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研究生:洪若維
研究生(外文):Hong, Ruo-Wei
論文名稱:開發結合細胞分群與化學分群技術之酪胺酸磷酸化蛋白質體研究
論文名稱(外文):An Integrated Subcellular and Chemical Fractionation Strategy toward In-depth Analyses of Tyrosine Phosphoproteome
指導教授:陳玉如陳玉如引用關係
指導教授(外文):Chen, Yu-Ju
口試委員:陳玉如陳皓君王少君
口試委員(外文):Chen, Yu-JuChen, Hauh-Jyun CandyWang, Shau-Chun
口試日期:2014-07-08
學位類別:碩士
校院名稱:國立中正大學
系所名稱:化學暨生物化學研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:英文
論文頁數:67
中文關鍵詞:酪胺酸磷酸化蛋白質體細胞分群連續的固定化金屬親和層析逆向層析分群技術
外文關鍵詞:Tyrosine phosphoproteomeSubcellular fractionationGa3+-Fe3+-IMAC enrichmentReverse phase fractionation
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磷酸化蛋白為非常重要的後轉譯修飾,調控細胞訊息傳遞的過程,在蘇胺酸磷酸化蛋白(phosphothreonine-modifiied protein)、絲胺酸磷酸化蛋白(phosphoserine-modifiied protein)和酪胺酸磷酸化蛋白(phosphotyrosine-modifiied protein) 三種磷酸化修飾中,酪胺酸磷酸化蛋白與許多疾病有著密切的關連,並且亦是許多疾病藥物治療目標。近年來,隨著質譜技術和純化技術的發展,酪胺酸磷酸化蛋白的萃取與鑑定有十足的進展,但由於酪胺酸磷酸化蛋白含量相當的低(1.8%),造成酪胺酸磷酸化蛋白在質譜的偵測困難,其訊號往往被其他高含量的磷酸化蛋白所抑制,造成質譜偵測上的困難。為了提高對於酪胺酸磷酸化蛋白的偵測,本論文開發出一項利用結合細胞分群(subcellular fractionation)和化學分群的技術(Ga3+- Fe3+-IMAC與reverse phase fractionation method),去降低磷酸化樣品當中的複雜度,進而提升酪胺酸磷酸化蛋白的偵測。
於第一部分的論文中,我們發展連續的固定化金屬親和層析(Ga3+- Fe3+-IMAC)方法,經由鎵 (Ga3+)和鐵 (Fe3+)兩項金屬離子對於磷酸化胜肽親和能力的差異,將相對含有較多負價鍵的磷酸化胜肽於第一維固定化金屬親和層析(1st Ga3+-IMAC)與親和能力較弱的鎵離子反應得到純化分析,接著於第二維固定化金屬親和層析(2nd Fe3+-IMAC)便可經由親和能力較為強的鐵離子將未能於第一維當中得到分析的磷酸化胜肽再做純化分析,進一步提升對於酪胺酸磷酸化胜肽(phosphotyrosine-modifiied peptide)純化上的效率。相較於利用單一鐵離子的固定化金屬親和層析 (Fe3+-IMAC)與連續的固定化金屬親和層析(Ga3+- Fe3+-IMAC)在Raji B 細胞當中的測試,可將磷酸化胜肽的偵測,由4553條磷酸化胜肽與其中421條酪胺酸磷酸化胜肽,提升至5288條磷酸化胜肽與599條酪胺酸磷酸化胜肽。如果再結合細胞分群(subcellular- Ga3+- Fe3+-IMAC)將Raji B 細胞分離為細胞核、細胞膜與細胞質,進而降低樣品的複雜度,則可將磷酸化胜肽的偵測由5288條磷酸化胜肽與其中599條酪胺酸磷酸化胜肽(11%),提升至5205條磷酸化胜肽與1388條酪胺酸磷酸化胜肽(26%),其酪胺酸磷酸化胜肽在磷酸化胜肽當中的比例便經由細胞分群的方法得到提升,並且也在Ga3+- Fe3+-IMAC的連續萃取過程中,分析出在1388條酪胺酸磷酸化胜肽當中,其中較為酸性的酪胺酸磷酸化胜肽(n=1088, 78%)大部分在第一維固定化金屬親和層析被萃取出,另外也在Raji B 細胞中,經由結合細胞分群(subcellular fractionation)和連續的固定化金屬親和層析(Ga3+- Fe3+-IMAC),在偵測到的1388條酪胺酸磷酸化胜肽當中,偵測到274條在過去文獻當中還未偵測到的酪胺酸磷酸化胜肽,並且亦有效的提升對於BCR訊息傳遞途徑的偵測範圍。
為了更進一步的做更為全面性並且靈敏的磷酸化蛋白質體學的偵測,在論文第二部分開發出一項微量並且高解析度的逆向層析的分群技術(reverse phase stage tip method)。在HeLa 細胞測試結果中,相較於單一鐵離子的固定化金屬親和層析(Fe3+-IMAC)方法,結合逆向層析的分群技術(Fe3+-IMAC-RP)可有效的將磷酸化胜肽的偵測由5205提升至7962條磷酸化胜肽,其中約15%為酪胺酸磷酸化胜肽。另外,我們亦去比較在細胞分群(subcellular fractionation)方法於兩項分群實驗(Fe3+-IMAC-RP與subcellular- Ga3+- Fe3+-IMAC)在HeLa 細胞偵測上的差異,此兩方法在磷酸化胜肽和酪胺酸磷酸化胜肽皆僅有20%的重疊偵測機率。
綜合本論文結果,可得知經由本三項技術(1)細胞分群(subcellular fractionation)、(2)連續性固定化金屬親和層析(Ga3+- Fe3+-IMAC),以及(3)高解析度逆向層析的分群技術(RP Stage tip fractionation method) ,可有效降低樣品的複雜度,進而提升磷酸化胜肽和酪胺酸磷酸化胜肽的萃取與鑑定,未來可經由三項分餾技術的結合,應用於疾病上做全面性的酪胺酸磷酸化胜肽的偵測與探索。

