臺灣博碩士論文加值系統

本論文的研究目的是發展一套對磺酸化蛋白質萃取和分離的新方法，以利在質譜上分析蛋白質的磺酸化位置和後續的蛋白質體學研究。在氧化還原的訊號機制中可逆半胱胺酸氧化修飾扮演很重要的角色，這篇論文在分析過氧化還原.上半胱胺酸的氧化狀態是一個挑戰。這裡我們提出一個新的方法使用修飾二氧化鈦的奈米鑽石從混和的消化蛋白質中選擇性分離萃取磺酸根胜.包含磺酸和亞磺酸，並經由質譜分析。這個方法不管在低濃度或高複雜的環境下都會選擇性的萃取磺酸化胜.，是一種高效率的萃取工具。實驗室有發表過使用修飾聚精胺酸的奈米鑽石去萃取磺酸化胜.從酵素消化過甲酸氧化的牛血清蛋白上，這裡是補充另外一種濃縮萃取的方法。在MALDI上，我們使用修飾二氧化鈦粒子所萃取到的不同半胱胺酸位置有22個，而在 LC-ESI-MS/MS上我們可以看到所有的半胱胺酸全部35個位置。最後我們應用這個方法搭配LC-MS/MS去辨識過氧化還原.和牛血清蛋白上高度氧化的半胱胺酸位置和數量。在過氧化還原.上的四個氧化半胱胺酸C52, C71, C83, C173，都可以藉由修飾二氧化鈦的奈米鑽石對磺酸的高選擇性萃取。由實驗證明，這個新的萃取方法應用在牛血清蛋白或過氧化還原.上，是可以幫我們了解半胱胺酸的氧化狀態，而進一步去研究探討。

Reversible oxidative cysteine modifications play a critical role in the redox-based signaling mechanism. Dynamic profiling oxidation state of the cysteine residues in protein such as Peroxiredoxin (Prx) presents a formidable challenge. Here, we present a novel approach to selectively isolate sulfopeptides (peptides containing cysteine sulfonic acid and sulfinic acid) from complex digests using TiO2-coated nanodiamonds (TiO2-coated NDs) prior to MS analysis. The method was applied to selectively concentrate sulfopeptides from either a highly dilute solution or a highly complex peptide mixture. This method allowed us to identify the 22 distinct cysteine oxidation status out of a total 35 present in performic-acid-oxidized BSA by MALDI-TOF MS and all the distinct cysteine oxidation status by LC-ESI-MS/MS. Finally, we applied the new approach to identify the cysteine oxidation status of hydrogen peroxide–treated Prx and BSA by LC-ESI-MS/MS. Cysteine residues were found to display in either cysteine sulfonic acid or cysteine sulfinic acid status after hydrogen peroxide treatment. Enhanced detection of low abundance sulfopeptides containing active cysteine positions (C52, C71, C83, and C173) was achieved due to the highly selective enrichment using TiO2-coate NDs.

謝誌
中文摘要.........................................................................Ⅰ
英文摘要.........................................................................Ⅱ
目錄............................................................................Ⅲ
表目錄...........................................................................Ⅳ
圖目錄...........................................................................Ⅴ
Chapter 1. 緒論..................................................................1
1.1前言..........................................................................1
1.2生物分子中的磺酸化作用............................................................2
1.3過氧化還原.(Peroxiredoxin) ....................................................4
1.4鑑定磺酸化胜.的方法..............................................................6
1.4.1同位素標記法..................................................................6
1.4.2 MALDI-TOF MS...............................................................6
1.4.3 基質添加物...................................................................7
1.5 過甲酸氧化蛋白質................................................................8
1.6奈米鑽石於磺酸化胜.萃取效果........................................................9
1.7 實驗目的......................................................................11
1.8 文獻探討......................................................................12
Chapter 2.實驗部分................................................................13
2.1 實驗藥品......................................................................13
2.2 儀器設備......................................................................15
2.3實驗步驟.......................................................................17
2.3.1 酸洗奈米鑽石.................................................................17
2.3.2 功能性奈米粒子合成(TiO2-coated NDs)...........................................18
2.3.3 牛血清蛋白(BSA)的過甲酸氧化....................................................20
2.3.4 微波輔助酵素消化蛋白質.........................................................21
2.3.5 牛血清蛋白(BSA)的雙氧水氧化....................................................21
2.3.6 過氧化還原.(Prx)的雙氧水氧化...................................................22
2.3.7 基質溶液的配置................................................................22
2.3.8選取最佳化環境條件的PH..........................................................23
2.3.9複雜環境下奈米鑽石萃取胰島素鏈A...................................................24
2.3.10估計TiO2-coated NDs對濃縮胰島素鏈A的有效吸附量...................................25
2.3.11 估計TiO2-coated NDs對分析胰島素鏈A的回收率.....................................26
2.3.12利用TiO2-coated NDs萃取過甲酸氧化牛血清蛋白......................................27
2.3.13利用TiO2-coated NDs萃取雙氧水氧化過氧化還原......................................28
