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系統識別號 U0002-2808201105301500
中文論文名稱 利用毛細管電泳及MALDI-TOF/MS技術分析蛋白質
英文論文名稱 Analysis of the protein by capillary electrophoresis and MALDI-TOF/MS
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 99
學期 2
出版年 100
研究生中文姓名 薛仁瑋
研究生英文姓名 Ren-Wei Syue
學號 697160660
學位類別 碩士
語文別 中文
口試日期 2011-07-15
論文頁數 177頁
口試委員 指導教授-吳俊弘
委員-吳俊弘
委員-鄭建中
委員-陳銘凱
中文關鍵字 毛細管電泳  雷射基質輔助脫附質譜  蛋白質 
英文關鍵字 capillary electrophoresis  MALDI-TOF/MS  phosphorylation 
學科別分類 學科別自然科學化學
中文摘要 本研究主要在開發以毛細管電泳(CE)及質譜(MALDI-TOF/MS)技術做為偵測磷酸化蛋白質的新方法。我們以HPLC和MALDI確認β-酪蛋白經胰蛋白酶消化後的胜肽片段之CE析出順序。在電泳緩衝溶液(10 mM H3BO4-Na2B4O7,pH 9.35)中添加兩價金屬離子Ca2+、Sr2+、Ba2+,利用磷酸化胜肽片段與金屬離子結合而造成較大電泳遷移率變動的現象,可有效區別α-酪蛋白及β-酪蛋白的磷酸化與非磷酸化胜肽片段,當添加3 mM Ba2+時,效果最佳。此外,蛋白質經酵素消化反應(在50 mM NH4HCO3樣品緩衝溶液中,於37 ℃反應18小時)時,常會產生多切或少切的錯誤切片段。我們發現,當改變反應條件時(10 mM NH4HCO3樣品緩衝溶液,添加0.1 mM Ba2+和0.1 mM Ca2+,在37 ℃反應18小時),可得較多正確切產物,所得MALDI質譜圖數據經蛋白質資料庫比對,進行peptide mapping時,亦可得到較佳吻合度。
此外,我們發現,磷酸化胜肽片段通常偏低的MALDI訊號,可藉由在製備MALDI樣品時將添加物(酸及其銨鹽)與樣品和基質混合,而得到明顯的改善。使用添加物100 mM HCl + 100 mM NH4Cl的混合溶液可大幅提高α-酪蛋白及β-酪蛋白上磷酸化胜肽片段的MALDI訊號。另外,以0.1 % PEO (Mwt:106)及100 mM蜜二糖(Melibiose)做為MALDI樣品添加物,亦能提高不同分子量的Polylysine在質譜上的訊號強度。最後,我們發展出新的MALDI樣品製備方法,將最被廣泛應用,但也卻最易形成不均勻結晶的2,5-Dihydroxybenzoic acid(2,5-DHB)基質所製備樣品之結晶均勻化;在基質及樣品混合點盤後,急速冷凍(-20 ℃),並經真空乾燥。此樣品製備方法可大幅提高MALDI訊號的再現性,並增加利用MALDI進行定量分析的應用性。
英文摘要 In this research we mainly aimed to develop capillary electrophoresis (CE) and MALDI-TOF/MS based techniques for the detection of phosphoproteins. We established the elution orders of the tryptic digest of β-casein for CE electropherogram by HPLC and MALDI. With the addition of divalent metal ion Ba2+, Ca2+, and Sr2+ into the CE buffer (10 mM H3BO4-Na2B4O7,pH 9.35), and owing to the preferential binding of metal ions onto phosphopeptides, larger mobility shifts could be observed for phosphopeptides, and thus it was feasible to distinguish between phosphorylated and non-phosphorylated peptide fragments for the tryptic digests of α-casein and β-casein. The best result could be obtained when 3 mM Ba2+ was added. In addition, in the reaction conditions of 50 mM NH4HCO3 sample buffer, 37 ℃ reaction temperature, and 18-hr reaction duration, some false peptide fragments of miss-cut or over-cut could be found from the products of enzymatic digestion of proteins. According to our experimental results, more correctly digested fragments could be produced when the reaction conditions were changed to 10 mM NH4HCO3 sample buffer with the addition of 0.1 mM Ba2+ and 0.1 mM Ca2+, 37 ℃ reaction temperature, and 18-hr reaction duration. Better agreement with the on-line protein data bank could be achieved when performing peptide mapping process for the MALDI spectrum obtained from the above-mentioned new reaction conditions.
Furthermore, we found that the usually low MALDI signals of the phosphorylated peptide fragments could be improved by mixing the additives, including acids and ammonium salts, with sample and matrix while preparing MALDI sample. The MALDI signals of the phosphorylated peptide fragments in trypic digest of α-casein and β-casein could be largely raised by the 100 mM HCl + 100 mM NH4Cl additive. Moreover, both MALDI sample additives, 0.1 percentage PEO (Mwt : 106) and 100 mM melibiose, proved to be able to increase the MALDI signals of polylysine samples with different molecular weights. Finally, we developed a new sample preparation method to make homogenous sample when using 2,5-Dihydroxybenzoic acid (2,5-DHB) as MALDI matrix, which usually resulted in hetergenous MALDI sample. After placing the mixture of matrix and sample on the sample plate, the sample was frozen quickly at -20 ℃ and then dried with the vacuum. This new sample preparation method could largely enhance the reproducibility of measured MALDI signals and increase the applicability of MALDI on quantitative analysis.
