臺灣博碩士論文加值系統

1.
葡萄糖的非酵素性共價加成產物或醛類衍生物和蛋白質的糖化反應是引起糖尿病的血管併發症的主要原因。體內的葡萄糖含量若長期維持高濃度,則體內的雙羰基醛類化合物，如：乙二醛和2-氧丙醛的含量亦會提高。乙二醛來自於醣類與核酸之氧化、脂質過氧化以及亞硝胺的代謝；而2-氧丙醛主要則來自於糖解的中間產物G-3-P之分解。這些具反應性的醛類化合物會與蛋白質、脂質以及胺基酸反應最後生成醣化最終產物。本研究利用液相層析奈米噴灑游離質譜法(LC/NSI/MS)探討乙二醛和2-氧丙醛修飾蛋白質上胺基酸的選擇性。首先我們利用LC/NSI/MS確認乙二醛和2-氧丙醛會與N?acetyl-cysteine以及N?acetyl-L-lysine側鏈反應形成交聯產物。接著，我們以由14個胺基酸組成且含有一組雙硫鍵的胜肽somatostatin分別與乙二醛和2-氧丙醛反應也都偵測到加成產物及交聯產物。最後，我們探討乙二醛與2-氧丙醛分別與人類血紅蛋白反應的選擇性。結果發現在ㄢJ白上的Lys-11、Arg-92以及β蛋白上的Lys-17、Lys-144及Cys-93上有加成單一乙二醛的修飾；而2-氧丙醛則修飾在ㄢJ白上的Arg-31以及β蛋白上的Lys-144、Arg-104。我們也證實有乙二醛會與ㄢJ白上的Arg-92形成環狀hydroimidazolone。類似的環狀hydroimidazolone也發生在2-氧丙醛與ㄢJ白上的Arg-31及Arg-92上；然而，目前尚未偵測到乙二醛與2-氧丙醛與人類血紅蛋白的交聯產物。
2.
在癌症的發展上，慢性發炎是一個重要的危險因子，當被活化的巨噬細胞及嗜中性白血球在慢性感染及發炎時會製造過多超氧負離子與一氧化氮，此二物種快速反應就會產生過氧化亞硝酸根。過氧化亞硝酸根可以誘導DNA鏈的斷裂及突變，也會與蛋白質反應，並生成3-硝基酪胺酸。有釵h發炎和神經退化性的疾病都與3-硝基酪胺酸有關聯，包含眼睛發炎、視網膜缺血、癌症等。2000年有文獻報導使用抗3-硝基酪胺酸抗體結合西方轉漬法搭配質譜法成左瘍痔w出老鼠視網膜中有含有3-硝基酪胺酸的蛋白質。
本研究利用二維電泳搭配質譜法來鑑定出人類尿液中含有3-硝基酪胺酸的蛋白質。我們首先將尿液樣品經過丙酮沉澱、薄膜濃縮等步驟除去大部分鹽類後，再以二維電泳將尿液蛋白進行分離。接著利用抗-3-硝基酪胺酸抗體結合西方轉漬法，找出尿液中含有3-硝基酪胺酸的蛋白質。
以質譜儀鑑定2D- PAGE中的蛋白質前，需將凝膠做水解步驟，因此我測試各種不同的凝膠微波水解技術，找出最佳的凝膠水解條件。而之後的尿液樣品將以此最佳水解條件進行水解再搭配質譜法鑑定蛋白質身份。目前實驗，已進展至利用抗3-硝基酪胺酸抗體結合西方轉漬法後可以成它a看到含有3-硝基酪胺酸的蛋白質，亦由質譜鑑定出三個含3-硝基酪胺酸的可能蛋白質身分，包括： protein complex 4, epsilon 1 subunit; adaptor related; LIM domain only 6[Homo sapiens] and Hypothetical protein FLJ32940 isoform 1。我們也發現一個含有硝化色胺酸的蛋白質: transmembrane protein 16 F[Homo sapiens]