2.4 濃縮萃取數據結果分析.............................................................29
2.4.1基質輔助雷射脫附法質譜儀（MALDI-TOF-MS）..........................................29
2.4.1.1 樣品製備...................................................................29
2.4.1.2 儀器設定...................................................................29
2.4.2 液相層析電噴灑式串聯質譜儀（LC-ESI-MS/MS）.......................................30
2.4.2.1 樣品製備...................................................................30
2.4.2.2 儀器設定...................................................................30
2.4.3 數據分析.....................................................................31
2.4.3.1 MALDI-MS 過甲酸牛血清蛋白磺酸化胜.數據分析......................................31
2.4.3.2 LC-MS/MS Protein ID 數據分析...............................................31
2.4.3.3 MaxQuant軟體對磺酸化胜.分析比對...............................................32
Chapter 3.結果與討論................................................................33
3.1檢查TiO2-coated NDs是否合成順利...................................................33
3.2不同pH值對濃縮萃取胜.標準品的效果....................................................36
3.3 TiO2-coated NDs對磺酸化與磷酸化胜.的親和性比較......................................38
3.4利用TiO2-coated NDs 分析胰島素鏈A的偵測極限.........................................41
3.5 TiO2-coated NDs對分析胰島素鏈A的有效吸附量.........................................42
3.6估計TiO2-coated NDs對胰島素鏈A的回收率..............................................43
3.7 TiO2-coated NDs在複雜環境下對磺酸化胜.的選擇性......................................44
3.8過甲酸氧化牛血清蛋白的磺酸化胜.萃取...................................................46
3.9使用兩種粒子對過甲酸氧化牛血清蛋白濃縮萃取的序列覆蓋率和比較...............................48
4.0 比較這兩種粒子對過甲酸氧化牛血清蛋白濃縮萃取的磺酸化胜.和半胱胺酸 氧化位置.................................................................................50
4.1利用雙氧水氧化的磺酸化胜.萃取牛血清蛋白-液相層析二次質譜分析...............................51
4.2雙氧水氧化過氧化還原.的磺酸化胜.萃取..................................................52
4.3利用雙氧水氧化的磺酸化胜.萃取過氧化還原.-液相層析二次質譜分析..............................53
Chapter 4. 結論....................................................................55
Chapter 5. 參考文獻.................................................................56
表目錄
表1.生物體中常見後轉錄修飾種類。.........................................................1
表2 各種酸性官能基pKa值18。............................................................10
表 3.β-casein的常見的磷酸根列表，s 的個數代表前一個胺基酸上的磷酸根數目。......................39
表4.以表面修飾二氧化鈦的奈米鑽石對酵素消化過甲酸氧化的牛血清蛋白進行濃縮萃取，所得到的胜.。.........48
表5. 表面修飾二氧化鈦的奈米鑽石對酵素消化雙氧水的過氧化還原.進行濃縮萃取，送
測LC-ESI-MS/MS的數據分析。............................................................54
圖目錄
圖1. 磺酸化在生物體內的重要性。1..........................................................3
圖2. Peroxoredoxin 氧化還原催化循環機制。................................................5
圖3. MALDI-TOF 雷射脫附游離的過程。......................................................7
圖4 .酵素消化過甲酸氧化的牛血清蛋白進行濃縮萃取。.............................................8
圖5. 表胍離子基團-磺酸錯合物的結構。.......................................................10
圖6. 本研究使用的金屬氧化物為二氧化鈦，推測其對磺酸根可能的鍵結模式。............................11
圖7. 本實驗利用修飾二氧化鈦的奈米鑽石去進行濃縮萃取3 。.......................................12
圖8. 強酸酸洗前和酸洗後鑽石表面官能基示意圖。................................................17
圖9. 還原前後奈米鑽石表面官能基示意圖。.....................................................18
圖10. 功能性奈米粒子的合成順序。...........................................................20
圖11. 修飾二氧化鈦奈米鑽石純化濃縮胰島素A流程圖。.............................................25
圖12. TEM (a,b) and HRTEM (c) images of the TiO2-coated NDs. Inset in figure a report the selected area diffraction pattern of anatase TiO2..................................................33
圖13. Number distribution of acid-treated NDs and TiO2-coated NDs. The mean diameter of acid-treated NDs and was 74.9± 23.3 nm while that of TiO2-coated NDs was 152.3± 41.5 nm....................................................................................34
圖 14. Zeta potentials of acid-treated NDs ( ) and TiO2-coated NDs (□) as a function of solution pH....................................................................................35
圖 15. Effect of solution pH on affinity capture of sulfopeptides using TiO2-coated NDs. (a) Direct negative mode MALDI mass spectrum of a peptide mixture containing 1 pmol of insulin chain A (oxidized) and 10 pmol of tryptic digest of BSA. (b-d) Negative mode MALDI mass spectra of the same peptide mixture extracted at (b) pH 2.9, and(c) pH 2.5with ND pretreatment. Asterisks denote the sulfopeptide ions.........37
