| Summary: | 碩士 === 國立臺灣大學 === 化學研究所 === 89 === We report a new on-line concentration approach for the analysis of trace proteins with UV-vis absorption detection using capillaries modified with neutral, cationic, and neutral polymers in sequence. Co-ions and pH both play significant roles in determining migration mobility of proteins, thereby affecting stacking efficiency. We have found the best conditions in terms of speed, resolution and stacking efficiency for the analysis of four model proteins were: proteins were prepared in 200 mM propanoate (PA) buffer, pH 2.8, capillary was filled with 200 mM PA buffer adjusted with Tris, pH 3.8; and after sample injection, the capillary was immersed in 200 mM PA buffer adjusted with Tris, pH 3.3. Proteins stacked at the interface between the sample zone and PA buffer because of reduced electrophoretic mobility and were separated in 200 mM PA buffer, pH 3.3. Linearity between peak height and injection volume up to 0.76 μL has been shown when using a 35-cm capillary. When injecting about 1.42 μL of proteins into a 60-cm capillary, the LOD for lysozyme, myoglobin, carbonic anhydrase, and α-lactalbumin were 1.98, 3.21, 11.30, and 6.54 nM, respectively. Good reproducibility (<2%) was achieved due to the modification of capillary. In addition, peptide mapping has also been successed by this method.
一、前言……………………………………………………………………1
二、毛細管電泳之歷史沿革………………………………………………2
三、毛細管電泳簡介………………………………………………………4
3.1電滲流的產生……………………………………………………………4
3.2流型………………………………………………………………………5
四、毛細管電泳濃縮技術…………………………………………………6
4.1濃縮之必要性……………………………………………………………6
4.2濃縮方法…………………………………………………………………8
4.2.1增加分析物吸收係數…………………………………………………8
4.2.2增大光徑………………………………………………………………8
4.2.3濃縮技術………………………………………………………………9
4.2.3.1利用電泳原理………………………………………………………10
A、電場加強效應……………………………………………………………10
B、酸鹼滴定效應……………………………………………………………12
C、利用毛細管等速電泳之原理……………………………………………15
D、利用微胞電動力層析法之原理………………………………………17
1、正常模式………………………………………………………………19
2、反向模式………………………………………………………………20
E、利用毛細管凝膠電泳之原理…………………………………………20
4.2.3.2利用層析原理……………………………………………………22
A、固相萃取法……………………………………………………………22
B、薄膜預濃縮法…………………………………………………………23
C、中空纖維………………………………………………………………23
五、蛋白質分析技術簡介………………………………………………24
5.1常用的分析技術………………………………………………………24
5.2蛋白質分析的困境與解決之道………………………………………27
5.2.1選擇極端pH條件……………………………………………………28
5.2.2管壁上塗覆聚合物…………………………………………………28
六、研究動機……………………………………………………………31
七、參考文獻……………………………………………………………32
第二章 利用pH接合進行毛細管電泳線上濃縮微量蛋白質
一、前言…………………………………………………………………38
二、實驗理論……………………………………………………………42
三、實驗方法……………………………………………………………46
3.1儀器…………………………………………………………………46
3.2藥品及毛細管……………………………………………………46
四、實驗步驟…………………………………………………………47
4.1毛細管之前處理…………………………………………………47
4.2儀器與藥品部份…………………………………………………48
4.3實驗過程…………………………………………………………48
五、實驗結果及討論…………………………………………………49
5.1毛細管內壁塗覆聚合物對蛋白質樣品分析所造成之影響……49
5.2塗覆聚合物之種類與方法………………………………………49
5.3緩衝溶液之組成…………………………………………………50
5.4分離及濃縮條件之選擇…………………………………………53
5.4.1兩端pH值相同之緩衝溶液……………………………………55
5.4.2兩端pH值不同之緩衝溶液：…………………………………56
5.4.2.1出口端緩衝溶液pH值固定3.8，改變入口端緩衝溶液pH值…56
5.4.2.2入口端緩衝溶液pH值固定3.3，改變出口端緩衝溶液pH值…57
5.4.3電流值的變化及其意義………………………………………63
5.4.4結論……………………………………………………………65
5.5系統峰之影響……………………………………………………65
5.6濃縮效果…………………………………………………………66
5.6.1峰高……………………………………………………………70
5.6.2半高峰寬………………………………………………………70
5.6.3遷移時間的再現性……………………………………………70
5.6.4偵測極限………………………………………………………71
5.7樣品內離子強度之影響…………………………………………71
5.8樣品前打入一小段溶液塞之影響………………………………78
5.9以酵素消化蛋白質後之胜片段圖譜分析……………………79
5.9.1以pepsin消化蛋白質…………………………………………79
5.9.2以trypsin消化蛋白質…………………………………………85
六、結論………………………………………………………………86
七、參考文獻…………………………………………………………87
八、論文發表…………………………………………………………90
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