微米級尺寸生物樣品之質譜化學分析方法開發

Abstract

許多化學及生化過程無論是尺寸或時間皆發生於微米級尺寸。因此，適用於微米級尺寸之生物樣品分析方法可幫助科學家解決過往無法解決的問題，而進一步幫助我們對於理解生命的現象。質譜為21世紀最重要的分析技術之一，其具有相當卓越的速度、靈敏度及選擇性。因此在本論文中將以質譜為主要化學分析平台，嘗試開發數種可用於分析微米級尺寸生物樣品之策略。在論文(第二章) 的第一部分，微米級尺寸的分析平台成功被開發，其採用同位素標記策略(結合基質輔助雷射脫附/游離質譜及螢光顯微鏡) 觀察真菌菌絲之生長及代謝。基質輔助雷射脫附/游離質譜的主要缺點為所謂的"最佳點效應" - 在樣品沉澱物上含有濃度升高的分析物分子分布於微米級尺寸區域。因此，在第三章我研究雷射脫附/游離質譜樣品製備中，樣品沉澱物內代謝體和有機/無機化合物分布不均勻之現象，其在樣品點發生的"咖啡環" 現象藉由影像質譜進行研究。基質輔助雷射脫附/游離質譜適合用於微米級尺寸之化學分析，當使用質譜分析複雜的生物樣品時，適當的樣品處理也具有其重要性。在第四章，我開發一自動微萃取系統結合質譜及螢光顯微鏡。此開發平台可使生物樣品(單一果蠅及茶葉的碎片) 在被萃取當下同時被螢光顯微鏡及質譜分析。而在第五章，我提出一種分析方法其可用於了解含有各種微生物(如酵母菌) 之益生菌飲料之代謝體的時間分布。此研究使用多種不同的技術，諸如氣相層析及液相層析結合質譜。總括來說，四種以質譜為分析平台用於微米級尺寸樣品的化學分析方法已成功被開發。於第二章及第三章所呈現的方法可針對微米級尺寸生物樣品之生化分析提供高空間解析度。除可用於監測生物合成幾丁質過程中之中間物並擁有單菌絲之解析度(~ 10 微米) 外，也針對咖啡環現象進行探討，此研究之結果可幫助防止基質輔助雷射脫附/游離質譜影像於樣品製備過程中因人為所產生的干擾。而於第四章及第五章所提出則可適用於不同種類的樣品分析。其中一個方法可使複雜的生物樣品直接進行分析且無需任何樣品前處理，而另一種則可針對微生物豐富的益生菌飲料以適當的時間解析度進行代謝體變化之研究。最後，我希望於此篇論文中所提出之方法可於生物科學領域上有所貢獻。

Many chemical and biochemical processes occur on the microscale level with respect to both dimension and time scale. Analytical methods targeting microscale biological samples can improve our understanding of life, by helping scientists to solve the problems which could not be solved before. Mass spectrometry (MS) is one of the leading analytical techniques in the 21st century. It features superior speed, sensitivity, and selectivity. Hence, in this thesis, I attempted to develop strategies for chemical analysis of biological samples at microscale based on the use of MS. In the first part of the thesis (Chapter 2), a microscale analytical platform was established, which uses isotope-labeling strategy (combing with matrix-assisted laser desorption/ionization (MALDI)-MS with fluorescence microscopy) for monitoring metabolism of fungal mycelium. One of the main disadvantages of MALDI-MS is the so-called “sweet-spot effect” – occurrence of microscale sites within the sample deposits that contain an elevated concentration of analyte molecules. Therefore, in Chapter 3, I studied heterogeneous distribution of metabolites and inorganic/organic compounds within dry deposits of samples preparated for laser desorption/ionization MS. The occurrence of “coffee rings” in sample spots was investigated by means of mass spectrometric imaging. While, MALDI-MS is suitable for chemical analysis at microscale, adequate sample preparation is very important when employing it in the analyses of complex biological matrices. In Chapter 4, an automated microextraction system – combined with MS and fluorescence microscopy – was developed. The proposed platform enables disruption/extraction of biological samples (individual fruit flies and fragments of tea leaves), and simultaneous analysis by fluorescence microscopy and MS. Chapter 5 presents an analytical approach to reveal temporal profiles of metabolites in probiotic drinks, which contain various microorganisms (e.g. yeast). This study was possible thanks to application of several techniques, including gas chromatography and liquid chromatography hyphenated with MS. In conclusion, four MS-based analytical platforms have been developed for chemical analysis of biological samples at microscale. The approaches presented in Chapters 2 and 3 provide high spatial resolution for biochemical analysis of microscale samples. Monitoring of an intermediate in the biosynthesis of chitin with single hypha resolution (~ 10 μm) was possible. The phenomenon of coffee-ring formation was studied. The outcome of this study may help to prevent the occurrence of artifacts during sample preparation for MALDI-MS imaging. The methods presented in Chapters 4 and 5 are suitable for analysis of different kinds of samples. One of those methods enabled analysis of complex microscale biological samples without any sample pretreatment. The other one provided sufficient temporal resolution to reveal metabolic changes in microorganism-rich probiotic drinks. I hope that the methods developed in this project can contribute to new discoveries in bioscience.