論文目次 第一章 緒論............................................................................................1
1-1研究動機與目的..........................................................................1
1-2 研究背景.....................................................................................3
1-2.1蛋白質簡介.........................................................................3
1-2.2 磷酸化蛋白(Phosphoprotein)簡介....................................6
1-2.3 蛋白質定序方法簡介.....................................................10
1-2.4 毛細管電泳簡介............................................................11
1-2.5 基質輔助雷射脫附游離質譜法(MALDI-TOF/MS)簡介
........................................................................................13
1-2.6 MALDI-TOF/MS基質簡介............................................16
1-2.7 聚離胺酸(Poly(L-lysine))簡介......................................18
1-3 本章參考資料..........................................................................20
第二章 實驗方法..................................................................................29
2-1實驗儀器..................................................................................29
2-2 實驗方法.................................................................................30
2-3 毛細管內壁塗覆方式.............................................................36
2-4 實驗方法與步驟.....................................................................38
2-5 本章參考資料.........................................................................51
第三章 結果與討論..............................................................................52
3-1 利用HPLC純化及MALDI鑑定β-酪蛋白(Tryptic digest)胜
肽片段以建構CE圖譜..........................................................52
3-1.1 鑑定磷酸化β-酪蛋白胜肽............................................52
3-1.2 建構β-酪蛋白胜肽片段之電泳圖譜............................55
3-2 利用毛細管電泳技術偵測磷酸化胜肽.................................73
3-2.1 金屬離子對β-casein(Tryptic digest)電泳遷移變動率影
響..................................................................................74
3-2.2 金屬離子對 α-casein(Tryptic digest)電泳遷移變動率影
響..................................................................................81
3-3 錯誤切Bovine β-casein tetraphosphopeptide(P4)樣品之胰蛋
白酶消化反應實驗進行........................................................91
3-3.1 利用電泳技術分析P4胜肽樣品經胰蛋白酶再消化反
應..................................................................................92
3-3.2 利用MALDI-TOF/MS偵測P4胜肽樣品經胰蛋白酶再
消化反應......................................................................99
3-4 提高β-casein(Tryptic digest)胜肽片段正確切比例之消化條
件..........................................................................................104
3-4.1 在不同種類銨鹽中進行消化反應.............................104
3-4.2 在不同濃度碳酸氫銨中進行消化反應......................105
3-4.3 以不同反應溫度和反應時間進行消化反應..............111
3-4.4 在10 mM NH4HCO3中添加Ba2+、Ca2+ in對β-casein
消化反應的影響........................................................112
3-4.5 利用線上資料庫分析比對MALDI數據鑑定蛋白質序
列................................................................................121
3-5 利用MALDI-TOF/MS技術偵測磷酸化胜肽.....................123
3-5.1 以酸類添加物提高β-casein磷酸化胜肽之MALDI訊
號................................................................................123
3-5.2 以銨鹽添加物提高β-casein磷酸化胜肽之MALDI訊
號................................................................................126
3-5.3 以酸加銨鹽添加物提高β-casein磷酸化胜肽之MALDI
訊號............................................................................130
3-5.4 以HCCA為MALDI基質偵測β-casein磷酸化胜肽.131
3-5.5 以酸及銨鹽添加物來提高α-casein磷酸化胜肽之
MALDI訊號...............................................................137
3-6 改良MALDI-TOF/MS樣品結晶方法以提高定量分析的再
現性......................................................................................139
3-6.1改良結晶方法偵測α-casein(Tryptic digest)胜肽片段.140
3-6.2改良結晶方法偵測胜肽標準品(PEP STD).................147
3-6.3改良結晶方法偵測蛋白質標準品(PRO STD)............157
3-7 利用MALDI-TOF/MS技術偵測聚離胺酸(Polylysine).....163
3-7.1 濃度效應對Polylysine在MALDI中偵測之探討.....163
3-7.2 利用不同分子量PEO做為基質添加物偵測Polylysine
....................................................................................165
3-7.3 改變不同MALDI基質偵測Polylysine樣品............169
3-7.4 利用蜜二糖(Melibiose)做為基質添加物偵測Polylysine
....................................................................................170
3-8 結論.......................................................................................174
