微生物在大自然環境中扮演著重要的角色，在生物系統能量循環中具有關 鍵作用，是不可或缺的一環。即便外型渺小卻和人類的關係相當密切，有些細菌對於人體是有益的；有些則對人體有害會造成生命危害，甚至死亡。許多科學家極力研究這群神秘的生物群，推導出多種檢測微生物的技術。傳統分析微生物的檢測技術相當耗時且必須依賴培養基進行培養，因為細菌沒有顏色需要透過染色使其顯色，進一步確定為革蘭氏陽性菌或革蘭氏陰性菌。然而，無法知道菌種之結構、生理功能以及判定多樣性高的菌種。現代檢測微生物常用的分析方法包括有抗原檢測、核酸分析等，該技術受限於前處理需要針對特定的基因序列進行分析較為冗長、繁瑣；而近代質譜技術可以縮短樣品前處理的時間，提升分析效率。近代質譜法中以大氣質譜法為例，該技術不受真空系統限制，可在大氣壓力下進行分析。現今，許多科學家致力於快速分析菌種之間的關係，減少分析流程、增加準確性。本研究主要是以熱脫附電噴灑游離質譜法 (Thermal Desorption Ionization Mass Spectrometry, TD-ESI/MS)，藉由此技術快速分析且不須樣品前處理之特性，針對12種標準菌株，其革蘭氏陰性菌與革蘭氏陽性菌分別各六株，以脂質的質譜訊號進行探討。以 TD-ESI/MS 技術分析範圍介於m/z 400-1000之間，不做任何前處理的情況下，以針灸針做為採樣探針直接分析培養皿上的菌落，可以觀察不同菌種間脂質訊號的差異。此分析方法也具有良好的重覆性其相對標準偏差值 (RSD) 小於7.11% (n=10)。另外，利用多變量分析法中主成分分析法 (Principle component analysis, PCA) 對於質譜上特異性的訊號進一步分群，也成功的區別不同菌種之分布，此技術也運用在胃部疾病相關菌種之分析，判別出正常標準菌株與疾病之菌株有顯著的差異性。

Recently, mass spectrometric profiling has been applied to distinguish different bacterial species through fingerprinting. The most successful example is using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF/MS) to detect lipid/protein components released from bacteria for classification. Ambient mass spectrometry has dramatically reduced analysis time for organic and biological compound analysis, as the method requires minimum or no sample pretreatment. Its most notable advantages are the abilities to manipulate samples under atmospheric conditions for high-throughput analysis, and fast sample replacement. Herein, a new strategy that combines thermal desorption electrospray ionization mass spectrometry (TD-ESI/MS), an ambient mass spectrometric technique, with principal component analysis (PCA) was developed to classify various bacterial species based on the lipid profiles obtained by TD-ESI/MS.
The biological compounds in the cultures of twelve species of bacteria (six gram positive and six gram negative) were collected and analyzed using TD-ESI/MS and MALDI-TOF, respectively. The TD-ESI source comprised of a metal sampling probe, thermal desorption unit, and electrospray ionization interface was coupled to a triple quadrupole mass spectrometer for MS analyses. The lipid ions detected in each bacterial species by TD-ESI/MS were analyzed by PCA. In addition, the bacterial cells were extracted with organic solvents to obtain peptide/protein profiling with MALDI-TOF/MS. The lipid and protein profiles were further processed by principal component analysis (PCA).To directly collect single colonies on the petri dish, a stainless steel inoculating loop and an acupuncture needle were tested to generate the optimal mass spectrum by TD-ESI/MS. As a result, memory effects were mostly avoided with an acupuncture needle acting as a very small sampling probe to collect the colonies. An acupuncture needle was also used to determine the reproducibility of the tests, with the relative standard deviation (RSD) resulting in values below 7.11 % (n=10). A number of lipid ion peaks distributed between m/z 400-1000 were detected from all of the single colonies. Lipopolysacchrides, characteristic biomarkers for gram negative bacteria, were detected at m/z 550-800. Other lipids include diacylglycerols, triacylglycerols, phosphocholines, and sphingomyelin were also detected. The representative TD-ESI mass spectra were processed by PCA to successfully classify 12 bacterial species. In addition, analyses of homogeneous bacteria were performed by MALDI-TOF/MS at the range of m/z 2000-20000 (peptide/protein profiling) and m/z 400-1500 (lipid profiling). The representative mass spectra were analyzed by PCA to assist the classification of bacterial species.

