标题: 开发质谱法侦测生物代谢体之方法及应用
Mass spectrometric method for the analysis of metabolite in in metazoan and human samples
作者: 曾德玮
Tseng, Te-Wei
帕伟鄂本
陈月枝
Urban, Pawel
Chen, Yu-Chie
应用化学系硕博士班
关键字: 质谱;果蝇;同位素标定;生理时钟;汗水;大气压下质谱法;快速取样;mass spectrometry;fruit fly;in vivo stable isotope label;biological clock;sweat;ambient mass spectrometry;facile sampling
公开日期: 2011
摘要: 在此论文中,主要是以探讨利用质谱法于果蝇卵子与人体汗水等生物检体代谢物之分析与应用。在论文第一部份,我们藉由果蝇喂食具有稳定同位素碳十三葡萄醣发展活体同位素标定技术并以基质辅助雷射脱附游离质谱法维分析技术,进而取得果蝇的代谢体变化之讯息,自研究之结果中我们发现此方法可获得果蝇单一卵子之半定量分析资讯。实验中经喂食全碳十三葡萄糖的果蝇,碳十三同位素可进入果蝇的代谢过程中,在此我们将以目标代谢物-尿苷二磷酸的碳十三标定程度代表代谢速度,藉此标定程度我们发现此方法可反映环境变化对果蝇代谢速度的影响。在分析结果中,我们发现实验组中调控短暂的日夜颠倒即可立即反应在果蝇卵子之代谢体,实验组之果蝇卵子中的代谢体与控制组的果蝇卵子的标定程度结果几乎一样。而论文的第二部份,开发了一分析人体汗水代谢体之方法,藉由新颖之奈米喷洒脱附电喷洒游离质谱技术侦测以医疗胶布快速可收集来自于皮肤汗水样品。此方法结合医疗胶布的快速取样,不需任何前处理步骤即以奈米喷洒脱附电喷洒游离质谱分析。我们首先以自制的奈米喷洒脱附电喷洒游离源分析标准品,以确立此游离源之可行性,接着进行真实样品汗液的分析,实验中可以看到和背景梨子有差异的几支离子峯,然而这些离子峯的确认还有待进一步的分析。并且我们将取样及分析的过程做最佳化调整,此方法之优点在于可在几秒钟即可将样品收集完成,且可在几分钟内完成质谱分析。
In this work, we describe the development of two analytical methods based on mass spectrometry (MS), which are applicable to analysis of metabolites in biological samples: single eggs of fruit flies and human sweat. In the first part, we present mass spectrometric analysis of small metazoan (fruit fly) samples collected after in-vivo labeling with a stable isotope (carbon-13). We have found that matrix-assisted laser desorption/ionization (MALDI)-MS, used in conjunction with the isotopic labeling, enables quasi-quantitative analysis of metabolism. Flies were initially fed with 13C6-glucose, and the carbon-13 label was readily incorporated to primary metabolites. The yields of isotopic labeling of one target metabolite (uridine diphosphate glucose, UDP-Glc) were used as a proxy for the metabolic rates. The labeling – assessed based on the MALDI mass spectra – reflected the treatment applied to the fly stocks. A connection between circadian clock and the egg metabolism could be established. The study shows that a short-term perturbation to the day/night cycle has little effect on the metabolic rates in eggs – as assessed based on the labeling of UDP-Glc. In the second part of this work, we proposed a method for the analysis of sweat metabolites. The method combines facile sampling of sweat and detection by nanospray desorption electrospray ionization (nanoDESI)-MS. The samples were collected by using adhesive plasters as sampling tools, which was followed by nanoDESI-MS detection without any sample pretreatment. In this study, we implemented a home-made nanoDESI source, and tested it using standard compounds. Following a brief optimization of the detection method, the real samples were analyzed. In the resulting nanoDESI mass spectra, we could find peaks, which may correspond to sweat metabolites; however, a thorough identification of these putative metabolites has yet to be conducted. The main advantage of this method is its simplicity: sweat samples can be collected within a few seconds, and the analysis by nanoDESI-MS takes only several minutes.
URI: http://140.113.39.130/cdrfb3/record/nctu/#GT079925551
http://hdl.handle.net/11536/49885
显示于类别:Thesis


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