3-9 本章參考資料.......................................................................176


圖表索引
表3-1 (a) P4樣品和標準品之CE UV吸收峰的積分陎積(由圖3-25 ~
3-28計算而得)
(b) P4和STD的CE UV吸收峰積分陎積比(由(a)計算而得)...98
表3-2 P4樣品和標準品之MALDI的積分陎積(由圖3-29 ~ 3-32計
算而得).......................................................................................103
表3-3 β-casein在不同組成樣品緩衝溶液中進行胰蛋白酶消化反應
(37 ℃、18小時)所得相關正確切和錯誤切胜肽百分比之比較
(a) CE分析數據;(b) MALDI分析數據....................................110
表3-4 β-casein在不同NH4HCO3濃度-溫度和反應時間進行消化
反應所得相關錯誤切和正確切胜肽百分比之比較
(a) CE分析數據;(b)MALDI分析數據.....................................120
表3-5 利用線上資料庫對不同反應條件所得MALDI分析數據進行
蛋白質序列比對之分數............................................................122
表3-6 以不同添加物和2,5-DHB基質製備樣品所得α-casein磷酸
化胜肽之訊號強度比較表........................................................138
表3-7 在不同方式所製備α-casein(Tryptic digest)樣品之MALDI訊
號的平均RSD*..........................................................................144
IX
表3-8 在不同方式所製備PEP STD樣品之MALDI訊號的平均
RSD*..........................................................................................149
表3-9 在不同方式所製備PRO STD樣品之MALDI訊號的平均
RSD*..........................................................................................160


圖3-1 磷酸化 (a) β-酪蛋白胜肽樣品的HPLC層析圖
(b)經去磷酸反應之β-酪蛋白胜肽樣品的HPLC圖....53
圖3-1 磷酸化 (c) β-酪蛋白胜肽樣品在鹼性緩衝溶液之電泳圖
(d)經去磷酸反應之β-酪蛋白胜肽樣品在鹼性緩衝溶液
之電泳圖..................................................................54
圖3-2 (a) HPLC樣品(胜肽片段 M.w. 646)質譜圖
(b) HPLC樣品(胜肽片段 M.w. 646)在酸性緩衝溶液之電泳圖
(c) HPLC樣品(胜肽片段 M.w. 646)在鹼性緩衝溶液之電泳圖
................................................................................................56
圖3-3 (a) HPLC樣品(胜肽片段 M.w. 742)質譜圖
(b) HPLC樣品(胜肽片段 M.w. 742)在酸性緩衝溶液之電泳圖
(c) HPLC樣品(胜肽片段 M.w. 742)在鹼性緩衝溶液之電泳圖
................................................................................................58
圖3-4 (a) HPLC樣品(胜肽片段 M.w. 748)質譜圖
(b) HPLC樣品(胜肽片段 M.w. 748)在酸性緩衝溶液之電泳圖
(c) HPLC樣品(胜肽片段 M.w. 748)在鹼性緩衝溶液之電泳圖
................................................................................................59
圖3-5 (a) HPLC樣品(胜肽片段 M.w. 780)質譜圖
(b) HPLC樣品(胜肽片段 M.w. 780)在酸性緩衝溶液之電泳圖
(c) HPLC樣品(胜肽片段 M.w. 780)在鹼性緩衝溶液之電泳圖
................................................................................................60
圖3-6 (a) HPLC樣品(胜肽片段 M.w. 830)質譜圖
(b) HPLC樣品(胜肽片段 M.w. 830)在酸性緩衝溶液之電泳圖
(c) HPLC樣品(胜肽片段 M.w. 830)在鹼性緩衝溶液之電泳圖
................................................................................................62
圖3-7 (a) HPLC樣品(胜肽片段 M.w. 1383)質譜圖
(b) HPLC樣品(胜肽片段 M.w. 1383)在酸性緩衝溶液之電泳圖
(c) HPLC樣品(胜肽片段 M.w. 1383)在鹼性緩衝溶液之電泳圖
................................................................................................63
圖3-8 (a) HPLC樣品(胜肽片段 M.w. 2061)質譜圖
(b) HPLC樣品(胜肽片段 M.w. 2061)在酸性緩衝溶液之電泳圖
(c) HPLC樣品(胜肽片段 M.w. 2061)在鹼性緩衝溶液之電泳圖
(d) HPLC樣品(胜肽片段 M.w. 2061)在鹼性緩衝溶液之電泳放
大圖........................................................................................65
圖3-9 (a) HPLC樣品(胜肽片段 M.w. 2966)質譜圖
(b) HPLC樣品(胜肽片段 M.w. 2966)在鹼性緩衝溶液之電泳圖
(c) HPLC樣品(胜肽片段 M.w. 2966)在鹼性緩衝溶液之電泳放
大圖........................................................................................66
圖3-10 (a) HPLC樣品(胜肽片段 M.w. 3122)質譜圖
(b) HPLC樣品(胜肽片段 M.w. 3122)在鹼性緩衝溶液之電泳圖
(c) HPLC樣品(胜肽片段 M.w. 3122)在鹼性緩衝溶液之電泳放
大圖........................................................................................68
圖3-11 (a) HPLC樣品(胜肽片段 M.w. 5316)質譜圖
(b) HPLC樣品(胜肽片段 M.w. 5316)在鹼性緩衝溶液之電泳
圖... ...................................................... ................................ 69
圖3-12 (a) HPLC樣品(胜肽片段 M.w. 6359)質譜圖
(b) HPLC樣品(胜肽片段 M.w. 6359)在鹼性緩衝溶液之電泳
圖..........................................................................................71
圖3-13 (a) β-酪蛋白(Tryptic digest)在鹼性緩衝溶液電泳圖
(b) β-酪蛋白(Tryptic digest)在酸性緩衝溶液電泳圖..............72
圖3-14 (a)在電泳緩衝溶液中添加不同濃度Sr2+所得之β-casein胜肽
電泳圖(b)根據(a)的結果計算所得Sr2+對β-casein之Mobility shift ratio的影響..........................................................................75
圖3-15 (a)在電泳緩衝溶液中添加不同濃度Ba2+所得之β-casein胜肽
電泳圖
(b)根據(a)的結果計算所得Ba2+對β-casein之Mobility shift
ratio的影響..........................................................................77
圖3-16 (a)在電泳緩衝溶液中添加不同濃度Ca2+所得之β-casein胜肽
電泳圖
(b)根據(a)的結果計算所得Ca2+對β-casein之Mobility shift
ratio的影響..........................................................................78
圖3-17 (a)比較在電泳緩衝溶液中添加相同濃度Ba2+、Ca2+及Sr2+所
得之β-casein胜肽電泳圖
(b)根據(a)的結果計算所得對β-casein之Mobility shift ratio的