圖 16. Comparison of the MALDI-TOF mass spectra of sulfonated product of insulin chain A (oxidized) and insulin chain B (oxidized) and tryptic digest of of β-casein mixture containing tryptic digest of BSA ( a) Direct negative ion-mode MALDI mass spectrum of the peptide mixtures without affinity purification.(b) after enrichment using 20 μg of TiO2-coated NDs (c) Direct positive ion-mode MALDI mass spectrum of the peptide mixtures without affinity purification. (d) after enrichment using 20 μg of TiO2-coated NDs .The spectra shown in (a –b) and (c –d) were acquired in the negative and negative positive ion modes, respectively. Asterisks denote the sulfopeptide ions. Circle denote the Phosphopeptide ions.................................40
圖17. MALDI mass spectra of insulin chain A (oxidized), obtained with the ND pretreatment using 20 μg of TiO2-coated NDs. Sample concentrations at (a) 1 μM, (1 μL), (b) 0.5 μM, (1 μL) , and (c) 0.1 μM, (1 μL)......................................................................................42
圖18. Sequential enrichment of sulfopeptides from 100 pmol of chain A by TiO2-coated NDs (20 μg per extraction). The relative intensities of sulfopeptides in the spectra were analyzed by three trials, with the peak area averaged.................................................................................43
圖19. The recovery rate of sulfopeptides standard (insulin chain A(oxidized), 10 pmol) from TiO2-coated NDs (20 μg) using 12.5% NH4OH elution solution (10 μL ). The relative abundance of the sulfopeptides standard was determined by five replicate runs, with the peak area averaged..................................................................................44
圖20. MALDI mass spectra of a peptide mixture containing tryptic digest of BSA and 1 pmol of insulin chain A (oxidized) (a), and after enrichment using 20 μg of TiO2-coated NDs (b-d). The molar ratio of insulin chain A (oxidized) to BSA in the mixture is (a) 1:100, (b) 1:10, (c) 1:50, and (d) 1:100. Asterisks denote the sulfopeptide ions........................................................................45
圖21. Selective extraction of sulfopeptides from the tryptic digest of perfomic acid-oxidized BSA (7 ug). (a) Direct positive ion-mode MALDI mass spectrum of the peptide mixtures without affinity purification. (b-c) Positive ion-mode MALDI mass spectrum of the peptide mixture extracted using (b) 100 μg of PA-coated NDs and (c) 100 μg of TiO2-coated NDs, respectively. Numbers of asterisks denote the oxidized cysteine residues on sulfopepitde ions.........................................................................47
圖22. Peptide mass fingerprint (PMF) analysis of perfomic acid-oxidized BSA, obtained with enrichment using (a) TiO2-coated NDs and (b) PA-coated NDs, by MALDI TOF MS........................................................................................49
圖23. Overlaps of (a) unique sulfopeptides and (b) unique trioxidation (C) sites identified by affinity enrichment and MALDI-TOF MS using TiO2-coated NDs (blue circle) and PA-coated NDs (green circle)...................................................................................50
圖24. Structural characterization of the oxidative cysteine modifications of BSA, pretreated with 500 μM of H2O2 solution, by LC-ESI-MS/MS. Tandem mass spectra of (a) double charged sulfopeptide containing cysteine sulfinic acid at m/z = 596.269 and (b) doubly charged sulfopeptide containing cysteine sulfonic acid at m/z = 590.939...................................................................................51
圖25. Selective detection of sulfopeptides from tryptic digest of Peroxiredoxin 1 (10 μg) , pretreated with 500 μM of H2O2 solution, using TiO2-coated NDs. (a) Direct positive mode MALDI mass spectrum of the peptide mixtures without enrichment. (b) Positive mode MALDI mass spectrum of the same peptide mixture obtained with enrichment using 100 μg of TiO2-coated NDs...........................................................52
圖26. Structural characterization of the oxidative cysteine modifications of peroxiredoxin, pretreated with 500 μM of H2O2 solution, by LC-ESI-MS/MS. Tandem mass spectra of (a) quadruply charged sulfopeptide containing cysteine sulfinic acid at m/z = 596.044 and (b) doubly charged sulfopeptide containing cysteine sulfonic acid at m/z = 799.721.............................................................................54

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