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研究生： 吳晉任
Wu, Chin-Jen
論文名稱： 免疫親和性層析與免疫轉漬法結合質譜偵測針對磷酸化蛋白質之分析研究
Analysis of Phospho-Proteins Using Immunoaffinity Chromatography and Immunoblotting Coupled with MS Detection
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： 2004
畢業學年度： 92
語文別： 中文
論文頁數： 141
中文關鍵詞： 免疫親和性層析 、磷酸化蛋白質 、質譜 、蛋白質體
外文關鍵詞： phospho-protein, immunoaffinity, mass, proteomics
相關次數： 點閱：113  下載：2 
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    • 　　本論文主要是利用免疫親和性管柱還有一維膠電泳結合吸附金屬親和性管柱針對磷酸化蛋白質進行純化和分離，最後再利用基質輔助雷射脫附游離質譜或電噴灑離子化質譜對磷酸化蛋白質進行分析和鑑定。由於人類生理系統中細胞間的訊息傳遞與蛋白質的磷酸化有非常重要的關係，特別是酪胺蛋白激酉每扮演著細胞生長、分化、遷移等重要的角色。故本實驗會先對酪胺酸磷酸化的蛋白進行純化及分析，其主要流程為事先拿標準的酪胺酸磷酸化蛋白，對所設計的實驗方法進行測試，直至分析方法達到一個最穩定的最佳狀態後，再把此技術應用到真實的生物樣品上。
    　　在第一類型免疫親和性管柱的部分，乃是使用一個和酪胺酸磷酸化蛋白有專一免疫反應特性的小珠作為管柱的靜相，用於質譜鑑定前蛋白質的純化和濃縮。在這個部分是利用酪胺酸磷酸化的胜肽結合牛的血清蛋白(P-BSA)作為一個標準樣品，藉此找尋適當的平衡液、洗滌液和沖提液，分別用於管柱使用前的潤濕、非特異性蛋白的移除及酪胺酸磷酸化蛋白跟抗體的分離。直至管柱達到最佳的分離效果後，再將其運用至真實樣品中，來進行磷酸化蛋白的純化。
    　　在第二類型一維膠電泳結合吸附金屬親和性管柱的部分，主要是先使用一維膠電泳將蛋白質根據分子量的不同進行分離，之後利用染料顯影，同時間將相同的樣品運用相同比例的聚丙烯醯胺之膠片根據免疫轉漬法，讓磷酸化蛋白在曝光底片上顯影。比對底片和膠片上的蛋白質標記，即可找出磷酸化蛋白的相對位置。將磷酸化蛋白的區域切下，進行膠體內消化(In-gel digest)後，利用一根能和磷酸化胜肽結合的金屬親和性管柱，把磷酸化胜肽從眾多胜肽裡分離出來，最後由質譜分析，幫助磷酸化蛋白質的鑑定。在此研究方法裡將會以β酪蛋白(β-casein)等磷酸化蛋白質，對所設計的實驗方法及實驗流程進行測試。由實驗結果可以看出，其所設計之分析方法的確可以有效的捕捉到磷酸化胜肽，且有利於質譜對磷酸化蛋白質的斷定。
    　　傳統免疫沉澱法用來純化磷酸化蛋白的過程中，其經由加熱來斷裂蛋白和抗體之間鍵結的方法，不僅破壞了原有蛋白的結構，更會有抗體訊號的干擾，最重要的是抗體無法被重複使用。免疫親和性管柱的發展將可解決這些問體的產生，並從磷酸化蛋白質的鑑定，可以推出更多的生理流程及獲知更多的生物知訊。此技術的開發，在生理應用方面將會有莫大的幫助。另外一維膠電泳結合吸附金屬親和性管柱的方法，也可幫忙一些脂溶性蛋白的消化和消除樣品中大量蛋白的干擾，對磷酸化蛋白的分析，同樣也深具影響。

    　　The phosphorylation of proteins is highly related to the intercellular signaling transduction of human body. Protein tyrosine phosphorylation plays important roles on cell proliferation, differentiation, and migration. Therefore, the purpose of this study is to purify the phospho-proteins by immunoaffinity chromatography (IAC) as well as one-dimensional polyacrylamide gel electrophoresis (1-D gel) with immobilized metal affinity chromatography (IMAC), and to characterize the phosphor-protein by matrix-assisted laser ionization mass and electrospray ionization mass. Initially, the standard phosphotyrosine protein was used to detect the efficiency of IAC and 1-D gel with IMAC. The biological samples were to be analyzed until the reach of the optimal condition of analyzing tools.

    　　In the analysis of IAC, the bead coated with anti-phosphotyrosine antibody as the stationary phase was used to purify and concentrate the phosphoproteins in prior to Mass analysis. Phosphotyrosine conjugated with bovine serum albumin was used as the standard to select the optimal binding buffer, wash buffer, and elute buffer, which humidifies the column, removes the nonspecific proteins, and separates phosphotyrosine proteins and the coated antibody, respectively. The purification of phosphoproteins in the biological sample was analyzed until the reach of the opitimal condition of IAC.