1.
The non-enzymatic conjugated addition product of glucose or aldehyde derivatives and glycation reaction of protein are the main cause of vascular complications of diabetes. If the concentration of glucose remains at high level in the body, the amount of ?dicarbonyl compounds, such as glyoxal and methylglyoxal, also increases. Glyoxal is obtained from glucose and amino acid oxidation, lipid peroxidation; methylglyoxal is mainly from glucose degradation intermediate product: G-3-P self-decompose. These reactive ?dicarbonyl compounds react with protein, lipids and amino acids to form saccharides as the final product. This study makes use of LC/NSI/MS to investigate the reaction of glyoxal and methylglyoxal with the amino acids on protein. First, we use LC/NSI/MS to confirm that glyoxal and methylglyoxal react with N?acetyl-cysteine and N?acetyl-L-lysine to form cross-linked products. Next, we use somatostain, which contains 14 amino acids and disulfide bond to react with glyoxal and methylglyoxal respectively and we have detected their addition and cross-linked products. Finally, we investigated the selectivity of glyoxal and methylglyoxal with human hemoglobin. We found that there was a single glyoxal addition on at Lys-11 of ?globin, and Lys-16, Lys-144 and Cys-93 of β-globin:, whereas methylglyoxal were found to add ?globin at Arg-31and β-globin at Lys-144 and Arg-104. We also confirm that glyoxal forms hydroimidazolone with ?globin at Arg-92. Similar hydroimidazolone also occurs with methylglyoxal and ?globin at Arg-31and Arg-92. However, the cross-linked products of glyoxal or methylglyoxal with human hemoglobin have not been identified.
2.
In cancer research, inflammation is a very crucial and dangerous factor. During infection and inflammation, the activated macrophages and neutrophils will produce excess superoxide anion and NO which will react rapidly to produce peroxynitrite. Peroxynitrite could lead to breakage and mutation of DNA, and it also reacts with protein to form 3-nitrotyrosine. Many inflammation and neural degradation diseases are related to 3-nitrotyrosine, including eye inflammation, retinal ischemia, and cancer. In 2000, a report showed that using anti-3-nitrotyrosine together with Western blotting and mass spectromety effectively identified proteins containing 3-nitrotyrosine in mouse’s retinal.
This study tried to determine proteins that contain 3-nitrotyrosine in human urine using 2D-PAGE and mass spectrometry. First, we eliminated most salts in our urine sample using acetone precipitation and centrifugal device, then separated the proteins by 2D-PAGE. Next, we used anti-3-nitrotyrosine antibody-base Western blotting to locate the nitrated protein.
Before identifying the proteins in 2D-PAGE, the gel was digested. Hence, different gel microwave digestion conditions were carried out to find the best conditions. The urine samples were then analyzed using this condition. Currently, we successfully identified 3 kinds proteins containing 3-nitrotyrosine, including, protein complex 4, epsilon 1 subunit; adaptor related; LIM domain only 6[Homo sapiens] and Hypothetical protein FLJ32940 isoform 1. We also found a protein, transmembrane protein 16 F[Homo sapiens], containing nitrotryptothan.

目錄
目錄
圖表目錄
附圖目錄

第一部分 乙二醛和2-氧丙醛對蛋白質的後轉譯修飾 1

英文摘要 2
中文摘要 3
第一章 緒論 4
第二章 實驗流程 9
2-1. 藥品 9
2-2. 儀器 9
2-3. 液相層析電噴灑游離串聯四極柱質譜儀 10
2-3-1. 液相層析儀系統條件 10
2-3-2. 質譜儀全掃描模式條件 10
2-4. 液相層析奈電噴灑游離串聯質譜儀分析系統 10
2-4-1. 液相層析儀系統條件 10
2-4-2. 質譜儀全質譜掃描模式條件 11
2-5. 實驗步驟 11
2-5-1. N?acetyl-cysteine 與 glyoxal或methylglyoxal 之反應 11
2-5-2. N?acetyl-cysteine及N?acetyl-L-lysine與 glyoxal或methylglyoxal 之交聯反應 11
2-5-3. Somatostatin 與 glyoxal或methylglyoxal 反應 12
2-5-4. 人類血紅蛋白與 glyoxal或methylglyoxal 反應 12
2-5-5. 胰蛋白酶消化蛋白質之反應 12
2-6. 利用液相層析奈電噴灑游離串聯質譜儀分析系統量測經胰蛋白酶消化過的人類血紅蛋白的定性分析 12
第三章 實驗結果與討論 14
3-1. 液相層析電噴灑游離串聯質譜儀分析N?acetyl-cysteine、N?acetyl-L-lysine與 乙二醛、2-氧丙醛反應 14
3-2. 液相層析奈電噴灑游離串聯質譜儀分析somatostatin與乙二醛、2-氧丙醛反應 15
3-3. 液相層析奈電噴灑游離串聯質譜儀分析經胰蛋白酶消化的人類蛋白與glyoxal或methylglyoxal反應 16
第四章 結論 24

