活性,並且進一步地透過這些蛋白質對基因的表現進行控制。由這些數據也可應證,我們所發展出來“定量及多維分離的磷酸化蛋白質體策略”,可以在使用很少細胞量的條件底下,對雌二醇所引起的磷酸化改變,做一個有系統且有效地分析。
英文摘要 Phosphorylation and dephosphorylation of proteins play a critical role in modulating a variety of biological processes, including signal transduction, cell proliferation and gene expression. Therefore, global analysis of protein phosphorylation and its variation are important to the comprehending of signal transduction pathways and understanding mechanisms of diseases. In recent years, mass spectrometry (MS) has been a valuable tool for characterization and relative quantitation of protein phosphorylation. However, mass spectrometric analysis of protein phosphorylation is often not successful due to certain limitations. For instance, many regulatory phosphoproteins are expressed in low stoichiometry. In addition, phosphopeptides exhibit lower ionization efficiency than most non-phosphorylated peptides in the positive mode. Therefore, the detection of the phosphopeptides will be influenced. To solve these problems, immobilized metal affinity chromatography (IMAC) has been widely utilized to enrich phosphopeptides in mixtures prior to MS but non-phosphorylated peptides containing acidic residues such glutamic acid, aspartic acid, histidine and cysteine tend to nonspecifically bind to the metal resin. On the other hand, the IMAC-enriched peptides population is also too complex to fully analyze by a single LC-MS/MS run. Thus, in my study, an improved IMAC base strategy coupling with hydrophilic interaction chromatography (HILIC) and dimethyl labeling are developed to overcome the above drawbacks. This strategy can efficiently qualitative and quantitative protein phosphorylation in MCF-cells.
In the first topic (Chapter 2): After collision-induced dissociation (CID), the enhanced a1 signal of dimethylated peptides served as a unique mass tag for unequivocal identification of the N-terminal amino acids. In this study, we demonstrate that the a1 ion could further assist in mapping the precise phosphorylation site near the N terminal region and allow the determination of the exact site and level of phosphorylation in one step by stable isotope dimethyl labeling. We show that the a1 ion signal was suppressed for dimethylated peptides with a phosphorylation site at the N-terminus Ser/Thr residue (N-p*Ser/Thr) but was still enhanced for N-terminus Tyr residue (N-p*Tyr) or internal Ser/Thr residues (-p*Ser/Thr). Based on the dominant de-phosphorylated molecular ions and b-H3PO4 ions for N-p*Ser/Thr, we propose that dimethyl labeling increases the basicity of the N-terminus and accelerates the de-phosphorylation for N-p*Ser/Thr precursors, which, however, suppresses the a1 ion enhancement due to the resulting unsaturated covalent bond on C of the N terminus amino acid. Using this method, we excluded three Ser/Thr phosphorylation sites in A431 cells, two of which, however, were previously reported to be phosphorylation sites; we confirmed 3 known phosphorylation sites in A431 cells and quantified their ratios upon EGF treatment. Notably, we identified a novel phosphorylation site on Ser43 residue at N-terminus of the tryptic peptide derived from SVH protein in pregnant rat uteri. SVH protein has not been reported or implied with any phosphorylation event, and our data show that the Ser43 of SVH is an intrinsic phosphorylation site in pregnant rat uteri and that its phosphorylation level was slightly decreased upon c-AMP treatment.
In the second topic (Chapter 3): 17-Estradiol (E2) regulates transcriptional activity partly by inducing protein-kinase cascades, leading to the phosphorylation of estrogen receptors (ERs) and other functional proteins. Many of these phosphorylation events, however, are also modulated by growth factors. To gain an integrated insight into E2-modulated protein phosphorylation, we applied quantitative phosphoproteomics to investigate global changes in protein phosphorylation induced by E2 in MCF-7 cells. Proteomic analyses, which were performed using stable isotope dimethyl labeling coupled with IMAC-HILIC fractionation and nanoLC/MS/MS along with 1 mg of total cellular protein, identified and quantified 2857 unique phosphorylation sites associated with 1338 phosphoproteins. Most phosphopeptides were identified from a single HILIC fraction without any isotopic effects. In addition to S118 of estrogen receptor  (ER), a 30-min treatment with E2 significantly altered the status of 428 phosphorylation sites, including 112 novel phosphorylation sites. Interestingly, the substrate motifs for ERK1/2 were largely enriched not only in the up-regulated but also in the down-regulated phosphorylation sites. The proteomic data revealed an increase in the phosphorylation on either the T202 or Y204 sites of ERK1 after E2 treatment, while dual phosphorylation on both sites were not detected after a 30-min treatment. These observations indicated that a feeback de-activation loop of MAPK signaling to balance the phosphorylation was achieved rapidly during a 30-min treatment with E2. The PKA and CKII substrate motifs, however, were majorly enriched among the up-regulated phosphorylation sites. Western blot analysis confirmed that E2 increased the phosphorylation level of S226-HSP90, a CKII motif, by a factor of 2- to 3-fold without changing the total protein expression level. E2 also up-regulated another phosphorylation site (S255) of HSP90 and the S353 phosphorylation site, a CKII motif, of HSP90. These results indicated that E2 may modulate gene transcription by affecting the stability, function, and activity of many regulators through a phosphorylation-mediated chaperoning process. This study, using a quantitative, multi-dimensional phosphoproteomic approach that required a relatively low amount of cells, provides new insights into the diversity, variability, and dynamic nature of the protein phosphorylation/dephosphorylation elicited by E2.
論文目次 Abstract I
中文摘要 IV
誌謝 VI
Table of Contents VII
List of Tables IX
List of Figures X
Abbreviation XIII
Text 1
自述 120

Chapter 1 Introduction 1
1.1 Protein phosphorylation and phosphoproteomics 2
1.2 Liquid Chromatography/Mass spectrometry 2
1.3 Difficulty for phosphopeptide identification 3
1.4 Phosphoproteome enrichment 3
1.5 MS-based approach for phosphopeptide analysis 6
1.6 Quantitative phosphoproteomics 8
1.7 Estrogen actions and pathways 11
1.8 Reference cited in Chapter 1 13
1.9 Figures 24

Chapter 2 Mapping N-Terminus Phosphorylation Sites and Quantitation by Stable Isotope Dimethyl Labeling 40
Introduction 41
Experimental section 42
Result and discussion 45
Conclusion 50
Reference cited in Chapter 2 51
Figures and tables 54

Chapter 3 Quantitative Phosphoproteomics Studies Using Stable Isotope Dimethyl Labeling Coupled with IMAC-HILIC-nanoLC/MS/MS for Estrogen-Induced Transcriptional Regulation 68
Introduction 69
Experimental section 70
Result 73
Discussion 77
Reference cited in Chapter 3 81
Figures and tables 86

Chapter 4 Conclusion and perspective 117
Overview of this study 118
Perspective 119
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