影響. ...................................... ...............................................80
圖3-18 (a)α-casein(Tryptic digest)磷酸化胜肽、去磷酸化胜肽和兩者混
合後之毛細管電泳圖
(b) (a)圖在6.3 min至17 min 之放大圖...................................82
圖3-19 (a) 0 mM ~ 0.75 mM Ba2+對α-casein(Tryptic digest)電泳遷移
影響(b) 0.75 mM ~ 3 mM Ba2+對α-casein(Tryptic digest)電泳遷移
影響........................................................................................86
圖3-20 (a) 0 mM ~ 0.75 mM Ba2+對α-casein(Tryptic digest)電泳遷移
影響放大圖
(b) 0.75 mM ~ 3 mM Ba2+對α-casein(Tryptic digest)電泳遷移
影響放大圖............................................................................87
圖3-21 根據圖3-19的結果計算所得Ba2+對α-casein之Mobility
shift ratio的影響........................................................................88
圖3-22 (a) 0 mM ~ 2 mM Ca2+對α-casein(Tryptic digest)電泳遷移影

(b) 2 mM ~ 5 mM Ca2+對α-casein(Tryptic digest)電泳遷移影
響..........................................................................................89
圖3-23 (a) 0 mM ~ 2 mM Ca2+對α-casein(Tryptic digest)電泳遷移影
響放大圖
(b) 2 mM ~ 5 mM Ca2+對α-casein(Tryptic digest)電泳遷移影
響放大圖................................................................................90
圖3-24 根據圖3-22的結果計算所得Ca2+對α-casein之Mobility
shift ratio的影響........................................................................91
圖3-25 P4(X)胜肽樣品溶於去離子水中放置於37 ℃ 18至90小時
之毛細管電泳圖........................................................................94
圖3-26 P4(X)胜肽樣品溶於50 mM醋酸中放置於37 ℃ 18至90小
時之毛細管電泳圖,實驗條件如圖3-25..................................95
圖3-27 20 μM P4(X)胜肽樣品(in 50 mM NH4HCO3)經由37 ℃ Tryptic digest反應18至90小時之毛細管電泳圖,實驗條件如圖3-25之電泳圖.... .................... .................... .....................................96
圖3-28 20 μM P4(X)胜肽樣品(in 50 mM NH4HCO3)經由50 ℃ Tryptic digest反應18至66小時之毛細管電泳圖,實驗條件如圖3-25之電泳圖....................................................................................97
圖3-29 P4胜肽樣品溶於ddH2O,置於37℃數天,且不經胰蛋白消
化反應的 MALDI圖。MALDI偵測條件如圖2-4.................101
圖3-30 P4胜肽樣品溶於50 mM 醋酸,置於37 ℃18 ~ 90小時且
不經胰蛋白酶消化反應的MALDI圖。實驗條件如圖3-29..101
圖3-31 P4胜肽樣品在37 ℃經胰蛋白酶消化反應18 ~ 90小時所得
產物之MALDI圖,實驗條件同圖3-29..................................102
圖3-32 P4胜肽樣品在50 ℃經胰蛋白酶消化反應18 ~ 66小時所得
產物之MALDI圖,實驗條件同圖3-29..................................102
圖3-33 β-casein在不同銨鹽水溶液中進行胰蛋白酶消化反應所得胜
肽片段之CE電泳圖。消化反應之步驟如2-4(3)所述............107
圖3-34 (a)在不同濃度NH4HCO3的樣品緩衝溶液中進行胰蛋白酶消
化反應所得產物之電泳圖
(b)圖(a)在4至12分鐘的放大圖.............................................108
圖3-35 β-casein在不同濃度NH4HCO3中進行胰蛋白酶消化反應所得
產物的MALDI圖,MALDI實驗條件同圖2-4
(a)為P4(X)和P4(O)之質圖譜比較
(b)為1383(X)和2186(O)之質譜圖比較.................................109
圖3-36 (a) β-casein在50 mM NH4HCO3中,於37 ℃經胰蛋白酶消
化反應6 ~ 18小時所得產物之電泳圖
(b)為(a)在4 ~ 12分鐘之放大圖..............................................114
圖3-37 (a) β-casein在50 mM NH4HCO3中,於50 ℃經胰蛋白酶消
化反應6 ~ 18小時所得產物之電泳圖
(b)為(a)在4 ~ 12分鐘之放大圖..............................................115
圖3-38 (a) β-casein在10 mM NH4HCO3中,於37 ℃經胰蛋白酶消
化反應6 ~ 18小時所得產物之電泳圖
(b)為(a)在4 ~ 12分鐘之放大圖..............................................116
圖3-39 (a) β-casein在10 mM NH4HCO3中,於37 ℃經胰蛋白酶消
化反應18 ~ 24小時所得產物之電泳圖
(b)為(a)在4 ~ 12分鐘之放大圖..............................................117

圖3-40 (a) β-casein在10 mM NH4HCO3中,於50 ℃經胰蛋白酶消
化反應6 ~ 18小時所得產物之電泳圖
(b)為(a)在4 ~ 12分鐘之放大圖..............................................118
圖3-41 (a) β-casein在添加Ba2+和Ca2+的10 mM NH4HCO3中,於37 ℃
進行消化反應18小時所得產物之電泳圖
(b)為(a)在4 ~ 12分鐘之放大圖................................................119
圖3-42 不同酸類添加物所得β-casein磷酸化胜肽之MALDI圖及
P4(X)之積分陎積..................................................................125
圖3-43 以NH4HCO3和(NH4)2C6H6O7樣品添加物所測得β-casein磷酸
化胜肽MALDI圖及P4(X)之積分陎積..................................127
圖3-44 以(NH4)2SO4和NH4Cl樣品添加物所測得β-casein磷酸化胜肽
MALDI圖及P4(X)之積分陎積..............................................128
圖3-45 以NH4H2PO4和(NH4)2HPO4樣品添加物所測得β-casein磷酸
化胜肽MALDI圖及P4(X)之積分陎積..................................129
圖3-46 以MDPNA混合各種銨鹽樣品添加物所測得β-casein磷酸化
胜肽MALDI圖及P4(X)之積分陎積......................................132
圖3-47 以HCl混合各種銨鹽樣品添加物所測得β-casein磷酸化胜肽
MALDI圖及P4(X)之積分陎積..............................................133
圖3-48 以H2SO4混合各種銨鹽樣品添加物所測得β-casein磷酸化胜
肽MALDI圖及P4(X)之積分陎積..........................................134
圖3-49 以不同組成HCl + NH4Cl樣品添加物所測得β-casein磷酸化
胜肽MALDI圖及P4(X)之積分陎積......................................135
圖3-50 以HCCA為基質添加酸及銨鹽所得β-casein磷酸化胜肽