第二部分 利用二維電泳搭配質譜法鑑定人類尿液中含3-硝基酪胺酸的蛋白質25
英文摘要 26
中文摘要 27
第五章 緒論 28
第六章 實驗流程 41
6-1. 藥品 41
6-2. 儀器 42
6-3. 液相層析奈電噴灑游離串聯質譜儀分析系統 43
6-3-1. 液相層析儀系統條件 43
6-3-2. 質譜儀全質譜掃描模式條件 43
6-4. 實驗步驟 43
6-4-1. 蛋白質的硝化反應 43
6-4-2. SDS-PAGE測試一級抗體的回收次數以及西方轉漬法的轉漬時間43
6-4-3. SDS-PAGE分離經過氧化亞硝酸根硝化後的蛋白質 45
6-4-4. 以傳統酵素水解法水解凝膠中的蛋白質 46
6-4-5. 以微波輔助酵素水法水解解凝膠中蛋白質 47
6-4-6. 利用液相層析奈電噴灑游離串聯質譜儀分析硝化後的蛋白質 47
6-5. 利用2D-PAGE搭配西方轉漬法分析尿液中被自然硝化的蛋白質 47
6-5-1. 尿液樣品前處理 47
6-5-2. 蛋白質定量 48
6-5-3. SDS PAGE 分離尿液蛋白 48
6-5-4. 二維電泳分離尿液蛋白 49
6-5-5. Silver stain 銀染法 50
6-5-6. 判定凝膠中含3-硝基酪胺酸蛋白質 51
6-5-7. 人類尿液中含3-硝基酪胺酸蛋白質的身份鑑定 51
第七章 實驗結果與討論 52
7-1. 十二烷基硫酸鈉凝膠電泳測試一級抗體的回收次數以及西方轉漬法的轉漬時間 52
7-2. 測試不同的凝膠微波水解條件 52
7-3. 二維電泳分析搭配西方轉漬法分析尿液中被硝化的蛋白質 67
7-3-1. 測定人類尿液中蛋白質的含量 67
7-3-2. 二維電泳分析人類尿液蛋白 68
7-3-3. 西方轉漬法測定人類尿液中3-硝基酪胺酸蛋白質 68
7-3-4. SDS PAGE 分離尿液蛋白及判定含3-硝基酪胺酸蛋白質的位置 69
7-3-5. 人類尿液中含3-硝基酪胺酸蛋白質的身份鑑定 69
第八章 結論 71
第九章 參考文獻 72
附圖 84
中英對照表 103




















圖表目錄

第一部分 乙二醛和2-氧丙醛對蛋白質的後轉譯修飾 1

圖一. 高等糖化最終產物與蛋白質產生褐色、螢光和交聯 4
圖二. 乙二主要來源及其反應途徑 6
圖三. 甘油醛-3-磷酸 (glyceraldehydes- 3-phosphate, G-3-P) 的自發性分解 6
圖四. 乙二醛及2-氧丙醛的去毒化 7
圖五. hydroimidazolone (MG-H1)及Nε-(carboxymethyl)lysine (CML)、S-(carboxymethyl)cysteine (CMC) 8
表一. 已偵測到被乙二醛或2-氧丙醛修飾的人類血紅蛋白上的胺基酸位置 17
表二. 人類血紅蛋白中所有半胱胺酸側鏈暴露的表面積 21
表三. 人類血紅蛋白中所有精胺酸側鏈暴露的表面積 21
表四. 人類血紅蛋白中所有離胺酸側鏈暴露的表面積 22