論文審定書 i
論文公開授權書 ii
誌謝 iii
中文摘要 v
英文摘要 vi
目錄 viii
圖目錄 xi
表目錄 xv
第一章、　緒論 1
第一節、　前言 1
第二節、　細菌的介紹 3
1. 細菌的外觀 3
2. 細菌的結構 4
第三節、　菌種檢測之方法 8
1 細菌演化分類與鑑定上的獨特性及應用 8
2 傳統檢測微生物之技術 10
3 現代檢測微生物之技術 13
第四節、　現代質譜法 18
1. 電噴灑游離法 (Electrospray ionization, ESI) 18
2. 大氣壓力化學游離法 (Atmosphere pressure chemical ionization, APCI) 20
3. 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) 22
4. 金屬氧化物雷射游離質譜法 23
第五節、　大氣質譜法 24
1. 傳統大氣質譜法 24
2. 現代大氣質譜法 25
第六節、　熱脫附電噴灑游離法 (Thermal Desorption Electrospray Ionization, TD-ESI) 27
第七節、　多變量統計於質譜分析數據之應用 28
1. 主成分分析 (principal component analysis, PCA) 29
2. 集群分析 (clustering) 30
第八節、　研究動機與目標 32
第二章、　實驗 33
第一節、　儀器設備與軟體 33
第二節、　主成分分析軟體介紹 36
第三節、　菌株種類 37
第四節、　實驗藥品與試劑 37
第三章、　結果與討論 40
第一節、　熱脫附電噴灑游離質譜技術在菌種分析之應用 40
1. 熱脫附電噴灑游離質譜法對菌種分析的最適化參數探討 40
2. 熱脫附電噴灑游離質譜法裝置之最適化參數探討 43
3. 以 TD-ESI/MS 對於菌種分析 45
4. 以負模式分析不同菌種 54
5. P. aeruginosa 生長周期分析 58
6. 以 TD-ESI/MS 分析臨床診斷之胃部相關疾病 60
第二節、　基質輔助雷射脫附游離飛行時間質譜法檢測菌種間脂質及大分子之訊號分析 63
1. 基質輔助雷射脫附游離飛行時間質譜法對菌種分析的最適化參數探討 64
2. 以50% CHCl3作為萃取溶劑，探討萃取時間的影響 67
3. 比較不同基質分析菌株萃取前後之性質 70
4. MALDI-TOF/MS 分析12株菌株之分析結果 71
5. 多變量分析軟體分析結果 74
6. 以 MALDI-TOF/MS 不同有機溶劑極性萃取之結果 80
第四章、　結論 86
第五章、　附錄 87
第六章、　參考文獻 97

[1] 陳淙偉, Arthrobacter AB -10 細菌生產新型抗生物質之研究, 2005. 國立高雄大學, 生物科技研究所碩士論文。
[2] Basile, F.; Zhang, S. F.; Shin, Y. S.; Drolet, B., Atmospheric pressure-thermal desorption (AP-TD)/electrospray ionization-mass spectrometry for the rapid analysis of Bacillus spores. The Analyst 2010, 135 (4), 797-803.
[3] Korneev, K. V.; Arbatsky, N. P.; Molinaro, A.; Palmigiano, A.; Shaikhutdinova, R. Z.; Shneider, M. M.; Pier, G. B.; Kondakova, A. N.; Sviriaeva, E. N.; Sturiale, L.; Garozzo, D.; Kruglov, A. A.; Nedospasov, S. A.; Drutskaya, M. S.; Knirel, Y. A.; Kuprash, D. V., Structural Relationship of the Lipid A Acyl Groups to Activation of Murine Toll-Like Receptor 4 by Lipopolysaccharides from Pathogenic Strains of Burkholderia mallei, Acinetobacter baumannii, and Pseudomonas aeruginosa. Frontiers in immunology 2015, 6, 595.