    　　In 1-D gel with IMAC, the separation of proteins depends on the molecular weight of each protein in 1-D gel and then stained by commassie blue. The following step is to characterize the expression of the phosphoproteins using the identical sample by immunoblot analysis. After comparing the locations of protein marker between the 1-D gel and X-ray film, the phosphoproteins were identified. After cutting the gel pieces including the phosphoproteins through in-gel digestion, IMAC was used to isolate the phosphopeptides. The final step is to identify the phosphoproteins by Mass analysis. In this study, beta-casein (β-casein), selected as the standard sample, was to test the designed approaches.

    　　As the data suggested, our designed experimental approaches can efficiently capture the phophopeptides and facilitate the analysis of phosphoproteins by Mass.

    　　In the conventional immunoprecipitation, the broken of the binding of the antibody and the targeting antigen by the high-temperature heat not only alternates the structure of the proteins but also obstructs the signal detection through Mass analysis in the presence of antibody. Most importantly, the antibody can not be reused. The development of IAC can resolve these existing problems and demonstrate more signaling transduction pathways from the identification of phosphoproteins. Additionally, 1-D gel combined with IMAC facilitates the digestion of hydrophobic proteins and the removal of the abundant nonspecific proteins. In conclusion, the development of IAC and 1-D gel with IMAC may contribute to analyze the phosphoproteins.

    第一章 序論 1.1前言.........................................................................2 1.2蛋白質的磷酸化...............................................................3 1.3抗體和抗原的結合.............................................................4 1.3.1抗體結構................................................................4 1.3.2抗體與抗原的親和作用力..................................................5 1.4親和性管柱...................................................................6 1.5質譜分析蛋白質的原理.........................................................7 1.5.1基質輔助雷射脫附離子化的原理............................................8 1.5.2電噴灑離子化的原理......................................................9 1.6實驗架構.....................................................................9 第二章 免疫親和性管柱的架設 2.1研究動機....................................................................12 2.2免疫親和性管柱..............................................................14 2.2.1前言...................................................................14 2.2.2免疫親和性管柱分離磷酸化蛋白質的原理...................................15 2.3管柱靜相的製備..............................................................17 2.3.1實驗部分...............................................................19 2.4免疫親和性管柱的組裝........................................................19 2.5管柱的進樣及收集系統........................................................20 2.6儀器裝置....................................................................20 第三章免疫層析法結合質譜偵測對酪胺酸磷酸化蛋白質的分析研究 3.1研究目的....................................................................24 3.2實驗部分....................................................................25 3.2.1藥品與試劑.............................................................25 3.2.2 儀器設備..............................................................27 3.2.3管柱的分離條件.........................................................29 3.2.4分離溶液的配製.........................................................30 3.2.5酪胺酸磷酸化蛋白質樣品的配製...........................................30 3.2.6消化管柱的製備.........................................................32 3.2.7標準品的添加...........................................................33 3.2.8去鹽管柱的製備.........................................................33 3.2.9樣品的濃縮.............................................................35 3.2.10基質輔助雷射脫附離子化質譜基質的配製..................................35 3.3結果與討論..................................................................35 3.3.1 phosphotyrosine conjugated to BSA(pTyr-BSA)對免疫親和性管柱的測試.....36 3.3.2溫度對管柱效率的影響...................................................38 3.3.3酸鹼度對管柱效率的影響.................................................39 3.3.4酪胺酸磷酸化抗體管柱回收率的測試.......................................41 3.3.5酪胺酸磷酸化抗體管柱對三個蛋白質混合物pTyr-BSA的分離...................42 3.3.6酪胺酸磷酸化抗體管柱對pTyr-BSA結合細胞萃取液的分離.....................43 3.3.7酪胺酸磷酸化抗體管柱對磷酸化EGF receptor的分離.........................44 3.3.8影響管柱效率的其他因素.................................................46 3.4小結........................................................................48 第四章一維膠電泳結合吸附金屬親和性管柱針對酪胺酸磷酸化蛋白質的分析研究 4.1研究目的....................................................................51 4.2一維膠電泳的原理............................................................53 4.3免疫轉漬法的原理............................................................54 4.4吸附金屬親和性管柱的原理....................................................55 4.5實驗部分....................................................................56 4.5.1藥品與試劑.............................................................56 4.5.2儀器設備...............................................................60 4.5.3標準品的配製...........................................................62 4.5.4膠體的製 備............................................................62 4.5.5樣品的分離.............................................................63 4.5.6免疫轉漬法的操作.......................................................63 4.5.7膠體的染色.............................................................65 4.5.8膠體內的消化...........................................................66 4.5.9吸附金屬親和性管柱的製備...............................................67 4.5.10質譜的偵測............................................................68 4.6結果與討論..................................................................69 4.6.1標準品對分析方法的測試.................................................69 4.6.2直接溶液消化結合吸附金屬親和性管柱及一維膠電泳結合吸附金屬親和性管柱對 β-casein混合細胞萃取液之分析探討......................................71 4.6.3利用本分析方法對A 431(EGF-stimulate)細胞萃取液中磷酸化EGF R的研究......72 4.6.4利用本分析方法對其他磷酸蛋白質的研究...................................74 4.7小結........................................................................75 第五章總結與未來展望 5.1總結........................................................................77 5.2未來展望....................................................................78 參考文獻.......................................................................80

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