利用具有親合性標籤的穩定同位素試劑針對蛋白質磷酸化反應程度的定量方法

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姓名

黃思喬(Szu-Chiao Huang) 查詢紙本館藏

畢業系所

化學學系

論文名稱

利用具有親合性標籤的穩定同位素試劑針對蛋白質磷酸化反應程度的定量方法
(Stable Isotope Phosphoprotein AffinityTagging (SIPAT) for Quantitation of ProteinPhosphorylation Degree)

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摘要(中)

在serine、threonine和tyrosine胺基酸上的蛋白質磷酸化，是調節蛋白質功能及表現很重要的因素，細胞在生物體中的增殖(proliferation)、分化(differentiation)或受到環境影響而產生的變化，都是受到蛋白質磷酸化作用的調控。因此，為了進一步以蛋白質的觀點瞭解複雜的生物體，發展一套靈敏可大規模分析蛋白質磷酸化的方法是必要的。在此論文中，我們發展一套以穩定同位素具親和性的標籤(Stable Isotope Phosphoprotein Affinity Tagging, SIPAT)的方法，將可針對蛋白質磷酸化程度作定量分析。
這個方法主要是針對磷酸化的serine和threonine來進行同位素的標定。我們在兩個不同狀態(控制組及實驗組)的磷酸化蛋白質的serine 以及threonine胺基酸上做衍生化，利用氫氧離子進行?-elimination將磷酸根從serine 以及threonine上移除，再以1,2-ethanedithiol (EDT) 進行Michel addition轉換成帶有硫醇的官能機(-SH)，接著，在衍生化後的兩個不同狀態的蛋白質上分別接上12C/13C cleavable isotope-coded affinity tag （cICAT）試劑。此時，樣品中裡的cysteine胺基酸也會同時接上cICAT試劑。將兩份樣品混合，經過酵素消化，樣品內就含有這些標定過的磷酸胜肽(簡稱為SIPAT-L/SIPAT-H)，以及含有cysteine並被標定過的胜肽(簡稱為cICAT-L/cICAT-H)，利用SCX以及immobilized avidin 將這些胜肽純化後，可以降低LC-MS/MS分析樣品的複雜程度。而磷酸化程度的改變，可以利用磷酸化蛋白質中，計算其同位素標定後磷酸化胜肽以及cysteine胜肽的離子強度得到。
在這篇論文中，主要分成三個部分來進行：(1) 第一部份工作是將SIPAT反應最佳化。在此部分是使用較為便宜的iodoacetylamine (IAM) 和EZ-Link® PEO-Iodoacetyl biotin來進行反應條件的最佳化，因為他們的反應基群(iodoacetyl-)都類似cICAT試劑。(2) 第二部分則是對磷酸化蛋白質進行鑑定及定量。在此部分中，我們使用不同濃度比例的蛋白質樣品，來驗證SIPAT方法的可行性，探討其對於鑑定磷酸化蛋白質以及對蛋白質磷酸化定量的結果。(3) 第三部分，則是針對不同磷酸化程度以及不同蛋白質含量的樣品做磷酸化程度的定量分析。在這部分，我們使用蛋白質去磷酸脢（alkaline protein phosphatase 1, PP1）製備含有不同磷酸化程度的樣品，以驗證SIPAT方法定量分析蛋白質磷酸化反應的可行性。
實驗結果顯示出這個SIPAT方法，不只可以鑑定出磷酸化的胜肽，更重要的是，可以定量分析少量磷酸化胜肽的磷酸化程度，而且這個方法也可以同時針對蛋白質作定量分析，將來可以應用在蛋白質體學中磷酸化蛋白質的大規模鑑定和定量分析。

摘要(英)

Phosphorylation on serine, threonine and tyrosine residues is an extremely important modulator of protein function. Cellular processes such as proliferation, differentiation, and adaptation to environmental changes are regulated by protein phosphorylation. Development of sensitive and comprehensive analytical methods for determination of protein phosphorylation is therefore a necessity in the pursuit of a detailed molecular view of complex biological processes. In this thesis, we developed a stable isotope phosphoprotein affinity tagging (SIPAT) strategy for quantitation of protein phosphorylation degree.
The strategy is based on the specific labeling on the phosphoserine and phosphothreonine. The proof-of-concept experiment was performed on casein mixture with various protein concentrations and different degree of phosphorylation, in which the phosphoseryl and phosphothreonyl residues were derivatized by hydroxide ion-mediated β-elimination followed by the Michael addition of 1,2-ethanedithiol (EDT). Then proteins containing the EDT moiety from two set of mixture were captured and separately labeled with cleavable isotope-coded affinity tag (abbreviated as cICAT) reagent. Meanwhile, cysteine-containing proteins from two sets of mixture are simultaneously labeled with cICAT reagent. After proteolytic digestion, the labeled modified phosphopeptides and cysteine-containing peptides can be isolated using SCX and immobilized avidin cartridges, which greatly reduces the sample complexity for subsequent LC-MS/MS analysis. The change in phosphorylation degree can be measured by the change in the ion intensity of phosphopeptides pairs and non-phosphorylated cysteine-containing peptides from the same protein.
The main theme of the thesis will have three parts: (1) Optimization of the reaction condition of SIPAT. We will use iodoacetylamine (IAM) and EZ-Link® PEO-Iodoacetyl biotin to mimic the cICAT reagent and to optimize the reaction condition. (2) Identification and quantification of protein phosphorylation. We will perform the SIPAT strategy on the sample of different protein ratio to demonstrate the performance of the SIPAT in the identification and quantification of protein phosphorylation. (3) Quantitation of protein phosphorylation degree. We will prepare sample of different protein expression levels as well as in phosphorylation degree to demonstrate the SIPAT for quantitation of protein phosphorylation degree.
The results illustrated the efficiency of the SIPAT strategy to not only purify the phosphopeptides, but also, more importantly, to permit the quantitation for phosphorylation degree of low-abundance phosphopeptides. This method is also capable of protein quantitation on a global basis. Overall, the results exemplify the application of the SIPAT approach and demonstrate its potential utility for proteome-wide phosphoprotein identification and quantitation.

