本論文主要分為三個部份，第一個部份是將毛細管電泳技術與Poiseuille’s Law及Taylor-Aris Dispersion方法相結合，用以研究蛋白質的構形變化。從實驗結果顯示，胰島素(Insulin, Ins)在pH8.4的緩衝溶液中，有聚集的行為，且緩衝溶液離子強度增高時會使其聚集行為更明顯。增加緩衝溶液的鋅離子濃度，則所測得的Ins流體動力半徑也隨之增大。當 [Zn2+]/[Ins]≧8.7時，聚合體Ins與單體Ins的體積比值顯示此時Ins已聚集形成六聚體。鋅離子誘發Ins形成聚合體的效應在較高離子強度的緩衝溶液中較為顯著。

    第二個部份是以不同種類的線性親水性高分子作為分離介質，以及在緩衝溶液中添加金屬離子，以期達到高解析度的聚胜肽毛細管電泳分離。我們以F127、dextran、PEG及纖維素等高分子對聚離胺酸(polylysine)混合樣品及經過胰蛋白酶進行消化反應後的蛋白質樣品進行毛細管電泳分離。其中以20%~30%的F127及15%~25%的Dextran有較佳的解析效果，並得到類似的消化樣品電泳圖譜，可見這兩種高分子解析聚胜肽的行為和機制是相似的。另外在緩衝溶液中添加金屬離子會改變聚胜肽的電泳行為。二價金屬離子中，鎂離子(Mg2+)的添加對聚胜肽的電泳解析度較佳。當添加高價金屬離子如鈷胺錯離子 時，會使聚胜肽的電泳圖譜產生較劇烈的改變。 

    第三個部份則是利用毛細管電泳技術，開發出新的分析方法，用以偵測磷酸化蛋白上的磷酸化位置。本實驗以牛奶中的β-酪蛋白作為主要的研究對象，將原本具有五個磷酸化位置的β-酪蛋白以酵素進行消化反應以及去磷酸化反應。利用電泳遷移率位移的現象，亦即從消化後的β-酪蛋白胜肽片段在有磷酸化修飾及去磷酸化後的電泳圖譜，可以分辨出具有磷酸化位置的片段。為了驗證結果的正確性，我們利用製備型的HPLC將在β-酪蛋白中具有磷酸化位置的胜肽片段純化分離，以質譜(MALDI-TOF)確定其序列，並進行毛細管電泳分析以確認具磷酸化胜肽之吸收峰。

This thesis consists of the results of three research subjects. In the first part we combined capillary electrophoresis (CE) technique with Poiseuille’s Law and Taylor-Aris Dispersion method to investigate the conformational changes of proteins. According to the results of this experiment, we found that insulin (Ins) would aggregate in tris/boric acid (TB, pH8.4) buffer and the aggregation of Ins would increase with increasing ionic strength. The hydrodynamic radius of Ins would increase gradually with increasing zinc concentration in TB buffer. When [Zn2+]/[Ins]≧8.7, the volume ratio of associated Ins to monomeric one showed that Ins had aggregated to form the hexameric conformation. The effect of Zn2+ induced polymeric conformation of Ins was more significant in buffer solution with higher ionic strength.
In the second part linear hydrophilic polymer and metal ion were added into CE buffer so as to enhance the separation resolution of polypeptide mixture. Both samples of polylysine mixtures and peptide fragments of myoglobin digested by trypsin were analyzed by using CE with different concentrations of F127, dextran(70K、500K、2000K), PEG, and celluloses as separation mediums. Higher separation resolutions were achieved when 20~30% F127 and 15~25% dextran were used. Moreover, the peak patterns of myoglobin digest in the electropherograms obtained from the two polymer systems were very similar. Therefore, we considered F127 and dextran to have similar resolving mechanism for the CE separation of polypeptides. We also found that the presence of metal ion in CE buffer would alter the electrophoretic migration behaviors of polypeptides. Among the divalent cations added, Mg2+ would result in better separation resolution. When multivalent cation like Co(NH3)63+ was added, the consequential peak pattern in the electropherogram of myoglobin digest differed very much from that of the other metal ions, which suggested some special binding affinities might occur between metal ion and peptides.
In the last part we developed a CE-based analytical method for the detection of phosphorylation sites in phosphoproteins. β-casein, the model phosphoprotein with five phosphorylation sites, was digested by trypsin and followed by the treatment of dephosphorylation reaction. According to the electropherograms obtained before and after dephosphorylation reaction for the digested peptide fragments, the peaks with mobility shifts indicated the peptides with phosphorylation sites. In order to confirm the result, we used preparative HPLC to purify the digested β-casein sample, MALDI-TOF to identify the sequence, and CE to differentiate the phosphorylated peptide peaks in the electropherogram.