[4] 尹開民、潘銓泰、顏榮華、楊喜男（2012年）：（病態大樓使人生病）。
[5] 湯雅芬、李禎祥（102年2月第二十三卷第一期）：（MALDI-TOF質譜儀在臨床微生物鑑定的應用）。
[6] 蔡竹固（2007）：（植物病原真菌分子檢測）頁6-10。
[7] Wright, W. E.; Piatyszek, M. A.; Rainey, W. E.; Byrd, W.; Shay, J. W., Telomerase activity in human germline and embryonic tissues and cells. Developmental genetics 1996, 18 (2), 173-9.
[8] Woese, C. R.; Fox, G. E., Phylogenetic structure of the prokaryotic domain: the primary kingdoms. Proceedings of the National Academy of Sciences of the United States of America 1977, 74 (11), 5088-90.
[9] Delsuc, F.; Brinkmann, H.; Philippe, H., Phylogenomics and the reconstruction of the tree of life. Nature reviews. Genetics 2005, 6 (5), 361-75.
[10] Doolittle, W. F., Phylogenetic classification and the universal tree. Science 1999, 284 (5423), 2124-9.
[11] Briglia, M.; Eggen, R. I.; de Vos, W. M.; van Elsas, J. D., Rapid and sensitive method for the detection of Mycobacterium chlorophenolicum PCP-1 in soil based on 16S rRNA gene-targeted PCR. Applied and Environmental Microbiology 1996, 62 (4), 1478-80.
[12] Duineveld, B. M.; Kowalchuk, G. A.; Keijzer, A.; van Elsas, J. D.; van Veen, J. A., Analysis of bacterial communities in the rhizosphere of chrysanthemum via denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA as well as DNA fragments coding for 16S rRNA. Applied and Environmental Microbiology 2001, 67 (1), 172-8.
[13] Giri Dhurba. Gram Staining : Principle, Procedure, Interpretation and Animation. 2016.
[14] Claus, D., A standardized Gram staining procedure. World Journal of Microbiology & biotechnology 1992, 8 (4), 451-2.
[15] Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M., Electrospray Ionization-Principles and Practice. Mass Spectrom Rev 1990, 9 (1), 37-70.
[16] Watrous, J.; Roach, P.; Alexandrov, T.; Heath, B. S.; Yang, J. Y.; Kersten, R. D.; van der Voort, M.; Pogliano, K.; Gross, H.; Raaijmakers, J. M.; Moore, B. S.; Laskin, J.; Bandeira, N.; Dorrestein, P. C., Mass spectral molecular networking of living microbial colonies. Proceedings of the National Academy of Sciences of the United States of America 2012, 109 (26), E1743-E1752.
[17] Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G., Mass spectrometry sampling under ambient conditions with desorption electrospray ionization. Science 2004, 306 (5695), 471-473.
[18] Watrous, J.; Hendricks, N.; Meehan, M.; Dorrestein, P. C., Capturing Bacterial Metabolic Exchange Using Thin Film Desorption Electrospray Ionization-Imaging Mass Spectrometry. Analytical Chemistry 2010, 82 (5), 1598-1600.
[19] Jackson, A. U.; Werner, S. R.; Talaty, N.; Song, Y.; Campbell, K.; Cooks, R. G.; Morgan, J. A., Targeted metabolomic analysis of Escherichia coli by desorption electrospray ionization and extractive electrospray ionization mass spectrometry. Analytical Biochemistry 2008, 375 (2), 272-281.
[20] Woese, C. R., Bacterial evolution. Microbiological reviews 1987, 51 (2), 221-71.
[21] Strittmatter, N.; Jones, E. A.; Veselkov, K. A.; Rebec, M.; Bundy, J. G.; Takats, Z., Analysis of intact bacteria using rapid evaporative ionisation mass spectrometry. Chemical Communications 2013, 49 (55), 6188-6190.
