摘 要

近年來微生物的鑑定主要朝向快速且準確的測試方式；質譜技術的新興發展，已開始應用於生化分子的研究，用來偵測複雜混合物，可作為有效分析生物指標的工具。實驗中利用靈敏度佳，準確性高，質量範圍廣，分析簡便的基質輔助雷射脫附游離飛行時間質譜法（Matrix-Assisted Laser Desorption Ionization/ Time of Flight Mass Spectrometry；MALDI- TOF/MS），已廣泛應用於化學及生命科學等領域，作為偵測與鑑定胜肽、蛋白質等生化分子的主要工具。
本論文第一部分使用易感染地區性腸胃疾病的細菌，以MALDI- TOF/MS直接分析菌屬、菌種、及亞種間的差異，尋找其中特有的生物標幟，建立具代表性的指紋質譜圖，當作微生物分類的參考。第二部分使用大小約為10 ~100 nm之噬菌體作為材料，由於噬菌體的發現，使得很多病毒學家相繼研究，因而刺激現代病毒學之快速發展。實驗中利用噬菌體MS2（bacteriophage MS2）及T4（bacteriophage T4）先進行宿主細胞的置備，再以液體培養法培養噬菌體，進行部份半純化後，以酸化萃取送入MALDI-TOF/MS進行偵測微量的蛋白質表現。
另外結合膠體電泳法進行in gel digestion及PSD胺基酸序列分析。
由實驗結果顯示，不同菌種間所得到之質譜圖離子訊號分佈不同。而噬菌體MS2經MALDI- TOF/MS分析，可發現m/z 5255強度較強之質譜訊號，且具有良好之再現性；噬菌體T4發現m/z 8600，經比對參考資料庫相似性為Tail fiber assembly helper protein。未來將比較不同噬菌體的質譜分析圖，以期找出不同噬菌體之個別生物指標，彙整成質譜資料庫。此分析方法的成功將有助於未來應用在病毒的偵測，以便快速鑑定疾病的感染來源。

Abstract

Rapid and reliable identification of microorganisms is of paramount importance for advancing homeland security. Mass spectrometry is emerging as an effective bioanalytical tool having unique capabilities in handing complex mixtures. Matrix-assisted layser desorption / ionization time-of-flight mass spectrometry （MALDI/TOF MS）is commonly used in the analysis of bio-molecules owing to its merits of high sensitivity, wide mass range, and ease-of-operation.
The objective of the first part of the thesis is to develop an analysis method to characterize the genus, strains, and subspecies of the infections gastroenteritis bacteria by using MALDI/TOF MS. MALDI/TOF MS can be applied to classify different microoganisms based on examination of ion patterns from different microoganisms and unique distinguishing protein ions as biomarkers for different strains. The second section, bacteriophages MS2 and T4 were used as materials for identification of their specific protein markers. Protocols including sample preparation, purification, combined in gel digestion and post source decay to which the mass spectrometer brought for analytical specificity were employed.
According to the experimental results, classification of different microorganisms based on examination of ion patterns is feasible. Furthermore, the peak at m/z 5255 in the MALDI mass spectra of MS2 analysis and the peak at m/z 8600 of T4 can provide the unique distinguishing signals. The experimental mass spectral peaks were submitted to the database search, and one of these peaks was matched to a tail fiber assembly helper protein. Thus, the method developed and the mass spectra presented in this thesis can be potentially applied to the practical virus analysis.