MALDI圖及P4(X)之積分陎積..............................................136
圖3-51 以不同添加物和2,5-DHB基質製備樣品所得α-casein磷酸化
胜肽的MALDI訊號強度之條狀圖……………………........139
圖3-52 以不同基質和結晶方式所製備α-casein(Tryptic digest)MALDI
樣品之結晶圖..........................................................................143
圖3-53 改變基質結晶方式偵測α-casein(Tryptic digest)所得MALDI
訊號強度之條狀圖………………………………………......145
圖3-54 改變基質結晶方式偵測α-casein(Tryptic digest)所得MALDI
訊號強度之條狀圖..................................................................145
圖3-55 改變基質結晶方式偵測α-casein(Tryptic digest)所得MALDI
訊號強度之條狀圖…………………………………………..146
圖3-56 改變基質結晶方式偵測α-casein(Tryptic digest)所得MALDI
訊號強度之條狀圖..................................................................146
圖3-57 以不同基質和結晶方式所製備PEP STD MALDI樣品之結晶
圖……………………………………………..........................148
XVIII
圖3-58圖3-58 改變基質結晶方式偵測PEP STD所得MALDI訊號強度之條狀圖..............................................................................150
圖3-59 改變基質結晶方式偵測PEP STD所得MALDI訊號強度之條狀圖..........................................................................................150
圖3-60 改變基質結晶方式偵測PEP STD(Bradykinin 1-7)訊號強度圖..............................................................................................152
圖3-61 改變基質結晶方式偵測PEP STD(Angiotensin II)訊號強度圖..............................................................................................152
圖3-62 改變基質結晶方式偵測PEP STD(Angiotensin I)訊號強度圖..............................................................................................153
圖3-63 改變基質結晶方式偵測PEP STD(Substance P)訊號強度圖..............................................................................................153
圖3-64 改變基質結晶方式偵測PEP STD(Bombesin)訊號強度圖…154
圖3-65 改變基質結晶方式偵測PEP STD(Renin Substrate)訊號強度圖..............................................................................................154
圖3-66 改變基質結晶方式偵測PEP STD(ACTH clip 1-17)訊號強度圖..............................................................................................155
圖3-67圖3-67 改變基質結晶方式偵測PEP STD(ACTH clip 18-39)
訊號強度圖..............................................................................155
圖3-68改變基質結晶方式偵測PEP STD(Somatostatin 28)訊號強度圖..............................................................................................156
圖3-69以SA和DHB基質在不同結晶方式製備PRO STD MALDI
樣品之結晶圖..........................................................................159
圖3-70 改變基質結晶方式偵測PRO STD所得MALDI訊號強度之
條狀圖......................................................................................160
圖3-71改變基質結晶方式偵測PRO STD(Insulin)訊號強度圖….....161
圖3-72 改變基質結晶方式偵測PRO STD(Ubiquitin I)訊號強度圖.161
圖3-73 改變基質結晶方式偵測PRO STD(Cytochrom C)訊號強度圖..............................................................................................162
圖3-74 改變基質結晶方式偵測PRO STD(Myoglobin)訊號強度圖.162
圖3-75 利用HCCA偵測不同濃度Polylysine (M.w. 4,000~15,000)之MALDI圖...............................................................................164
圖3-76 利用HCCA偵測不同濃度Polylysine (M.w. 20,000~30,000)之MALDI圖...............................................................................164
圖3-77 利用HCCA添加PEO 60萬偵測Polylysine (M.w. 4,000 ~ 15,000)之MALDI圖................................................................166
圖3-78 利用HCCA添加PEO 60萬偵測Polylysine (M.w. 20,000 ~ 30,000)之MALDI圖................................................................166
圖3-79 利用HCCA添加PEO 100萬偵測Polylysine (M.w. 4,000 ~ 15,000)之MALDI圖................................................................167
圖3-80 利用HCCA添加PEO 100萬偵測Polylysine (M.w. 20,000 ~ 30,000)之MALDI圖................................................................167
圖3-81 利用HCCA添加PEO 800萬偵測Polylysine (M.w. 4,000 ~ 15,000)之MALDI圖................................................................168
圖3-82 利用HCCA添加PEO 800萬偵測Polylysine (M.w. 20,000 ~ 30,000)之MALDI圖................................................................168
圖3-83 利用HCCA與DHB偵測Polylysine (M.w. 4,000 ~ 15,000)
之MALDI圖............................................................................169
圖3-84 利用HCCA與DHB偵測Polylysine (M.w. 20,000 ~ 30,000)
之MALDI圖............................................................................170
圖3-85 利用DHB添加Melibiose偵測Polylysine (M.w. 4,000 ~
15,000) 之MALDI圖..............................................................172
圖3-86 利用DHB添加Melibiose偵測Polylysine (M.w. 20,000~
30,000)之MALDI圖................................................................173
