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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 羅禮強(Lee-Chiang Lo) | |
| dc.contributor.author | Ya-Ping Chen | en |
| dc.contributor.author | 陳雅萍 | zh_TW |
| dc.date.accessioned | 2021-06-08T04:14:43Z | - |
| dc.date.copyright | 2010-08-18 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-08-10 | |
| dc.identifier.citation | (1) Venter, J. C.; et al. Science 2001, 291, 1304-1351.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22272 | - |
| dc.description.abstract | 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。 | zh_TW |
| dc.description.abstract | 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. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T04:14:43Z (GMT). No. of bitstreams: 1 ntu-99-R96223204-1.pdf: 1738862 bytes, checksum: c7e792cd0250589ec683c57c693d4582 (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 目錄
目錄 I 圖表目錄 IV 英文縮寫 i 摘要 ii Abstract iii 第一章 緒論 1 1.1 前言 1 1.2 蛋白質體學之研究動機 2 1.2.1 蛋白質分析方法 2 1.2.2 活性導向之蛋白質標定技術(ABPP) 3 1.3 研究對象 4 1.4 Fluorophosphonate (FP)探針之發展與應用 5 第二章 材料與方法 12 2.1 分析方法 12 2.1.1 SDS聚丙醯胺凝膠電泳(SDS-PAGE) 12 2.1.2 西方點墨法(Western bloting) 13 2.1.3 螢光影像分析方法 13 2.1.4 銀染(silver stain) 13 2.2 蛋白質定量 14 2.3 細胞萃取液之製備 14 2.3.1 大腸桿菌之轉植 14 2.3.2 大腸桿菌細胞萃取 14 2.3.3 酵母菌細胞萃取 15 2.4 標定反應 15 2.4.1 Click chemistry ([3+2]成環反應) 15 2.4.2 探針反應條件測試 15 2.4.3 活性探針標定濃度測試 16 2.4.4 活性探針靈敏性測試 16 2.4.5 活性探針之專一性與選擇性測試 16 2.4.6 B2C探針標定蛋白質之純化 16 2.4.7 以活性探針標定細胞之蛋白質萃取液 17 2.4.8 測試N2C探針可否進入大腸桿菌 17 2.4.9 測試N2C探針可否進入酵母菌 18 2.5 乙醯膽鹼酯酶(acetylcholinesterase，AchE)活性測試 18 2.6 細胞培養 18 2.6.1 測試探針可否進入細胞 18 2.6.2 測試探針與細胞反應之濃度 19 2.6.3 測試探針與細胞反應之時間 19 2.6.4 Subcellular fractionation 19 第三章 實驗結果 20 3.1 以B2C探針測試標定條件 20 3.2 不同發報端探針其靈敏性之比較 21 3.3 探針標定蛋白質之專一性與選擇性 21 3.4乙醯膽鹼酯酶標定 22 3.5 活性中心標定測試 22 3.6 探針標定蛋白質之選擇性 22 3.7 B2C探針標定lipases from hog pancreas之純化與身分鑑定 23 3.8 比較辨識端不同之探針其標定效率 24 3.9 細胞萃取液之探針標定 24 3.10 Subcellular fractionation之標定 25 3.11 探針對大腸桿菌、酵母菌與人類細胞之通透性 25 3.12 細胞內標定(in vivo)之條件 26 3.13 In vitro與in vivo標定之比較 26 第四章 討論 28 參考文獻 35 圖表目錄 Figure 1-1. The relation between genome and proteome. 2 Figure 1-2. Schematic of a proteome reacting with a probe. 4 Figure 1-3. The structures of fluorophosphonate-based inhibitors. 5 Figure 1-4. Proposed mechanism for serine hydrolase labeling by FP. 6 Figure 1-5. Different reporter groups of FP probe. 7 Figure 1-6. The structures of benzyl fluorophosphonate-based probes. 11 Figure 3-1. pH profile and time course of B2C probe labeling reaction. 39 Figure 3-2. Labeling of trypsin with probes by varying concentrations of probes. 40 Figure 3-3. Labeling sensitivity of probes to trypsin. 41 Figure 3-4. Specificity of the biotin probes. 42 Figure 3-5. Specificity of the rhodamine probes. 43 Figure 3-6. Specificity of the azide probes. 44 Figure 3-7. Labeling of proteases with probes. 45 Figure 3-8. Confirm the active site labeling. 46 Figure 3-9. Labeling different species of lipases with the biotin probes. 47 Figure 3-10. Labeling different species of lipases with the rhodamine probes. 48 Figure 3-11. Labeling different species of lipases with the azide probes. 49 Figure 3-12. Enrichment of B2C-labeled proteins. 50 Figure 3-13. Comparison of labeling efficiency of benzyl fluorophosphonate-based probes. 51 Figure 3-14. Cell lysates labeling. 52 Figure 3-15. Subcellular fractionation of 293T cells. 53 Figure 3-16. Cell permeability of benzyl fluorophosphonate-based probes for human cell. 54 Figure 3-17. Cell permeability of N2C probe for E. coli and yeast. 55 Figure 3-18. The conditions of in vivo labeling. 56 Figure 3-19. Comparison of in vitro and in vivo labeling. 57 Figure 4-1. Staudinger ligation. 32 Table 1-1. The codes of benzyl fluorophosphonate-based probes. 11 Table 4-1. The labeling signals of probes. 31 Table 4-2. Comparison of background signals of different reactions. 32 | |
| dc.language.iso | zh-TW | |
| dc.subject | 細胞內標定 | zh_TW |
| dc.subject | 苯甲基氟化磷酸酯 | zh_TW |
| dc.subject | 化學探針 | zh_TW |
| dc.subject | 標定效率 | zh_TW |
| dc.subject | PMSF | zh_TW |
| dc.subject | 絲胺酸水解酶 | zh_TW |
| dc.subject | benzyl fluorophosphonate | en |
| dc.subject | in vivo labeling | en |
| dc.subject | serine hydrolases | en |
| dc.subject | PMSF | en |
| dc.subject | labeling efficiency | en |
| dc.subject | probes | en |
| dc.title | 苯甲基氟化磷酸酯類化學探針在蛋白質體學上的應用 | zh_TW |
| dc.title | Utilization of Benzyl Fluorophosphonate-based Probes for Proteomic Applications | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林敬哲(Jing-Jer Lin),吳世雄(Shih-Hsiung Wu) | |
| dc.subject.keyword | 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, | zh_TW |
| dc.subject.keyword | benzyl fluorophosphonate,probes,labeling efficiency,PMSF,serine hydrolases,in vivo labeling, | en |
| dc.relation.page | 57 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2010-08-10 | |
| dc.contributor.author-college | 理學院 | zh_TW |
| dc.contributor.author-dept | 化學研究所 | zh_TW |
| Appears in Collections: | 化學系 | |
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