標題: 發展一結合流式微陣列之蛋白質體分析方法以作為泛素(Ubiquitin)相關之生物分子交互作用確認平台
Development of a Proteomic Approach Combining Suspension Microarray to Enable the Profiling and Multiplexed Validation of the Ubiquitin-Associated Interactome
作者: 許馨云
HSU HSIN-YUN
國立交通大學應用化學系(所)
公開日期: 2011
摘要: æ³›ç´ åŒ–(ubiquitination)是一種蛋白質轉譯後的修飾作用,原本被èª�為僅是細胞內一種 負責蛋白質é™�解的信號,é€�é�Žæ­¤é�Žç¨‹ä¾†æŽ§åˆ¶ç´°èƒžä¸­é—œé�µä¿¡è™Ÿè›‹ç™½å’Œèª¿ç¯€è›‹ç™½çš„濃度,å�Œ 時å�¯ä»¥åŽ»é™¤é�Žå¤šã€�å�—æ��ã€�折疊錯誤或çª�變的蛋白質。但目å‰�越來越多的研究指出這種修 飾作用在細胞中還å�¯ç”¨ä¾†èª¿ç¯€è¨±å¤šä¸�å�Œçš„生物途徑以維æŒ�正常生ç�†åŠŸèƒ½çš„é�‹ä½œï¼Œè‹¥æ­¤ç¨® 修飾作用發生å•�題,將å�¯èƒ½å°Žè‡´ç–¾ç—…,例如人類退化性的神經疾病ã€�細胞凋亡ã€�å…�ç–«(如 NF-B)與發炎å��應ã€�細胞週期ã€�DNA的修補和抑癌基因(如p53)等,都被發ç�¾èˆ‡æ³›ç´ çš„ 修飾作用有關。因此,發展高通é‡�方法用以有效的分æž�這些å�—到精細調控的訊æ�¯å‚³å°Žç¶² 絡,找到å�—此修飾作用影響的蛋白質分å­�,將有助於疾病診斷試劑的開發。質譜技術是 ç›®å‰�用以分æž�泛素化蛋白質分å­�最具潛力的方法。然而,許多目å‰�在此領域的研究ä»�å�� é‡�在定性的分æž�這些修飾作用。蛋白質分å­�的確èª�也ä»�然需è¦�相當ç¹�複的實驗æµ�程。此 外大多數生物體內細胞的應答是é€�é�Žä¸�å�Œé�µçµ�å½¢å¼�ã€�程度的泛素修飾,因此,建立一個 能夠定é‡�分æž�這些修飾作用的方法是必è¦�的。 本計畫將çµ�å�ˆè³ªè­œæŠ€è¡“與æµ�å¼�懸浮微陣列發展一高通é‡�的泛素化蛋白質分å­�分æž�å¹³å�° 並使這些候é�¸ç›®æ¨™åˆ†å­�能夠有效的大è¦�模定é‡�分æž�確èª�。å°�特定泛素é�µçµ�å½¢å¼�有親和性 的蛋白質çµ�å�ˆå�€ä½�(ubiquitin binding domains, UBDs)將作為泛素化蛋白質分å­�çš„åˆ�步分 類之質譜增è�š(enrichment)工具,使這些泛素修飾的蛋白質分å­�根據ä¸�å�Œé�µçµ�å½¢å¼�而能夠 分別的平行分æž�並研究ä¸�å�Œé�µçµ�å�¯èƒ½å°�應的相關功能。轉錄因å­�NF-kBå�ƒèˆ‡åœ¨è¨±å¤šç”Ÿç�† 功能é�‹ä½œåŒ…括å…�疫發炎å��應ã€�細胞的增生分化與癌化等。泛素修飾作用在其訊æ�¯å‚³å°Žèª¿ 節上扮演關é�µæ€§çš„角色並且已廣泛被研究。本計畫將以NF-kB作為模å¼�分å­�以此訊æ�¯é€” 徑來驗證此分æž�方法的å�¯è¡Œæ€§ï¼Œå€™é�¸ç›®æ¨™åˆ†å­�å°‡é€�é�Žæµ�å¼�懸浮微陣列作有效確èª�。 在此為期三年的計畫中,待完æˆ�的工作分列如下: 第一年:特定泛素é�µçµ�親和性分å­�篩é�¸èˆ‡è©•ä¼° 建立pull-down assay與實驗æ¢�件之最佳化 第二年:建立轉錄因å­�NF-kB細胞內活化模å¼� 細胞培養與泛素化蛋白質樣本製備 親和性液相層æž�質譜分æž� 第三年:整å�ˆæµ�å¼�懸浮微陣列平å�°ï¼š 候é�¸ç›®æ¨™åˆ†å­�確èª� 設計微陣列檢測æµ�程與標的組å�ˆä»¥å»ºç«‹è¨ºæ–·æ‡‰ç”¨å·¥å…·
Post-translational modifications (PTMs) of proteins by the covalent conjugation of ubiquitin (also called ubiquitination), which originally implicated as a signal for proteolytic degradation, have now been discovered to regulate diverse biological processes. Considering the importance of ubiquitination in normal cellular physiology, and their roles in disease etiology and progression, it will be critical to develop high-throughput methods capable of mapping the delicate networks to efficiently identify proteins modified by these small molecules. For the characterization of ubiquitinated proteins, the mass spectrometry offers great promise. However, much of current analysis focused on identifying modifications, rather than on quantifying them. The laborious validation procedure for the identified targets is another bottleneck. In addition, biological effects are often due to changes in the linkage types and the level of the ubiquitination, and thus there is intense necessity in the improvement of the methodology in the quantitative study of these modifications. This proposal aims to develop an approach combining mass spectrometry and suspension microarray for ubiquitin- modified proteins profiling and allows high throughput quantitatively validation of the candidates. The selective ubiquitin binding domains (UBDs) specific to individual types of linkages will be applied for pull down assays as an initial tool for cataloging ubiquitome profiling to discover protein panels with corresponding ubiquitin linkages and the associated functions. The nuclear factor B (NF-kB) signaling will be used as a model pathway to demonstrate the feasibility of the approach and the identified molecules will be used as the target proteins for the effective validation by the suspension microarray. In this three-year project, it is anticipated that the following work will be accomplished: (1) Selection of the affinity binders, setup and the optimization of the pull down assay (2) Establishment of the NF-kB activation protocol as a model pathway and the setup of affinity-based LC-MS/MS to demonstrate the feasibility of the approach to profile the ubiquitin-associated interactome (3) The prototype of the suspension microarray panel to enable the multiplexed validation of the hits and its potential applications as diagnostic tools
官方說明文件#: NSC99-2113-M009-016-MY2
URI: http://hdl.handle.net/11536/99108
https://www.grb.gov.tw/search/planDetail?id=2216901&docId=354968
顯示於類別:研究計畫