關鍵字(中)

★ 定量
★ 磷酸化

關鍵字(英)

★ phosphorylated protein
★ quantitation

論文目次

中文摘要…………………………………………………………............ I
ABSTRACT……….………………………………...…...……............... III
謝誌………………...…………………………………………...…….…. V
CONTENTS…………………………………………………….............. VI
LIST OF FIGURES………………………………….…………………... IX
LIST OF TABLES……………………...…………………………...….. XIV
ABBERVIATIONS…………………………………………………....... XV
CHAPTER 1 INTRODUCTION……………….……………....…........ 1
1.1 Significance of Protein Phosphorylation…………………...………... 1
1.2 Complexity of Protein Phosphorylation……………...………...……. 2
1.3 Traditional Methods for Analysis of Protein Phosphorylation…...…….. 3
1.4 Mass Spectrometry-Based Strategies for Qualitative and Quantitation
Analysis of Protein Phosphorylation..……….…………...……..…... 3
1.4.1 Determination of Protein Phosphorylation Sites……….……….. 4
1.4.2 Protein Quantitation by Cleavable Isotope-Coded Affinity Tag....... 6
1.4.3 Quantitation of Protein Phosphorylation………….....….……... 7
1.5 Limitation of Current Methods and the Purpose of this Study…............... 9
CHAPTER 2 EXPERIMENTAL METHODS…………....................... 10
2.1 Materials........................................................................................................ 11
2.2 Instrumentation…………………………….…….................…...............…. 12
2.2.1 MALDI-TOF…………………….…………..............…..............… 12
2.2.2 LC-MS/MS……………….……………….…………….................. 12
2.3 Protein Quantitation…………….............……………………………….... 13
2.3.1 BCATM Protein Assay Kit………………..………………….……... 13
2.3.2 Coomassie (Bradford) Protein Assay Kit………..............…....……. 14
2.3.3 SDS-PAGE…………………………......…………………............... 14
2.4 Peptide Desalting Method………….………………………..……............... 15
2.4.1 Zip-Tip……………………………….…………..………………… 15
2.4.2 Acetone Precipitation………………………………....…................. 15
2.5 Protein Dephosphorylation…………………………………..……..…..….. 16
2.6 Labeling Reaction………………………………………………...…...….... 16
2.6.1 ?-Elimination and Michel Addition………………………..….….... 16
2.6.2 Reduction/Acrylation………………………………………..……... 17
2.6.2.1 Iodoacetamide………………….………..………………..... 17
2.6.2.2 EZ-Link® PEO-Iodoacetyl Biotin……………..………….... 18
2.6.2.3 Cleavable ICAT Reagent…………………..………….……. 18
2.7 Protein Digestion………….………………………..............……….……... 19
2.8 Peptide Purification and Acidic Cleavage...................................................... 19
2.8.1 Cation Ion-exchange Cartridge…..................................………........ 20
2.8.2 Avidin Cartridge……………………….…………….……….…….. 21
2.8.3 Acidic Cleavage………………………..…….……………….……. 22
2.9 Capillary LC-MS Analysis and Data Analysis………………..…………..... 23
2.9.1 Capillary LC-MS Analysis…………………...………………..…… 23
2.9.2 Protein Identification......................................................................... 23
2.9.3 Protein Quantification…….......................................…………...….. 24
CHAPTER 3 RESULTS AND DISUSSION…………………………... 25
3.1 SIPAT Strategy for the Determination of Protein Phosphorylation Degree.. 25
3.2 Optimization of the ?-Elimination and Michael Addition…………...….. 27
3.2.1 Identification of Phosphorylation Sites in Casein Mixture............. 27
3.2.2 Removal of Excess EDT………………..……….…………... 29
3.2.3 Optimization of labeling with iodoacetamide (IAM)………..……. 29
3.2.4 Reaction Temperature…………………..…….……………... 31
3.2.5 Reaction Time…………………….….………….…………. 31
3.2.6 Denaturation of Proteins…………………….…….………… 32
3.2.7 Optimization of Labeling with EZ-Link® PEO-Iodoacetyl Biotin.... 33
3.3 cICAT Labeling Test with Laminin………….………………….….... 33
3.4 Performance of SIPAT Strategy……………….……….…….…….... 34
3.5 Relative Quantitation of Protein Phosphorylation…….………….......... 36
3.6 Protein De-Phosphorylation - ?S2-casein…...….................................... 37
3.7 Quantitation for the Level of Phosphorylation with Different Protein
Concentration………………………...…………..…...…..………. 38
CHAPTER 4 CONCLUSION…………….…………..…...……….….. 40
REFERENCES......................................................................................... 41
FIGURES.................................................................................................. 45
TABLES………………............…………….……...…....………….…… 86
APPENDIXS…………………………………...……....……………….. 90

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指導教授

陳玉如、丁望賢
(Yu-Ju Chen、Wang-Hsien Ding)

審核日期

2005-7-18

推文