The Development of Applying Photoreactive Cross-Linking Reaction in Proteomics

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Project title

Code/計畫編號

NSC100-2113-M019-002-MY2

Translated Name/計畫中文名

開發光活化之交聯鍵結反應於蛋白質體學上之應用

Project Coordinator/計畫主持人

Pang-Hung Hsu

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Bioscience and Biotechnology

Website

https://www.grb.gov.tw/search/planDetail?id=2313522

Year

2011

Start date/計畫起

01-08-2011

Expected Completion/計畫迄

31-07-2012

Bugetid/研究經費

1928千元

ResearchField/研究領域

化學
生物技術(理)

化學交聯鍵結反應能在具有交互作用的蛋白分子間形成共價鍵結，特別是對於具有功能性的蛋白質複合物。經由化學交聯鍵結的蛋白分子能維持其原有的結構與空間上的位相，而此分子複合物之結構可由不同的分析方法分析，而不會造成不同蛋白分子的瓦解。質譜技術在對於具交聯鍵結的蛋白分子分析上為一極有效的工具，然而因具交聯鍵結的蛋白分子其分子裂解的模式過於複雜，因此直到2010 年才有具自動資料分析功能的方法以鑑定交聯鍵結的氨基酸位置。但目前能使用的分析方法只能鑑定已知的氨基酸單位，因所使用的交聯鍵結反應必須選擇性地與已知官能基反應，例如N-羥基硫代琥珀酰亞胺僅與蛋白或胜肽的N 端一級胺基或是離氨酸的側鏈反應產生鍵結。使用具有選擇性的交聯鍵結反應限制了蛋白分子上形成鍵結的氨基酸數目，也同時侷限了此方法在結構生物學上的應用。因此我們提出了一個對於蛋白複合物結構研究上新的構想，整合使用具有非選擇性之光反應活化之交聯鍵結反應與以質譜技術為基礎的蛋白質體學分析方法。為達成此一目標，我們提出三階段的實驗構想。(1)開發一全新的資料分析演算法以鑑定被非選擇性之光反應活化試劑交聯鍵結的蛋白分子。(2)以細胞外的蛋白複合物為模型，驗證此全新的質譜數據分析演算法。(3)將此一方法實際應用於細胞內蛋白複合物的結構研究。我們相信此方法不但可提供目前使用具選擇性的交聯鍵結所遇到的瓶頸，同時也能填補傳統結構生物學上使用核磁共振光譜或是x- 射線繞射結晶法研究蛋白複合物所面臨的限制。 The chemical cross-linking reaction is able to convert the information of the non-covalent interactions between protein molecules, especially within the functional protein complex, into the covalent bonds between the interaction sites. The cross-linked protein molecules will retain their natural conformation and orientation in space; therefore, they can be analyzed by different analytical methods which normally disrupt the complex structure. Mass spectrometry technology has been shown to be a powerful tool to analyze the cross-linked protein molecules. However, due to the high complexity of the fragmentation patterns of the cross-linked protein molecules, there is no automatic data analysis tool available for identification of the cross-linked amino acid residues until 2010. Moreover, the current available method can only characterize the linkages between certain amino acid residues because the cross-linking reagents work to selectively connect to certain functional groups, such as the N-hydroxysulfosuccinimide esters reacts with the primary amine group in protein/peptide N-terminus or the lysine side chain. The property of selective cross-linking limits the numbers of cross-linked amino acid residue between protein molecules and also restricts the application in structural biology. Here I propose a new approach to study the protein complex structure by integrating the non-selective photoreactive cross-linking strategy and mass spectrometric based proteomics tool. In order to establish this novel method for structural biology study, three specific aims will be pursued in this study. (1) To develop a new database search algorithm for identification of protein molecules linked by the non-selective photoreactive cross-linking reagent. (2) To validate the search algorithm by in vitro model protein systems. (3) To apply this method for in vivo protein complex structure study. I believe this new approach will provide solutions to overcome the limitations of the applications of chemical cross-linking methods and also to fill the gap in structural biology for studying protein complexes that NMR spectroscopy or x-ray crystallography methods cannot solve.

Keyword(s)

Photo-reactive Cross-Linking
Mass Spectrometry

DSpace CRIS

The Development of Applying Photoreactive Cross-Linking Reaction in Proteomics

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