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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22272
標題: | 苯甲基氟化磷酸酯類化學探針在蛋白質體學上的應用 Utilization of Benzyl Fluorophosphonate-based Probes for Proteomic Applications |
作者: | Ya-Ping Chen 陳雅萍 |
指導教授: | 羅禮強(Lee-Chiang Lo) |
關鍵字: | 苯甲基氟化磷酸酯,化學探針,標定效率,PMSF,絲胺酸水解酶,細胞內標定, benzyl fluorophosphonate,probes,labeling efficiency,PMSF,serine hydrolases,in vivo labeling, |
出版年 : | 2010 |
學位: | 碩士 |
摘要: | 隨著後基因體時代的來臨,已逐漸建立研究蛋白質體學的方法,而各式各樣的探針可以在複雜系統中快速篩選出標的蛋白,並進一步分析及身分鑑定。本論文針對水解酵素的一系列苯甲基氟化磷酸酯類化學探針,分別在氟化磷酸酯位置修飾不同基團(ethyl、butyl、octyl和cyclohexyl)進行初步篩選。接著針對此類化學探針研究其生化反應條件,包含酸鹼值、反應時間和探針濃度的使用,並探討不同修飾的探針之標定效率。由結果可知在單一酵素標定效率依序為butyl>ethyl>octyl≒cyclohexyl。接著探討帶有不同發報基團探針的標定效率,如生物素、螢光團(rhodamine和BODIPY)和疊氮基彼此之差異性。發報端會影響探針對蛋白質的標定,而在不同發報基團中以rhodamine探針的靈敏性最好。修飾ethyl與butyl基團之探針分別與293T細胞萃取液反應,特定訊號會經PMSF不可逆抑制而消失,而這些蛋白質須進一步確認是否為絲胺酸水解酶。最後,帶有不同發報基團探針中只有疊氮基和BODIPY可運用於細胞內標定。 With the postgenome era rapidly approaching, a number of approaches have been proposed for the studies of the proteome. There are several kinds of chemical probes for the screening of target proteins in complex system, followed by analysis and identification of the target proteins. Here, we utilized a series of benzyl fluorophosphonate-based probes with different substituents (ethyl, butyl, octyl and cyclohexyl) connected to fluorophosphonate for the screening of target proteins. The labeling profiles of serine hydrolases were determined. First, the optimized conditions for the probes were performed by varying pH, labeling time and the concentrations of probes. In addition, the labeling efficiency of the probes was also investigated. The highest labeling efficiency in purified serine hydrolases (i.e., trypsin) could be observed for the probe with butyl group (butyl>ethyl>octyl≒cyclohexyl). Furthermore, the labeling efficiency and profiles of different reporter groups, such as biotin, fluorophore (rhodamine and BODIPY) and azide were compared. The results showed that the labeling profiles of various reporter’s probes were different, and the highest sensitivity was observed for rhodamine probe. The probes with ethyl or butyl group were also analyzed for their labeling profile using 293T cells. Probe-labeled proteins were indirectly identified by irreversible profiling with PMSF, indicating that these proteins could be the probes target serine hydrolases. Based on the results, I concluded that the azide and BODIPY probes are capable of in vivo labeling serine hydrolases. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22272 |
全文授權: | 未授權 |
顯示於系所單位: | 化學系 |
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