參考文獻 第一章參考資料
1. Hochstrasser, D.F. “Proteome in perspective”., Clin. Chem. Lab. Med.1998, 36, 825-836.
2. Kuyama, H.; Sonomura, K. ; Nishimura, O., “Sensitive detection of phosphopeptides by matrix-assisted laser desorption/ionization mass spectrometry:use of alkylphosphonic acids as matrix additives”., Rapid Commun. Mass Spectrom. 2008, 22,109-1116.
3. Choi, H.; Lee, H. ; Park, Z., “Detection of multiphosphorylated peptides in LC-MS/MS analysis under low pH conditions”., Anal. Chem. 2008, 80, 3007-3015.
4. Nelson, D. L. ; Cox, M. M., “Lehninger Principles of Biochemistry
3rd ed.”., 2000, New York : Worth Publishers.
5. Kyte, J. ; Doolittle, R. F., “A simple method for displaying the
hydropathic character of a protein”., J. Mol. Biol. 1982, 157(1),
105-132.
6. Decker, R. V. ; Foster, J. F., “The interaction of bovine plasma albumin with detergent anions. Stoichiometry and mechanism of binding of alkylbenzenesulfonates”., Biochemistry 1966, 1242–1249.
7. Reynolds, J. A. ; Herbert, S. H. ; Polet ; Steinhardt, J., “The binding of divers detergent anions to bovine serum albumin”.,
Biochemistry 1967, 937–943.
8. Ikai, A., “Stepwise degradation of serum low density lipoprotein by sodium dodecyl sulfate”., J. Biochem. 1976, 679–688.
9. Konigsberg, W., “Reduction of disulfide bonds in proteins with
dithiothreitol”., Methods Enzymol 1972, 25, 185-188.
10. Tanford, C., “Protein denaturation Theoretical models for the mechanism of denaturation”., Adv. Protein Chem. 1970, 1-95.
11. Randle, P. J.,”Mechanism in regulation : protein phosphrylation”., 1997, 203-237, Greenwich : JAI Press.
12. Niall, H., “Automated Edman degradation: the protein sequenator”., Meth. Enzymol. 1973, 27, 942-1010.
13. Taisuke, O. ; Qian, Z. ; Qingyuan, G. ; Thomas, V. ; John, A. B.,
“Protein phosphorylation in neutrophils monitored with
phosphospecific antibodies”., J. of Immun. Methods 2003, 281, 79-94.
14. Nemerya, N. S. ; Zemskova, M. A. ; Nyukhalkina, I. A.; Khailova, L. S., ”Chemical modification of the essential arginine residues of pyruvate dehydrogenase prevents its phosphorylation by kinase”., FEBS Letters 1996, 394, 96-98.
15. Shaohui, S. ; Jinglan, W. ; Zhuang, L. ; Yun, C. ; Yangjun, Z. ; Wenfeng, Y. ; Xiaohong, Q., “Phosphopeptide enrichment strategy based on strong cation exchange chromatography”., Chinese Journal of Chromatography 2008, 26, 195-199.
16. Yan, J. X. ; Packer, N. H. ; Gooley, A. A. ; Williams, K. L., “Protein phosphorylation: technologies for the identification of phosphoamino acids”., J. Chromatogr., A 1998, 808, 23-41.
17. Ficarro, S. B. ; McCleland, M. L. ; Stukenberg, P. T. ; Burke, D. J.;
Ross, M. M. ; Shabanowitz, J. ; Hunt, D. F. ; White, F. M. Nat.
Biotechnol. 2002, 20, 301-305.
18. Liu, H. ; Stupak, J. ; Zheng, J. ; Keller, B. O. ; Brix, B. J. ; Fliegel, L. ; Li, L., “Open Tubular Immobilized Metal Ion Affinity Chromatography Combined with MALDI MS and MS/MS for Identification of Protein Phosphorylation Sites”., Anal. Chem. 2004, 76, 4223-4232.
19. Stensballe, A. ; Jensen, O. N., “Phosphoric acid enhances the performance of Fe(III) affinity chromatography and matrix-assisted laser desorption/ionization tandem mass spectrometry for recovery,
detection and sequencing of phosphopeptides”., Rapid Commun. Mass Spectrom. 2004, 18, 1721-1730.
20. Jin, W. ; Dai, J. ; Zhou, H. ; Xia, Q. ; Zou, H. ; Zeng, R., “Phosphoproteome analysis of mouse liver using immobilized metal affinity purification and linear ion trap mass spectrometry”., Rapid Commun. Mass Spectrom. 2004, 18, 2169-2176.