第二部分 利用二維電泳搭配質譜法鑑定人類尿液中含3-硝基酪胺酸的蛋白質25
圖六. 亞硝酸根與次氯酸生成硝基氯的反應 30
圖七. 超氧負離子與一氧化氮生成過氧化亞硝酸的反應 30
圖八. biuret method 的反應機制 35
圖九. PAGE的形成反應 37
圖十. 西方轉漬法轉漬簡圖 39
圖十一. 西方轉漬法中抗體結合模式及放光反應 40
表五. 經過氧化亞硝酸根硝化後的5種混合蛋白質在凝膠中於37 ℃下，水解18小時的水解效率 54
表六. 經過氧化亞硝酸根硝化後的5種混合蛋白質在凝膠中於不同微波必v相同時間的微波水解條件的水解效率 55
表七. 經過氧化亞硝酸根硝化後的5種混合蛋白質在凝膠中於相同微波必v不同時間的微波水解條件的水解效率 56
表八. 經過氧化亞硝酸根硝化後的5種混合蛋白質在水溶液中於37 ℃下，水解18小時的水解效率 58
表九. 經過氧化亞硝酸根硝化後的5種混合蛋白質水溶液在水溶液中於不同微波必v相同時間的微波水解條件的水解效率 59
表十. 經過氧化亞硝酸根硝化後的5種混合蛋白質在水溶液中於相同微波必v不同時間的微波水解條件的水解效率 61
表十一. 經過氧化亞硝酸根硝化後的5種混合蛋白質在凝膠中經微波水解後由不同萃取pH值萃取的效率 62
表十二. 經過氧化亞硝酸根硝化後的蛋白質在凝膠中添加30 % ACN的有機溶劑進行微波輔助酵素水解條件的水解效率 64
表十三. 經過氧化亞硝酸根硝化後的蛋白質在凝膠中添加50 % MeOH的有機溶劑進行微波輔助酵素水解條件的水解效率 65
表十四. 經過氧化亞硝酸根硝化後的蛋白質在水溶液中添加30 % ACN的有機溶劑進行微波輔助酵素水解條件下的水解效率 66
表十五. 經過氧化亞硝酸根硝化後的蛋白質在水溶液中添加50 % MeOH的有機溶劑進行微波輔助酵素水解條件下的水解效率 67


附圖目錄

第一部分 乙二醛和2-氧丙醛對蛋白質的後轉譯修飾

附圖一. N?acetyl-cysteine與glyoxal修飾反應 (上圖)的液相層析電噴灑游離
質譜儀層析圖 (SIM)及質譜圖 (下圖) 84
附圖二. N?acetyl-cysteine、N?acetyl-L-lysine與glyoxal交聯反應 (上圖)的液相層析電噴灑游離質譜儀層析圖 (SIM)及質譜圖 (下圖) 84
附圖三. N?acetyl-cysteine與 methylglyoxal修飾反應 (上圖)的液相層析電噴灑游離質譜儀層析圖 (SIM)及質譜圖 (下圖) 85
附圖四. N?acetyl-cysteine、N?acetyl-L-lysine與 methylglyoxal交聯反應 (上圖)的液相層析電噴灑游離質譜儀層析圖 (SIM)及質譜圖 (下圖) 85
附圖五. somatostatin (上圖) 在 m/z 820、somatostatin A-gx、somatostatin K-gx (中圖) 在 m/z 849 以及somatostatin A-gx-K (下圖) 在 m/z 840下的液相層析奈電噴灑游離質譜儀層析圖。 86
附圖六. somatostatin (上圖) 在 m/z 820、somatostatin A-gx、somatostatin K-gx (中圖) 在 m/z 856 以及somatostatin A-gx-K (下圖) 在 m/z 847下的液相層析奈電噴灑游離質譜儀層析圖。 86
附圖七. somatostatin (上圖) 在 m/z 820、somatostatin A-gx、somatostatin K-gx (中圖) 在 m/z 849 以及somatostatin A-gx-K (下圖) 在 m/z 840下的碰撞誘導解離質譜圖 (LTQ)。 87
附圖八. somatostatin (上圖)在 m/z 820、somatostatin A-Mgx在 m/z 856、somatostatin K-Mgx (中圖) 以及somatostatin A-Mgx-K (下圖) 在 m/z 847下的碰撞誘導解離質譜圖 (LTQ)。 88
附圖九. 人類血紅蛋白a-蛋白上含Ne-(carboxymethyl)lysine之胜肽TNV11KgxAAWGK在m/z 517.4下的碰撞誘導解離質譜圖。 89
附圖十. 人類血紅蛋白a-蛋白上含L92RVDPVNFK胜肽的碰撞誘導解離質譜圖(上圖)及Ne-(carboxymethyl)arginine之胜肽L92RgxVDPVNFK在m/z 573.6下的碰撞誘導解離質譜圖(下圖)。 89
附圖十一. 人類血紅蛋白b-蛋白上含Ne-(carboxymethyl)lysine之胜肽SAVTALWG17KgxVNVDEVGGEALGR在m/z 1144下的碰撞誘導解離質譜圖。 90
附圖十二. 人類血紅蛋白b-蛋白上含Ne-(carboxymethyl)lysine之胜肽VVAGVANALAH144KgxYH在m/z 754.3下的碰撞誘導解離質譜圖。
90
附圖十三. 人類血紅蛋白b-蛋白上含S-(carboxymethyl)cysteine之胜肽GTFATLSELH93CgxDK在m/z 740.6下的碰撞誘導解離質譜圖。 91
附圖十四. 人類血紅蛋白a-蛋白上含Ne-(carboxyethyl)arginine之胜肽GAEALE31RMgxMF在m/z 548.2下的碰撞誘導解離質譜圖。(以chymotrypsin 水解) 91
附圖十五. 人類血紅蛋白b-蛋白上含LHVDPENF104R胜肽的碰撞誘導解離質譜圖(上圖)及Ne-(carboxyethyl)arginine之胜肽LHVDPENF104RMgx在m/z 591下的碰撞誘導解離質譜圖。 92
附圖十六. 人類血紅蛋白b-蛋白上含Ne-(carboxyethyl)lysine之胜肽VVAGVANALAH144KMgxYH在m/z 762下的碰撞誘導解離質譜圖。
93
附圖十七. 人類血紅蛋白a-蛋白上與乙二醛、2-氧丙醛含hydro-
imidazolone之胜肽L92RVDPVNFK 在m/z 571.3下的碰撞誘導解離質譜圖。 94
附圖十八. 人類血紅蛋白a-蛋白上GAEALE31RMF之胜肽與2-氧丙醛含hydroimidazolone之胜肽GAEALE31RH-MgxMF在m/z 539.3下的碰撞誘導解離質譜圖。 95

