本論文使用自行合成的金屬氧化物奈米粒子(metal oxide nanoparticles)，並利用其在紫外光範圍337 nm有良好吸收的光學性質，與基質輔助雷射脫附游離質譜法(MALDI-MS)結合，發展出以奈米金屬氧化物萃取蛋白質與胜肽之偵測方法。
本論文第一部分：以氧化鋅奈米粒子(ZnO NPs)作為磷酸化胜肽(phosphopeptide)之親和探針(affinity probe)，第一步驟利用ZnO NPs吸收微波輻射，將其熱能傳遞到蛋白質溶液中，使酵素消化反應發生，此方法可以大幅縮短消化時間，並透過ZnO NPs與磷酸化胜肽(phosphopeptide)間親和力，將磷酸化胜肽從胜肽混合液中萃取出來。第二步驟進行30分鐘incubation，使磷酸化胜肽與ZnO NPs間親和吸附力達到平衡、飽和，再將ZnO NPs離心下來，取出奈米粒子並與基質(2,5-DHB)混合乾燥後，進入質譜進行偵測。結果顯示不需經過繁複的清洗步驟即可以利用ZnO NPs成功萃取出磷酸化胜肽，因此可避免樣品損失，並能在牛奶真實樣品中偵測到磷酸化胜肽。
第二部分：以ZnO NPs 作為蛋白質(lysozyme、BSA、myoglobin)親和探針，透過ZnO NPs吸收微波輻射使其快速加熱，能輔助酵素消化反應，不需經過繁複的清洗步驟即可直接與基質混合，以MALDI-MS偵測，或直接注入ESI-MS中進行分析。
第三部分：利用ZnO NPs作為基質偵測蛋白質，並比較與傳統有機基質偵測結果，氧化鋅奈米粒子能成功偵測到lysozyme、myoglobin、trypsin、BSA等蛋白質訊號，觀察到解析度比用傳統有機基質提升約2~3倍，並且能以ZnO NPs成功測得真實樣品牛奶中蛋白質訊號。探討各式條件研究ZnO NPs基質之游離機制，得到結論為電荷轉移機制(charge transfer)，當雷射轟擊ZnO NPs基質時電子激發到導帶(conduction band)，再與氣相分析物發生反應產生電荷轉移，因此能夠偵測到分析物之分子離子訊號(M+˙)。將ZnO NPs結合高溫燒結技術應用在鋁箔紙基板上，能將偵測的質量上限提升到80 kDa (transferrin)及150 kDa (IgG)。第四部分：利用氧化鋅(ZnO)奈米粒子作為基質偵測小分子(perylene、pyrene、acenaphthene)，結果不僅有效增強小分子訊號強度，並能在鹽類(NaCl 1 M)干擾下偵測到分析物訊號，並再次證明ZnO NPs作為基質游離機制為電荷轉移機制。

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摘要........................................................................................................................I
謝誌…………………………………………………………...………………..III
目錄.....................................................................................................................IV
圖表目錄……………………………………………………………………….VI
壹、序論............................................................................................................. 1
1-1 質譜簡介..................................................................................................... 1
1-1-1 基質輔助雷射脫附游離質譜法.......................................................... 1
1-1-2 電噴灑游離質譜法.............................................................................. 3
1-2 磷酸化蛋白質萃取..................................................................................... 4
1-2-1 磷酸化胜肽[17] ...................................................................................... 4
1-2-2 Immunoaffinity Chromatography......................................................... 6
1-2-3 Immobilized Metal Affinity Chromatography (IMAC) ....................... 7
1-2-4 Metal Oxide Affinity Chromatography (MOAC)................................. 8
1-2-5 Strong Cation Exchange Chromatography (SCX) ............................... 8
1-3 研究動機..................................................................................................... 9
貳、氧化鋅奈米粒子作為磷酸化胜肽之親和探針...................................... 10
2-1 目的...........................................................................................................10
2-2 實驗部份...................................................................................................10
2-2-1 儀器....................................................................................................10
2-2-2 藥品................................................................................................... 11
2-2-3 氧化鋅奈米粒子製備[12] ...................................................................12
2-2-4 蛋白質微波消化反應.......................................................................13
2-3 結果與討論...............................................................................................16
2-3-1 氧化鋅奈米粒子性質........................................................................16
2-3-2 磷酸化蛋白質微波消化...................................................................18
2-3-3 探討蛋白質消化反應中微波及酵素效應.......................................20
2-3-4 探討奈米粒子吸收微波能量特性...................................................22
2-3-5 氧化鋅奈米粒子作為磷酸化胜肽之探針.......................................23
2-3-6 氧化鋅奈米粒子偵測磷酸化胜肽之偵測極限(LOD) ....................29
2-3-7 真實樣品(牛奶)中萃取磷酸化胜肽.................................................32
V
2-4 結論...........................................................................................................34
叁、 氧化鋅奈米粒子作為蛋白質親和探針................................................. 35
3-1 目的...........................................................................................................35
3-2 實驗部份...................................................................................................35
3-2-1 儀器...................................................................................................35
3-2-2 藥品...................................................................................................36
3-2-3 蛋白質微波消化反應： ...................................................................37
3-3 結果與討論...............................................................................................38
3-3-1 Lysozyme 以ZnO NPs 輔助微波消化..............................................38
3-3-2 Bovine Serum Albumin (BSA)以ZnO NPs 輔助微波消化..............41
3-3-3 Myoglobin 以ZnO NPs 輔助微波消化............................................46
3-4 結論...........................................................................................................50
肆、 氧化鋅奈米粒子偵測蛋白質之電荷轉移質譜及提升偵測解............ 51
4-1 目的...........................................................................................................51
4-2 實驗部份...................................................................................................51
4-2-1 儀器...................................................................................................51
4-2-2 藥品...................................................................................................52
4-2-3 氧化鋅奈米粒子作為基質偵測分析物...........................................53
4-3 結果與討論...............................................................................................54
4-3-1 氧化鋅奈米粒子作為基質偵測各種蛋白質...................................54
4-3-2 pH 對偵測蛋白質的影響..................................................................62
4-3-3 氧化鋅奈米粒子結合高溫燒結並以鋁箔紙作為基板材料...........67
4-3-4 探討ZnO NPs 基質中添加氧化試劑偵測蛋白質..........................69
4-3-5 在負離子模式下偵測蛋白質...........................................................74
4-3-6 氧化鋅奈米粒子作為基質之CT-SALDI ........................................76
4-3-7 氧化鋅奈米粒子作為基質偵測牛奶中蛋白質...............................76
4-4 結論...........................................................................................................78
伍、 利用氧化鋅奈米粒子作為基質偵測有機小分子之電荷轉移............ 79
5-1 目的...........................................................................................................79
5-2 實驗部份...................................................................................................80
5-2-1 儀器...................................................................................................80
5-2-2 藥品...................................................................................................80
5-2-3 氧化鋅奈米粒子作為基質偵測分析物...........................................81
VI
5-3 結果與討論...............................................................................................82
5-3-1 偵測有機小分子探討氧化鋅奈米粒子之電荷轉移機制...............82
5-3-2 添加氧化試劑對氧化鋅奈米粒子電荷轉移機制之影響...............86
5-4 結論...........................................................................................................88
陸、總結........................................................................................................... 89
柒、參考文獻................................................................................................... 90

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