21. Cao, P. ; Stults, J. S., “Mapping the phosphorylation sites of proteins using on-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization multiple stage tandem mass spectrometry”., Rapid Commun. Mass Spectrom. 2000, 14,
1600-1608.
22. Zhou, L. ; Kang, G. Y. ; Kim, K. P., “A binary matrix for improved detection of phosphopeptides in matrix-assisted laser desorption/ionization mass spectrometry”.,Rapid Commun Mass Spectrom. 2009, 23, 2264-2272.
23. Lin, B. ; Li, T. ; Zhao, Y. ; Huang, F. K. ; Guo, L. ; Feng, Y. Q., “Preparation of a TiO2 nanoparticle-deposited capillary column by liquid phase deposition and its application in phosphopeptide analysis”., J. Chromatogr. A 2008, 1, 95-102.
24. Wu, J. H. ; Li, X. H. ; Zhao, Y. ; Zhang, W. ; Guo, L. ; Feng, Y. Q., “Application of liquid phase deposited titania nanoparticles on silica spheres to phosphopeptide enrichment and high performance liquid chromatography packings”., J. Chromatogr., A 2011, 20, 2944-2953.
25. Huang, Y. ; Shi, Q. ; Tsung, C. K. ; P., H. ; Gunawardena.; Xie, L. ; Yu, Y. ; Liang, H. ; Yang, P. ; Stucky, G. D. ; Chen, X., “An optimized magnetite microparticle-based phosphopeptide enrichment strategy for identifying multiple phosphorylation sites in an immunoprecipitated protein”., Anal. Bio. 2011, 1, 19-31.
26. Karas, M.; Hillenkamp, F., “Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons”., Anal. Chem. 1988, 60, 2299-2301.
27. Tanaka, K. ; Waki, H. ; Ido, Y.; Akita, S. ; Yosida, Y. ; Yoshida, T., ”Protein and polymer analysis up to m/z 100000 by laser ionization time-of-flight mass spectrometry”., Rapid Commun. Mass Spectrom. 1988, 2, 151-158.
28. Claydon, M. A. ; Davey, S. N. ; EdwardsJones, V. ; Gordon, D. B., ”The rapid identification of intact microorganisms using mass spectrometry”., Nat. Biotechnol. 1996, 14, 1584-1586.
29. Holland, R. D. ; Wilkes, J. G. ; Rafii, F. ; Sutherland, J. B. ; Persons, C. C. ; Voorhees, K. J. ; Lay, J. O., ” Rapid identification of intact whole bacteria based on spectral patterns using matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry”., Rapid Commun. Mass Spectrom. 1996, 10, 1227-1232.
30. Krishnamurthy, T. ; Ross, P. L. ; Rajamani, U., “Detection of pathogenic and non-pathogenic bacteria by matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry”., Rapid Commun. Mass Spectrom. 1996, 10, 883-888.
31. Van Breemen, R. B. ; Snow, M. ; Cotter, R. J., “Time-resolved laser desorption mass spectrometry”., Int. J. Mass Spectrom.Ion Phys. 1983, 49, 35-50.
32. Liebler, D. C., “Introduction to proteomics: tools for the new biology”., 2002, Washington : Humana press.
33. Weickardt, C. ; Schmid, R. P., “Design reflectron time-of-flight mass spectrometry with and without grids a direct comparison”., Int. J. Mass Spectrom. 2001, 206, 181-190.
34. Spengler, B. ; Kirsch, D. ; Kaufmann, R., “Peptide sequencing by matrix-assisted laser desorption mass spectrometry”., Rapid Commun. Mass Spectrom. 1992, 6, 105-108.
35. Chernushevich, I., “An introduction to time-of-flight mass spectrometry”., Journal of Mass Spectrometry 2001, 36, 849.
36. Kjellstrom, S. ; Jensen, O. N., ”Phosphoric acid as a matrix additive for MALDI MS analysis of phosphopeptides and phosphoproteins”., Ana. Chemistry 2004, 17, 5109-5117.
37. Chapman, J. R., “Protein and peptide analysis ”., NY: Humana press.
38. Tang, K. ; Allman, S. L. ; Chen C. H., “Matrix-assisted laser desorption ionization of oligonucleotides with various matrices”., Rapid Communications in Mass Spectrometry 1993, 10, 943-948.
39. Tholey, A., “Ionic liquid matrices with phosphoric acid as matrix
additive for the facilitated analysis of phosphopeptides
by matrix-assisted laser desorption/ionization mass spectrometry”., Rapid Communications in Mass Spectrometry 2006, 20, 1761-1768.
40. Kuyama, H. ; Kazuhiro, S. ; Osamu, N., “Sensitive detection of
phosphopeptides by matrix-assisted laser desorption/ionization mass
spectrometry: use of alkylphosphonic acids as matrix additives”., Rapid Commun Mass Spectrom. 2008, 22, 1109-1116.
41. Nabetani, T. ; Miyazaki, K. ; Tabuse, Y. ; Tsugita, A., “Analysis of acidic peptides with a matrix-assisted laser desorption/ionization mass spectrometry using positive and negative ion modes with additive monoammonium phosphate”., Proteomics 2006, 6, 4456-4465.
