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–ï¼ˆubiquitination)是一種蛋白質轉è¯å¾Œçš„修飾作用,原本被èª�為僅是細胞內一種
è² è²¬è›‹ç™½è³ªé™�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‰�è¶Šä¾†è¶Šå¤šçš„ç ”ç©¶æŒ‡å‡ºé€™ç¨®ä¿®
飾作用在細胞ä¸é‚„å�¯ç”¨ä¾†èª¿ç¯€è¨±å¤šä¸�å�Œçš„生物途徑以ç¶æŒ�æ£å¸¸ç”Ÿç�†åŠŸèƒ½çš„é�‹ä½œï¼Œè‹¥æ¤ç¨®
修飾作用發生�題,將�能導致疾病,例如人類退化性的神經疾病�細胞凋亡��疫(如
NF-B)與發炎å��應ã€�細胞週期ã€�DNAçš„ä¿®è£œå’ŒæŠ‘ç™ŒåŸºå› (如p53)ç‰ï¼Œéƒ½è¢«ç™¼ç�¾èˆ‡æ³›ç´ çš„
ä¿®é£¾ä½œç”¨æœ‰é—œã€‚å› æ¤ï¼Œç™¼å±•é«˜é€šé‡�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�—æ¤ä¿®é£¾ä½œç”¨å½±éŸ¿çš„蛋白質分å�,將有助於疾病診斷試劑的開發。質èœæŠ€è¡“是
ç›®å‰�用以分æž�æ³›ç´ åŒ–è›‹ç™½è³ªåˆ†å�最具潛力的方法。然而,許多目å‰�在æ¤é ˜åŸŸçš„ç ”ç©¶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。æ¤
外大多數生物體內細胞的應ç”是é€�é�Žä¸�å�Œé�µçµ�å½¢å¼�ã€�ç¨‹åº¦çš„æ³›ç´ ä¿®é£¾ï¼Œå› æ¤ï¼Œå»ºç«‹ä¸€å€‹
èƒ½å¤ å®šé‡�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‡�çš„æ³›ç´ åŒ–è›‹ç™½è³ªåˆ†å�分æž�å¹³å�°
並使這些候é�¸ç›®æ¨™åˆ†å�èƒ½å¤ æœ‰æ•ˆçš„å¤§è¦�模定é‡�分æž�確èª�。å°�ç‰¹å®šæ³›ç´ é�µçµ�å½¢å¼�有親和性
的蛋白質çµ�å�ˆå�€ä½�(ubiquitin binding domains, UBDs)å°‡ä½œç‚ºæ³›ç´ åŒ–è›‹ç™½è³ªåˆ†å�çš„åˆ�æ¥åˆ†
類之質èœå¢žè�š(enrichment)å·¥å…·ï¼Œä½¿é€™äº›æ³›ç´ ä¿®é£¾çš„è›‹ç™½è³ªåˆ†å�æ ¹æ“šä¸�å�Œé�µçµ�å½¢å¼�而能å¤
分別的平行分æž�ä¸¦ç ”ç©¶ä¸�å�Œé�µçµ�å�¯èƒ½å°�æ‡‰çš„ç›¸é—œåŠŸèƒ½ã€‚è½‰éŒ„å› 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ç‰ã€‚æ³›ç´ ä¿®é£¾ä½œç”¨åœ¨å…¶è¨Šæ�¯å‚³å°Žèª¿
節上扮演關é�µæ€§çš„è§’è‰²ä¸¦ä¸”å·²å»£æ³›è¢«ç ”ç©¶ã€‚æœ¬è¨ˆç•«å°‡ä»¥NF-kB作為模å¼�分å�以æ¤è¨Šæ�¯é€”
徑來驗è‰æ¤åˆ†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æ¤ç‚ºæœŸä¸‰å¹´çš„計畫ä¸ï¼Œå¾…完æˆ�的工作分列如下:
ç¬¬ä¸€å¹´ï¼šç‰¹å®šæ³›ç´ é�µçµ�親和性分å�篩é�¸èˆ‡è©•ä¼°
建立pull-down assay與實驗�件之最佳化
ç¬¬äºŒå¹´ï¼šå»ºç«‹è½‰éŒ„å› å�NF-kB細胞內活化模å¼�
ç´°èƒžåŸ¹é¤Šèˆ‡æ³›ç´ åŒ–è›‹ç™½è³ªæ¨£æœ¬è£½å‚™
親和性液相層æž�質èœåˆ†æž�
第三年:整���懸浮微陣列平�:
候é�¸ç›®æ¨™åˆ†å�確èª�
è¨è¨ˆå¾®é™£åˆ—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 |