Protein phosphorylation is an important post-translational modification since it plays a key process in many cellular processes. Although MS technology has been commonly used for phosphorylation site identification in a complex phosphoproteome, achieving comprehensive coverage of the human phosphorylation network remains a challenge. Among the serine, threonine and tyrosine phosphorylation in human, tyrosine phosphorylation is most well-known therapeutic target, yet its infrequent occurrence (1.8%) and low substiochmetric levels of modified form low abundance cause difficulty for the currently under-represented tyrosine phosphorproteome. To address this challenge, in this thesis, we have developed an integrated subcellular and chemical fractionation strategy to enhance the number of the phosphotyrosine component of the phosphoproteome.
Taking advantage of the distinct binding affinities of Ga3+ and Fe3+ for phosphopeptides, in the first part, we designed a metal-directed immobilized metal ion affinity chromatography (IMAC) for the sequential enrichment of phosphopeptides. To further reduce the sample complexity, subcellular fractionation was performed on cell lysate to separate into membrane, nuclear and cytosol components. 5288 phosphopeptides and 599 phosphotyrosine peptides from the model cell line (Raji B) were identified using sequential Ga3+-Fe3+-IMAC, which is higher than identifications obtained using only a single Fe3+-IMAC enrichment, with only 4553 phosphopeptides and 421 phosphotyrosine peptides identified. This corresponds to an increase of 1.4 folds in the identification of tyrosine phosphopeptides. With incorporation of subcellular fractionation before Ga3+-Fe3+-IMAC phosphopeptide enrichment, we identified 5205 phosphopeptides and 1388 phosphotyrosine peptides, including 274 previously unidentified tyrosine phosphorylation sites. We also found that 1088 phosphotyrosine peptides (78%) can be effectively purified in 1st Ga3+-IMAC fraction. The increase in the numbers of identified phosphotyrosine peptides is due to the increase in the ratio of phosphotyrosine peptides obtained from all the detected phosphopeptides. 26% phosphotyrosine peptides was obtained with subcellular-Ga3+-Fe3+-IMAC method while only 11% with Ga3+-Fe3+-IMAC method. In addition, subcellular-Ga3+-Fe3+-IMAC method also effectively increased the BCR pathway (B cell receptor signaling pathway) identification coverage in Raji B cell.
For deep phosphoproteome analysis with high sensitivity, on the second part of thesis, a small-scale high-pH RP (reverse phase) stage tip was developed for further phosphopeptides fractionation after IMAC enrichment. The number of phosphopeptides can be increased from 5205 to 7962 (15 % tyrosine sites) in 400 ug HeLa cell lysate. Comparing the two integrated fractionation approaches, Subcellular-Ga3+-Fe3+-IMAC and Fe3+-IMAC-RP from HeLa cell line, more tyrosine phosphopeptide can be detected by Fe3+-IMAC-RP (n=1225) compared to Subcellular-Ga3+-Fe3+-IMAC (n=545) and only 20% overlap of identified tyrosine phosphopeptides were observed between these two approaches. We demonstrated that this strategy can be applied for more comprehensive characterization of the tyrosine phosphoproteome.