目錄
第一章	緒論………………………………………………………….1
 1-1研究背景..………………………………………………………...1	
  1-1.1蛋白質簡介……………………………………………………1
  1-1.2變性蛋白質……………………………………………………4
  1-1.3 pH值對蛋白質的影響………………………………………..4
  1-1.4胰島素（Insulin）簡介……………………………………….5
  1-1.5磷酸化蛋白(Phosphoprotein)簡介……………………………8
  1-1.6毛細管電泳分離介質的簡介…………………………………10
 1-2相關實驗方法…………………………………………………….18
  1-2.1流體動力半徑………………………………………………....18
 1-3本章參考資料…………………………………….........................21第二章  實驗方法…………………………………………………….28
 2-1實驗原理………………………………………………………….28
  2-1.1黏度之測量…………………………………………..………..28
  2-1.2擴散係數之測量………………………………..……………..30
  2-1.3Taylor-Aris Dispersion…………………………..……………..32
 2-2實驗儀器……………………..…………………………………...36
 2-3實驗藥品…..……………………………………………………...36
 2-4毛細管內壁塗覆方式…………………………………………….39
 2-5實驗方法與步驟………………………………………………. 42
 2-6本章參考資料…………………………………………………..50
第三章  結果與討論………………………………………………..52
 3-1蛋白質物理性質之探討………………………………..............52
  3-1.1胰島素聚集行為………………………..................................52
 3-2高分子分離介質對聚胜肽毛細管電泳解析之影響..................54
  3-2.1 Polylysine在PEG中的解析行為..........................................54
  3-2.2 Polylysine在不同濃度F127中的解析行為.........................55
  3-2.3 Polylysine在Dextran中的解析行為....................................56
3-2.4緩衝溶液效應.........................................................................56
  3-2.5比較Polylysine在不同高分子中的解析行為......................57
 3-3高分子分離介質對Mb(Trypsin digest)解析之影響..................58
  3-3.1不同濃度F127的分離效果...................................................58
  3-3.2 Dextran的分子量效應...........................................................59
  3-3.3添加PA、PEO、PEG.............................................................60
  3-3.4 Agarose及Metaphor agarose的分離效果............................61
  3-3.5纖維素及其衍生物之分離效果.............................................62
 3-4離子效應對解析行為影響之探討..............................................63
  3-4.1 改變緩衝溶液的離子強度....................................................63
  3-4.2在緩衝溶液中添加不同金屬離子.........................................64
 3-5利用CE技術偵測磷酸化蛋白...................................................66
  3-5.1在酸性條件中偵測β-酪蛋白胜肽樣品.................................67
  3-5.2 在鹼性條件中偵測β-酪蛋白胜肽樣品................................68
  3-5.3 HPLC純化及質譜鑑定..........................................................70
  3-5.4以毛細管電泳做雙重確認.....................................................71
  3-5.5 β酪蛋白磷酸化及去磷酸化胜肽片段之質譜鑑定………..73
 3-5.6定義HPLC純化β-酪蛋白的胜肽片段……………………74
3-5.7以CE鑑定α-酪蛋白的磷酸化位置……………………….75
3-6 結論...............................................................................................76
3-7 本章參考資料...............................................................................78
















圖表索引

表3-1 Insulin在不同pH值的濃度效應與流體動力半徑……………80
表3-2 Insulin在不同離子強度緩衝溶液的流體動力半徑……………80
表3-3 Insulin在pH=8.4, I=7.6mM的緩衝溶液中，添加不同濃度
      鋅離子對流體動力半徑的影響………………………………..81
表3-4 Insulin在pH=8.4, I=100mM的緩衝溶液中，添加不同濃度
      鋅離子對流體動力半徑的影響………………………………..81
表3-5磷酸化β-酪蛋白胜肽樣品已定義出的胜肽片段之資料表…..82
圖3-1以PEG為分離介質對Polylysine毛細管電泳解析度之影響..83
圖3-2 不同濃度的F127對Polylysine解析度之影響……………….84
圖3-3 不同濃度的Dextran(70K)對Polylysine解析度之影響……….85
圖3-4 不同分子量的Dextran對Polylysine解析度之影響………….86
圖3-5 不同分子量的Dextran對Polylysine之解析度……………….87
圖3-6 不同濃度的F127對Polylysine之解析度…………………….88
圖3-7 Mb(Trypsin digest)在不同濃度F127中分離之電泳圖……….89
圖3-8 Mb(Trypsin digest)在不同分子量Dextran中分離之電泳圖…90
圖3-9 Mb(Trypsin digest)在PA、PEO、PEG中分離之電泳圖…….91
圖3-10 Mb(Trypsin digest)在Agarose中分離之電泳圖…………….92
圖3-11 Mb(Trypsin digest)在不同種類纖維素中分離之電泳圖…….93
圖3-12改變離子強度對Mb(Trypsin digest)解析之影響……………94
圖3-13添加金屬離子對Mb(Trypsin digest)解析之影響……………95
圖3-14 β-酪蛋白胜肽樣品在Tris-Phosphate ( pH2.00, I=100mM )
        緩衝溶液中的電泳圖譜…………………………………….96
圖3-15 β-酪蛋白胜肽樣品在pH9.35, I=10mM中的電泳圖譜…......97
圖3-16 (a) β-酪蛋白胜肽樣品的HPLC層析圖
       (b)具有一個磷酸化位置的胜肽片段之MS圖譜…………. 98
圖3-17 β-酪蛋白胜肽樣品與HPLC-P1之電泳圖譜………………..99
圖3-18 β-酪蛋白胜肽樣品與HPLC-P4之電泳圖譜………………100
圖3-19 (a) 磷酸化β-酪蛋白胜肽樣品的HPLC層析圖
       (b) 去磷酸化β-酪蛋白胜肽樣品的HPLC層析圖……….101
圖3-20 HPLC-P4(去磷酸化)與HPLC-P4之電泳圖譜…….............102
圖3-21 β-酪蛋白磷酸化(P1)及去磷酸化(D1)之質譜鑑定...............103
圖3-22 β-酪蛋白磷酸化(P4)及去磷酸化(D4)之質譜鑑定...............104
圖3-23 (a) 磷酸化β-酪蛋白胜肽樣品的HPLC層析圖
       (b) HPLC純化樣品對照CE電泳圖之peak相對位置圖
       (c) HPLC純化樣品對照CE電泳圖之peak相對位置圖..105
          
圖3-24 α-酪蛋白磷酸化及去磷酸化之電泳圖譜.............................106

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