42. Kang, J. ; Toita, R. ; Oishi J. ; Niidome T. ; Katayama Y., “Effect of the Addition of Diammonium Citrate to α-Cyano-4-Hydroxycinnamic Acid (CHCA) Matrix for the Detection of Phosphorylated Peptide in Phosphorylation Reactions Using Cell and Tissue Lysates”., J. Am. Soc. Mass Spectrom. 2007, 18, 1925-1931.
43. Asara, J. ; Allison, J., “Enhanced Detection of Phosphopeptides in
Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Using Ammonium Salts”., J Am Soc Mass Spectrom. 1999, 10, 35-44.
44. Soltwisch, J. ; Berkenkamp, S. ; Dreisewerd, K., “A binary matrix of 2,5-dihydroxybenzoic acid and glycerol produces homogenous sample preparations for matrix-assisted laser desorption/ionization
mass spectrometry”., Rapid Commun Mass Spectrom. 2008, 22, 59-66.
45. Segura, T. ; Shea, L.D., “Surface-Tethered DNA Complexes for Enhanced Gene Delivery”., Bioconjugate Chem. 2002, 13, 621-629.
46. Wagner, E. ; Zatloukal, K. ; Cotton, M. ; Kirlappos, H. ; Mechtler, K. ; Curiel, D.T. ; Birnstiel, M.L.,“Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes”., Proc. Natl. Acad. Sci. USA 1992, 89, 6099-6103.
47. Schlosser, G.. ; Jakab, A. ; Pocsfalvi, G. ; Vekey, K. ; Hudecz, F. ; Mezo, G., “Matrix/analyte ratio influencing polymer molecular weight distribution in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry”., Rapid Commun Mass Spectrom. 2009, 23, 1249-1254.

第二章參考資料
1. Soren, N.; Michael, D. W.; Rainer, C., “Proteomics-post-genomic cartography to understand gene function”., Trends in Pharmacological Sciences 2001, 22, 376-384.
2. Hjerten, S., “High-performance electrophoresis. Elimination of electroendosmosis and solute adsorption”., J. Chromatogr. 1985, 347, 191-198.
3. 李能佳,「測量界面活性劑的臨界微胞濃度及蛋白質的有效電荷」,碩士論文,淡江大學化學系,2001。
4. Jing-Tao, W.; Peiqing, H.; Michael, X. L.; Mark, G. Q.; David, M. L., “Open-Tubular Capillary Electrochromatography with an On-Line Ion Trap Storage/Reflectron Time-of-Flight Mass Detector for Ultrafast Peptide Mixture Analysis”., Anal.Chem. 1997, 69, 320-326.
5. Darewicz, M.; Dziuba, J.; Caessens, P. W. J. R.; Gruppen, H., “Dephosphorylation-induced structural changes in β-casein and its amphiphilic fragment in relation to emulsion properties”., Biochimie. 2000, 82, 191-195.

第三章參考資料

1. 蘇稜雅,「利用毛細管電泳分離蛋白質及測量其物性」,碩士論文,淡江大學化學系,2008。
2. Miquel, E. ; Gomez, J. A. ; Alegria, A. ; Barbera, R. ; Farre, R. ; Recio, I. , “Identification of casein phosphopeptides in β-casein and commercial hydrolysed caseinby mass spectrometry”., Food Science and Tech. International 2006, 5, 379-384.
3. Tholey, A., “Ionic liquid matrices with phosphoric acid as matrix
additive for the facilitated analysis of phosphopeptides
by matrix-assisted laser desorption/ionization mass spectrometry”., Rapid Communications in Mass Spectrometry 2006, 20, 1761-1768.
4. Kuyama, H. ; Kazuhiro, S. ; Osamu, N., “Sensitive detection of
phosphopeptides by matrix-assisted laser desorption/ionization mass
spectrometry: use of alkylphosphonic acids as matrix additives”., Rapid Commun Mass Spectrom. 2008, 22, 1109-1116.
5. Nabetani, T. ; Miyazaki, K. ; Tabuse, Y. ; Tsugita, A., “Analysis of acidic peptides with a matrix-assisted laser desorption/ionization mass spectrometry using positive and negative ion modes with additive monoammonium phosphate”., Proteomics 2006, 6, 4456-4465.
6. Kang, J. ; Toita, R. ; Oishi J. ; Niidome T. ; Katayama Y., “Effect of the Addition of Diammonium Citrate to α-Cyano-4-Hydroxycinnamic Acid (CHCA) Matrix for the Detection of Phosphorylated Peptide in Phosphorylation Reactions Using Cell and Tissue Lysates”., J. Am. Soc. Mass Spectrom. 2007, 18, 1925-1931.
7. Asara, J. ; Allison, J., “Enhanced Detection of Phosphopeptides in
Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Using Ammonium Salts”., J Am Soc Mass Spectrom. 1999, 10, 35-44.
8. Schlosser, G.. ; Takats, Z. ; Vekey, K. ; Hudecz, F. ; Mezo, G., “Polylysine characterization using mass spectrometry”., Pro. of the European Peptide Sym. 2002, 23, 404-405.
9. Jaskolla, T. K. ; Papasotirious, D. G. ; Karas, M., “Comparison between the matrices alpha-cyano-4-hydroxycinnamic acid and 4-chloro-alpha-cyanocinnamic acid for trypsin and chymotrypsin, and pepsin digestions by MALDI-TOF mass spectrometry”., Journal of proteome research 2009, 7, 3588-3597.
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