研究生: |
賴承煜 LAI CHEN-YU |
---|---|
論文名稱: |
固定化鐵離子親合層析質譜技術之蛋白質磷酸化研究 Phosphoproteome Profiling Using Immobilized Fe(Ⅲ) Affinity Chromatography with Mass Spectrometry |
指導教授: |
陳玉如
Chen, Yu-Ju |
學位類別: |
碩士 Master |
系所名稱: |
化學系 Department of Chemistry |
論文出版年: | 2005 |
畢業學年度: | 93 |
語文別: | 英文 |
論文頁數: | 123 |
中文關鍵詞: | 磷酸化蛋白質 、質譜 、固定化鐵離子親合層析 |
英文關鍵詞: | Phosphoproteome, Mass Spectrometry, Immobilized Fe(Ⅲ) Affinity Chromatography |
論文種類: | 學術論文 |
相關次數: | 點閱:284 下載:12 |
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蛋白質磷酸化在細胞訊息傳遞及功能調控上扮演關鍵角色。雖然目前已發展數種鑑定蛋白質磷酸化的方法,但要觀察到細胞內所有的蛋白質的磷酸化仍有一段很長的距離。這是由於蛋白質的磷酸化是一個動態現象,在細胞內的含量低,並且位置也會有所不同。為了要解決這樣的困難,發展更有效的分析蛋白質磷酸化的方法仍是一個重要的課題。
在本論文中,我們結合次細胞分離法(subcellular fractionation),凝膠電泳(SDS-PAGE),金屬親和層析 ( immobilized metal affinity chromatography,IMAC),質譜 (mass spectrometry, MS)等技術分析複雜蛋白質樣品中所含的磷酸化蛋白,並針對其中IMAC 對於磷酸化胜的容量,專一性,沖提體積以及條件作細部的探討以及最佳化。為了證明此分析平台的可用性,我們將其應用在分析T細胞中的磷酸蛋白上。實驗中在細胞質中在603 個蛋白質中發現了782 個磷酸化胜及891個磷酸化的位置,在所有鑑定出的胜中,磷酸化胜佔百分之九十。此平台能廣泛應用在分析大量蛋白質磷酸化的研究上。
The protein phosphorylation is key step in cellular signaling to initiate various cellular functions. Despite the advances of various powerful analytical methods is
available, global characterization of the site specific phosphorylation remains far from routine practice. The characterization of phosphorylation has been challenged by the technical difficulty associated with their abundance in cell, dynamic modification pattern, and heterogeneous forms of phosphoproteins. The challenge warrants the need to develop methods capable of accurately elucidating sites of protein phosphorylation.
In this study, a high throughput platform combining subcellular fractionation, SDS-PAGE, immobilized metal affinity chromatography (IMAC) and mass spectrometry
(MS) was introduced for the analysis of phosphoprotein form complex protein mixture.The loading capacity, binding specificity, sample recovery, elution volume and elution
reagent of the IMAC were optimized and studied in detail. To demonstrate the feasibility for the analysis of complex protein mixture, the phosphopeptide from the crude protein
extract of the human jurkat T-cell was subjected to the SDS-PAGE separation, IMAC purification, followed by the nLC-ESI-MS analysis. The preliminary result identified 782
phosphopeptides with 891 phosphorylation sites from 603 proteins in cytosolic fraction, about 90% of the identified peptides were found to be phosphorylated using optimized
IMAC parameters. This technology platform can be applied to profiling protein phosphorylation on the proteome level.
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