Table of Contents
致謝.....................................................................................i
Abstract................................................................................ii
Table of Contents.......................................................................vi
List of Figures.........................................................................ix
List of Tables..........................................................................xi

Chapter 1 Introduction
1-1 Significance of phosphoproteome......................................................1
1-1.1 Protein phosphorylation of cellular signaling......................................1
1-1.2 The importance of tyrosine phosphorylation.........................................2
1-2 Current methods and challenges for tyrosine phosphoproteomics analysis...............3
1-2.1 Phosphopeptide enrichment method in phosphoproteome................................3
1-2.2 Sample fractionation strategy for large-scale proteomics...........................4
1-3 Current techniques for phosphotyrosine proteome characterization.....................6
1-3.1 Current fractionation strategy for phosphotyrosine peptide purification............7
1-3.2 Antibody-based strategy for tyrosine phosphoproteomics analysis....................8
1-4 Objective of this study..............................................................9

Chapter 2 Materials and Methods
2-1 Material.............................................................................10
2-2 Method...............................................................................11
2-2.1 Cell culture and sample preparation................................................11
2-2.2 Subcellular fractionation method...................................................11
2-2.3 Gel-assisted digestion.............................................................12
2-2.4 In solution digestion for cytosolic proteins.......................................12
2-2.5 IMAC procedure.....................................................................13
2-2-6 High-pH reverse phase stage tip method.............................................14
2-2.7 MALDI-TOF MS analysis..............................................................14
2-2.8 LC-MS/MS analysis..................................................................15
2-2.9 Database searches..................................................................16
2-2.10 Motif analysis....................................................................16

Chapter 3 Results and Discussion
3-1 Experimental design..................................................................17
3-2 Subcellular fractionation can increase the identification of low abundant tyrosine
phosphoproteins......................................................................17
3-3 Incorporation of sequential phosphopeptides enrichment by Ga3+-Fe3+-IMAC.............18
3-3.1 The principle of Ga3+-Fe3+-IMAC....................................................19
3-3.2 Phosphopeptide enrichment result by Ga3+-Fe3+-IMAC.................................19
3-3.3 Number of tyrosine phosphorylation sites can be increased by Ga3+-Fe3+ IMAC........20
3-3.4 Enhanced BCR pathway mapping in Raji B cell by integration of subcellular
fractionation and Ga3+-Fe3+-IMAC...................................................21
3-4 Stage tip based reverse phase chromatography for identification of tyrosine
phosphoproteome......................................................................21
3-4.1 Comparison of reverse phase chromatography (RP) and strong anion exchange
chromatography (SAX) stage tip fractionation method................................22
3-4.2 Effect of particle size in the separation efficiency of RP stage tip method for
fractionation of HeLa membrane peptides............................................23
3-4.3 Evaluation of the separation efficiency of RP stage tip method for fractionating
of HeLa phosphoproteome............................................................24
3-4.4 Comparison of coverage of phosphopeptides from HeLa cell...........................26
Chapter 4 Conclusion.....................................................................28
Reference................................................................................29
Figures..................................................................................33

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