目錄

壹、緒論...............................................................................................1
一、前言...........................................................................................1
二、基質輔助雷射脫附游離質譜法之背景介紹...........................4
1.MALDI/TOF的發展歷史........................................................4
2.MALDI的樣品製備與基質的特性.........................................5
（1） 樣品製備............................................................................5
（2） 基質的特性........................................................................6
3.MALDI離子形成的機制.........................................................7
4.飛行時間偵測器（Time of flight mass spectrum）................9
三、蛋白質電泳（SDS-PAGE）...................................................12
四、In-gel digestion.........................................................................14
五、細菌背景簡介..........................................................................16
1.細菌的特徵...............................................................................16
2.細菌的生理學...........................................................................16
3.腸道內之常在菌與病源菌.......................................................17
（1）大腸桿菌（Escherichia coli）..........................................18
（2）志賀氏桿菌（Shigella flexneri）......................................19
（3）枸櫞酸桿菌（Citrobacter freundii）..................................19
（4）沙門氏桿菌（Salmonella）................................................20
（5）腸炎弧菌（Vibrio parahaemolyticus）.............................. 20
（6）金黃色葡萄球菌（Staphylococcus aureus）.......................20
（7）仙人掌桿菌（Bacillus cereus）...........................................21
六、噬菌體背景簡介..........................................................................22
七、論文目標......................................................................................24
貳、材料與方法.......................................................................................26
一、菌株培養......................................................................................26
1.菌種來源.....................................................................................26
2.冷凍乾燥管之開封.....................................................................26
3.細菌菌株培養.............................................................................27
4.細菌菌種之保存.........................................................................27
5.噬菌體培養法.............................................................................27
6.噬菌體的保存..............................................................................29
7.噬菌體效價測定法......................................................................29
二、MALDI-TOF分析.......................................................................31
1.儀器裝置......................................................................................31
2.基質及基質溶劑配製..................................................................31
3.樣品前處理方法..........................................................................32
4. MALDI/TOF分析......................................................................34
三、SDS-PAGE膠體電泳..................................................................34
四、銀染（silver staining）................................................................36
五、In gel digestion..............................................................................37
參、結果與討論.......................................................................................40
一、選擇適當的MALDI-TOF基質..................................................40
二、MALDI-TOF MS分析細菌性腸胃炎疾病的菌種....................41
1.菌種的前處理.............................................................................41
2.以MALDI分析培養細菌的液體培養基...................................42
3.以MALDI分析不同的菌屬......................................................43
4.以MALDI分析相同菌屬不同的菌種.......................................44
5.以MALDI分析相同菌種不同的亞種.......................................45
三、MALDI-TOF MS分析“侵襲細菌的病毒--噬菌體(MS2、T4)，
探討生化分子的表現”.............................................................45
1.噬菌體的前處理.........................................................................45
2.利用MALDI分析噬菌體MS2..................................................46
（1）宿主細胞E. coli蛋白質對噬菌體質譜訊號的影響...........47
（2）宿主細胞E. coli培養時間長短之影響...............................48
（3）以MALDI鑑定經膠體電泳分離後的蛋白質...................49
3.利用MALDI分析噬菌體T4.....................................................51
肆、結論...................................................................................................53
伍、參考文獻...........................................................................................55






























圖表目錄

表一、常用的MALDI基質及適用的分析物........................................64
表二、實驗室參考菌株...........................................................................65
圖1.基質輔助雷射脫附游離飛行時間質譜儀（MALDI-TOF/MS）儀
器構造圖..........................................................................................66
圖2.利用MALDI-TOF分析細菌細胞的實驗策略...............................67
圖3.利用MALDI-TOF分析噬菌體的實驗策略...................................68圖4.E. coli-DH5α經30%ACN/70%H2O-0.25%TFA萃取後，利用（a）
α-cyano-4-hydroxycinnamic acid（b）2,5-dihydroxybnzoicacid （c）
sinapinic acid三種不同的基質進行MALDI-TOF分析................69
圖5.E. coli DH5α使用三種不同方式前處理.......................................70
圖6.培養細菌的液體培養基，經30﹪ACN / 70﹪H2O / 0.25﹪TFA處
理，所得之MALDI質譜.................................................................71
圖7.利用MALDI-TOF分析7株不同的菌屬，經30﹪ACN / 70﹪
H2O / 0.25﹪TFA處理，所得之質譜圖........................................72
圖8.相同的沙門氏菌（Salmonella）菌屬，但不同菌種的細菌經30
﹪ACN / 70﹪H2O / 0.25﹪TFA前處理後，所得之質譜圖..........73
圖9.不同E. coli亞種：（a）DH5a,（b）JM109,（c）VCS257,（e）
XL-1經30﹪ACN / 70﹪H2O / 0.25﹪TFA前處理後，所得之質譜
圖......................................................................................................74
圖10.將半純化的噬菌體MS2懸浮液先添加冰醋酸前處理後，混以基
質α-cyano-4-hydroxycinnamic acid分析，所得之MALDI質譜
圖....................................................................................................75
圖11. E. coli-F2與噬菌體MS2共同隔夜培養後，與醋酸進行前處理，
混以基質α-CHCA分析，所得之MALDI質譜圖....................76
圖12.呈現宿主細胞E. coli經培養後6-86小時，與醋酸前處理後，混
以基質α-CHCA分析，所得之MALDI質譜圖.......................77
圖13. 噬菌體MS2懸浮液經醋酸及超音波處理後所得之unknow
protein (箭頭所指之處)..............................................................78圖14.將圖13之SDS-PAGE中箭頭所指位置（unknow protein）經in
gel digestion所得之MALDI質譜圖..........................................79
圖15.將圖14 b中之in gel digestion所獲得特有的胜肽離子訊號，進
行質譜儀離子源後衰變（PSD）分析，所得之質譜圖...............80
圖16.噬菌體T4與E. coli 13055共同隔夜培養後，與醋酸進行前處
理，混以基質α-CHCA分析，所得之MALDI質譜圖.............81

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