一氧化氮分子藉由與半胱胺酸上的硫基形成可逆共價鍵結，可調節蛋白質的活性和功能進而影響多種生理機制。本論文的目標是發展便捷的方式從複雜樣品中純化硫基亞硝基胜肽。我們將文獻中兩種硫酯基磷探針與兩種固相載體磁性奈米粒子及珠狀瓊脂糖結合，經由追蹤式還原接合亞硝基化硫基反應，將一級RSNO轉換成相對穩定的雙硫鍵產物，應用於純化和鑑定RSNOs。將亞硝基化標準胜肽PTP1B與BSA複雜樣品混合後，探針可成功的純化出PTP1B，並得知珠狀瓊脂糖在複雜樣品中能夠減少分專一性吸附。使用2nd_TEP@agarose探針，已成功從COS-7細胞間質液中純化出能被亞硝基化的胜肽。硫酯基磷探針在研究亞硝基化蛋白上具有極大的潛力，因此我們接著改變探針結構，合成出另一種硫酯基磷探針，探討結構對RSNO反應性的影響。

Nitric oxide (NO) is covalently attached to cysteine thiols of proteins resulting in the formation of S-nitrosothiols (RSNOs), and regulation of protein activity and function in a wide range of physiological processes. The objectives of this thesis are development of probes for the enrichment of S-nitrosylated peptides. We conjugated two types of thioester phosphine ligand to either magnetic nanoparticle or agarose bead, respectively for identification and purification of RSNOs. Furthermore, through traceless reductive ligation of S-nitrosothiol mechanism the unstable primary RSNOs were converted to stable disulfide-iminophosphorane products. The S-nitrosylated PTP1B peptide mixed with tryptic BSA mixture was successfully captured by developed probe and identified by MALDI-TOF. In addition, the agarose beads show less non-specific interaction to tryptic BSA peptide. The enrichment of S-nitrosylated peptides from COS-7 cell lysate was achieved by 2nd_TEP@agarose. Thus, This strategy is a potential tool for investigate of S-nitrosylation proteins. Furthermore, we synthesized other type of thioester phosphine ligand by changing the original structure and investigate the effect of probe structure on the reactivity to RSNO.

第一章- 序論 1
1.1一氧化氮 1
1.1.2人體內一氧化氮的生成 3
1.2硫基亞硝基化蛋白 7
1.2.1蛋白質亞硝基化 8
1.3奈米粒子載體 9
1.3.1奈米科技的原理 10
1.3.2奈米材料的特性 11
1.3.3磁性奈米粒子 13
1.3.3.1磁性奈米粒子的特性 14
1.3.3.2氧化鐵磁性奈米粒子表面修飾 15
1.3.4珠狀瓊脂糖（Agarose beads） 17
1.4偵測硫基亞硝基化蛋白的方法 18
1.4.1間接檢測法 19
1.4.1.1化學發光法 (Chemiluminescence methods) 19
1.4.1.2比色法 (Colormetry) 20
1.4.1.3螢光法 (Fluorescence) 21
1.4.1.4生物素轉換法(Biotin-switch based methods) 22
1.4.2直接檢測法 (Direct methods) 24
1.4.3直接檢測法-非衍生物法: 25
1.4.3.1質譜法 (Mass spectrometry) 25
1.4.3.2抗體法 (Anti-S-nitrosocysteine antibody methods) 25
1.4.4直接檢測法-衍生物法 26
1.4.4.1金奈米粒子法 (Gold nanoparticles) 26
1.4.2.4有機汞樹酯法 (Organomercury resin capture, MRC) 27
1.4.2.5有機磷直接鍵結法 (Organophosphine based SNO direct labeling) 29
1.4.2.5.1四種反應機制 31
第二章- 專一性探針製備與應用 38
2.1研究動機 38
2.1第一代與第二代磁性奈米探針之合成 (1ST_TEP@MNP & 2nd_TEP@MNP) 40
2.1.1第一代磁性奈米探針的設計 (1ST_TEP@MNP) 41
2.1.2第二代磁性奈米探針的設計 (2nd_TEP@MNP) 42
2.1.2.1連接子鏈之合成（Linker） 44
2.1.2.2第二代磁性探針與第二代珠狀瓊脂糖的製備 46
2.1.2.3磁性奈米粒子表面濃度定量 47
2.2磁性奈米型探針偵測結果與討論 48
2.2.1一般型探針 49
2.2.2標定型磁性探針 52
2.2.2.1標定型磁性探針反應性測試 53
2.2.2.2專一性測試 54
2.2.2.3非專一性吸附討論 55
2.3製備第一代與第二代珠狀瓊脂糖探針(1st_TEP@agarose & 2nd_TEP@agarose) 58
2.3.1第一代珠狀瓊脂糖探針(1st_TEP@agarose) 59
2.3.1.1表面定量 59
2.3.2第二代珠狀瓊脂糖探針(2nd_TEP@agarose) 61
2.3.2.1表面濃度定量 62
2.4珠狀瓊脂糖型探針偵測結果與討論 62
2.4.1專一性測試比較 63
2.4.2偵測極限(Limit of detection) 67
2.4.3 Cos7細胞裂解液(Cell lysate)中反應性測試 68
2.4.4 2nd_TEP@Agarose討論 70
2.5第三代探針設計與製備 71
2.5.1合成第三代-一般型探針 73
2.5.2合成第三代生物素探針 (3rd_TEP@biotine) 74
2.5.3反應性測試 76
2.5.4 還原性測試 77
2.6第四代探針設計與製備 79
2.6.1 利用取代反應策略合成第四代探針 80
2.6.2 開環策略合成第四代探針 81
2.6.3第二代、第三代及第四代探針反應性測試 84
2.7合成二次標定的探針 84
第三章- 結果與討論 86
第四章- 結論與未來展望 89
第五章- 實驗方法 90
5.1 Chemical synthesis 90
5.2 Probes synthesis 104
5.3 Enrichment of nitrosylated the standard peptide 108
參考文獻 111

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