[22] Balog, J.; Sasi-Szabo, L.; Kinross, J.; Lewis, M. R.; Muirhead, L. J.; Veselkov, K.; Mirnezami, R.; Dezso, B.; Damjanovich, L.; Darzi, A.; Nicholson, J. K.; Takats, Z., Intraoperative tissue identification using rapid evaporative ionization mass spectrometry. Science Translational Medicine 2013, 5 (194), 194ra93.
[23] Wilm, M., Principles of electrospray ionization. Molecular & Cellular Proteomics : MCP 2011, 10 (7), M111 009407.
[24] Gaskell, S. J., Electrospray: Principles and practice. J Mass Spectrom 1997, 32 (7), 677-688.
[25] Rohner, T. C.; Lion, N.; Girault, H. H., Electrochemical and theoretical aspects of electrospray ionisation. Phys Chem Chem Phys 2004, 6 (12), 3056-3068.
[26] Byrdwell, W. C., Atmospheric pressure chemical ionization mass spectrometry for analysis of lipids. Lipids 2001, 36 (4), 327-346.
[27] Andrade, F. J.; Shelley, J. T.; Wetzel, W. C.; Webb, M. R.; Gamez, G.; Ray, S. J.; Hieftje, G. M., Atmospheric pressure chemical ionization source. 1. Ionization of compounds in the gas phase. Analytical Chemistry 2008, 80 (8), 2646-2653.
[28] Ferguson, E. E., Ion-Molecule Reactions. Annu Rev Phys Chem 1975, 26, 17-38.
[29] Croxatto, A.; Prod'hom, G.; Greub, G., Applications of MALDI-TOF mass spectrometry in clinical diagnostic microbiology. FEMS Microbiology reviews 2012, 36 (2), 380-407.
[30] McAlpin, C. R.; Voorhees, K. J.; Corpuz, A. R.; Richards, R. M., Analysis of Lipids: Metal Oxide Laser Ionization Mass Spectrometry. Analytical Chemistry 2012, 84 (18), 7677-7683.
[31] Voorhees, K. J.; Saichek, N. R.; Jensen, K. R.; Harrington, P. B.; Cox, C. R., Comparison of metal oxide catalysts for pyrolytic MOLI-MS bacterial identification. J Anal Appl Pyrol 2015, 113, 78-83.
[32] Huang, M. Z.; Cheng, S. C.; Cho, Y. T.; Shiea, J., Ambient ionization mass spectrometry: A tutorial. Analytica Chimica acta 2011, 702 (1), 1-15.
[33] Huang, M. Z.; Zhou, C. C.; Liu, D. L.; Jhang, S. S.; Cheng, S. C.; Shiea, J., Rapid Characterization of Chemical Compounds in Liquid and Solid States Using Thermal Desorption Electrospray Ionization Mass Spectrometry. Analytical Chemistry 2013, 85 (19), 8956-8963.
[34] 數據分析介紹<I> 主成分分析法 華聯快訊 (2013)。
[35] Pearson, K. On lines and planes of closest fit to systems of points in space. Philosophical Magazine 1901, 2: 559-572.
[36] 卓怡孜. 開發現代質譜法在藥物分析、疾病檢驗及蛋白質研究上的新技術, 2011. 國立中山大學, 化學系研究所 博士論文。
[37] Barnard, J. M.; Downs, G. M., Clustering of Chemical Structures on the Basis of 2-Dimensional Similarity Measures. J Chem Inf Comp Sci 1992, 32 (6), 644-649.
[38] Agilent Technologies, Inc. Class Prediction with Agilent Mass Profiler Professional. Revision A, August 2014.
[39] Liebisch, G.; Lieser, B.; Rathenberg, J.; Drobnik, W.; Schmitz, G., High-throughput quantification of phosphatidylcholine and sphingomyelin by electrospray ionization tandem mass spectrometry coupled with isotope correction algorithm. Biochimica et Biophysica acta 2004, 1686 (1-2), 108-17.
[40] 吳明賢, 人體消化第一站重鎮-捍「胃」你的健康. 22期, 財團法人全民健康基金會。