Mass spectrometric method for the analysis of metabolite in in metazoan and human samples
|關鍵字:||質譜;果蠅;同位素標定;生理時鐘;汗水;大氣壓下質譜法;快速取樣;mass spectrometry;fruit fly;in vivo stable isotope label;biological clock;sweat;ambient mass spectrometry;facile sampling|
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.
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