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研究生:詹雅庭
研究生(外文):Ya-ting Chan
論文名稱:以薄層層析結合電噴灑輔助雷射脫附游離質譜法分離及鑑定有機及蛋白質混合物
論文名稱(外文):Applications of Thin-Layer Chromatography/Electrospray-Assisted Laser Desorption Ionization Mass Spectrometry for Small Molecule Analysis and Protein Identification
指導教授:謝建台
指導教授(外文):Jen-Taie Shiea
學位類別:碩士
校院名稱:國立中山大學
系所名稱:化學系研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:127
中文關鍵詞:薄層層析法高通量分子影像蛋白質鑑定電噴灑輔助雷射脫附游離質譜法
外文關鍵詞:Protein identificationELDI/MSTLCHigh throughputMolecular imaging
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薄層層析法 (Thin Layer Chromatography,TLC) 具有操作簡單、成本低廉、樣品需求量少及快速等優點,是一種已廣泛應用於有機、無機和生化混合物的定性分析方法。電噴灑輔助雷射脫附游離質譜法(Electrospray-Assisted Laser Desorption Ionization Mass Spectrometry,ELDI/MS) 可直接在一大氣壓下,且不需添加任何基質,即可對樣品進行快速分析。本篇論文的研究目的是利用電噴灑輔助雷射脫附游離質譜法結合薄層層析法,對分離後之分析物進行快速掃描分析,並鑑定其成分。
本研究的第一部分是利用 TLC-ELDI/MS 應用在高通量和快速掃描分析。分析樣品為染劑標準品、胺類化合物及感冒糖漿,以適合的 C18 或 silica gel 材質的 TLC 片分離後,進行 ELDI/MS 分析,其是利用脈衝雷射將分析物從 TLC 片上脫附出,再與電噴灑產生的帶電液滴融合,分析物隨之進行電噴灑過程而被游離並以質譜偵測,可連續偵測數片相同或不同材質的 TLC 片;在快速掃描偵測研究中可以 0.024 cm/s 的掃描速度進行偵測,分析長度為 3.1 cm 的 TLC 片時間為 2.2 分鐘。
本研究的第二部分是以 TLC-ELDI/MS 結合影像質譜分析法的開發。分析物經由 TLC 分離後,置於自動控制的樣品平台上,以來回掃描的方式,紀錄整個掃描區域的質譜訊號,並選定特定分析物的分子離子訊號,再以軟體轉換成影像圖,可由影像圖觀察經 TLC 分離後,分析物分離之分佈並得知其成分。
本研究的第三部分是以 TLC-ELDI/MS 分析技術進行蛋白質鑑定,蛋白質利用 TLC 分離後,將每種蛋白質分別挖取出,並加入胰蛋白脢 (trypsin) 進行酵素消化反應,再以 ELDI/MS 鑑定消化後所得的胜肽訊號,並利用 Database 比對,搜尋可能之蛋白質種類。
藉由 TLC-ELDI/MS 分離且偵測有興趣之分子,具有快速、方便、操作過程簡單且不會在質譜偵測時造成干擾之優點。利用此技術對分離及鑑定有機小分子及蛋白質混合物將有極大的助益。
論文摘要..................................................................................................... I
目錄...........................................................................................................III
圖目錄..................................................................................................... VII
表目錄.................................................................................................. XVII
壹、緒論.....................................................................................................1
一、前言.................................................................................................1
二、薄層層析法之介紹........................................................................2
1.薄層層析法之發展.........................................................................2
2.薄層層析法之原理.........................................................................3
3.傳統薄層層析的偵測方法.............................................................4
三、薄層層析法結合質譜法的發展....................................................5
四、電噴灑輔助雷射脫附游離質譜法之介紹..................................10
1.電噴灑輔助雷射脫附游離質譜法之發展...................................10
2.電噴灑輔助雷射脫附游離質譜法之裝置設計...........................11
3.電噴灑輔助雷射脫附游離質譜法之原理...................................13
4.電噴灑輔助雷射脫附游離質譜法之游離過程及游離機制.......14
五、TLC 結合ELDI/MS 介面之設計................................................17
六、研究目標......................................................................................18
貳、實驗...................................................................................................21
一、電噴灑輔助雷射脫附游離質譜法實驗裝置..............................21
1.質譜儀............................................................................................21
2.電噴灑游離源...............................................................................21
(1)熔融矽毛細管............................................................................21
(2)注射式幫浦................................................................................21
(3)高電壓電源供應器....................................................................22
3.雷射脫附游離源...........................................................................22
4. XYZ 移動平台.............................................................................22
(1) XY 自動控制平台....................................................................22
(2) XYZ 手動平台..........................................................................22
二、其他實驗設備..............................................................................24
1. CCD 攝影機................................................................................24
2.紫外光燈源...................................................................................24
3.數位相機.......................................................................................25
4.恆溫控制水浴裝置.......................................................................25
5.離心機............................................................................................25
三、影像處理軟體..............................................................................25
四、化學藥劑與分析樣品..................................................................26
五、藥品配製......................................................................................29
六、薄層層析片..................................................................................30
七、電噴灑輔助雷射脫附游離質譜法實驗條件..............................30
八、薄層層析分離條件......................................................................30
1.染劑標準品的分離.......................................................................31
2.胺基化合物標準品的分離...........................................................31
3.感冒糖漿的分離...........................................................................31
4.蛋白質標準品的分離...................................................................32
九、以酵素消化蛋白質之步驟..........................................................32
參、結果與討論......................................................................................33
第一部份、薄層層析結合電噴灑輔助雷射脫附游離質譜法應用在高
通量與快速分析....................................................................................34
一、參數探討....................................................................................34
1.不同雷射能量對 TLC 脫附訊號的影響..................................34
2.不同脫附面積對 TLC 脫附訊號的影響..................................44
二、高通量分析................................................................................59
三、快速掃描分析............................................................................65
第二部份、以薄層層析結合電噴灑輔助雷射脫附游離質譜法進行分
子影像質譜法分析................................................................................73
一、參數探討....................................................................................74
1.雷射能量對 TLC-ELDI/MS 分子影像分析的影響................75
2.雷射頻率對 TLC-ELDI/MS 分子影像分析的影響................76
3.掃描速度對 TLC-ELDI/MS 分子影像分析的影響................77
二、以 TLC-ELDI/MS 進行分子影像質譜法分析.......................79
1.染劑標準品的分析.....................................................................79
2.胺基化合物的分析.....................................................................82
第三部份、薄層層析結合電噴灑輔助雷射脫附游離質譜法應用於蛋
白質的分離及鑑定................................................................................86
一、蛋白質的分離............................................................................86
1.以逆相 C18 TLC 片分離蛋白質................................................86
2.以正相 silica gel TLC 片分離蛋白質......................................90
二、蛋白質的鑑定............................................................................91
1.參數探討.....................................................................................91
2.經 TLC 分離後的蛋白質進行酵素消化並鑑定之結果..........95
肆、結論...................................................................................................99
伍、參考資料.........................................................................................100
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