第二部分 利用二維電泳搭配質譜法鑑定人類尿液中含3-硝基酪胺酸的蛋白質

附圖十九. 抗體回收次數及不同轉漬時間的條件測試 96
附圖二十. 抗體回收次數及不同轉漬時間的條件測試柱狀圖 96
附圖二十一. 以SDS-PAGE分析五種經過硝化反應的蛋白質 97
附圖二十二. 不同牛血蛋白質含量對UV吸收值所構成的校正曲線圖 97
附圖二十三. 人類尿液蛋白樣品經acetone沈澱、超濃縮過濾及定量後進行IEF及SDS-PAGE之後，以silver stain將凝膠染色 98
附圖二十四. 人類尿液蛋白樣品經ACN沈澱、超濃縮過濾及定量後進行IEF及SDS-PAGE之後，以silver stain將凝膠染色 99
附圖二十五. 西方轉漬法測定人類尿液中含3-硝基酪胺酸蛋白質的位置 100
附圖二十六. 人類尿液經SDS PAGE分離後以Coomassie blue染色後凝膠與經西方轉漬法之PVDF膜 100
附圖二十七. 人類尿液中含3-硝基酪胺酸蛋白質： protein complex 4, epsilon 1 subunit 上的胜肽KAVLALYNO2K在m/z 476.3下的碰撞誘導解離質譜圖。 101
附圖二十八. 人類尿液中含3-硝基酪胺酸蛋白質：LIM domain only 6上的胜肽PHCCACYNO2EAR在m/z 652.3下的碰撞誘導解離質譜圖。 101
附圖二十九. 人類尿液中含3-硝基酪胺酸蛋白質：Hypothetical protein FLJ 32940 isoform 1上的胜肽NIQKLYNO2K在m/z 476.3下的碰撞誘導解離質譜圖。 102
附圖三十. 人類尿液中含3-硝基色胺酸蛋白質transmembrane protein 16 F [Homo sapiens]上的胜肽LVFVKVHAPW NO2 EVLCTYAEIMHIK在m/z 943.5下的碰撞誘導解離質譜圖。 102

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