Analysis of carbon and nitrogen co-metabolism in yeast by ultrahigh-resolution mass spectrometry applying 13C- and 15N-labeled substrates simultaneously
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- 作者：Lars M. Blank (12)
Rahul R. Desphande (13)
Andreas Schmid (1)
Heiko Hayen (45) hayen@uni-wuppertal.de - 关键词：13C ; and 15N ; labeling experiment – ; Mass spectrometry – ; Yeast – ; Saccharomyces cerevisiae – ; Amino acid metabolism
- 刊名：Analytical and Bioanalytical Chemistry
- 出版年：2012
- 出版时间：June 2012
- 年：2012
- 卷：403
- 期：8
- 页码：2291-2305
- 全文大小：466.5 KB
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- 刊物类别：Chemistry and Materials Science
- 刊物主题：Chemistry
Analytical Chemistry
Food Science
Inorganic Chemistry
Physical Chemistry
Monitoring, Environmental Analysis and Environmental Ecotoxicology - 出版者：Springer Berlin / Heidelberg
- ISSN：1618-2650
文摘
Alternative metabolic pathways inside a cell can be deduced using stable isotopically labeled substrates. One prerequisite is accurate measurement of the labeling pattern of targeted metabolites. Experiments are generally limited to the use of single-element isotopes, mainly 13C. Here, we demonstrate the application of direct infusion nanospray, ultrahigh-resolution Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) for metabolic studies using differently labeled elemental isotopes simultaneously—i.e., 13C and 15N—in amino acids of a total protein hydrolysate. The optimized strategy for the analysis of metabolism by a hybrid linear ion trap-FTICR-MS comprises the collection of multiple adjacent selected ion monitoring scans. By limiting both the width of the mass range and the number of ions entering the ICR cell with automated gain control, sensitive measurements of isotopologue distribution were possible without compromising mass accuracy and isotope intensity mapping. The required mass-resolving power of more than 60,000 is only achievable on a routine basis by FTICR and Orbitrap mass spectrometers. Evaluation of the method was carried out by comparison of the experimental data to the natural isotope abundances of selected amino acids and by comparison to GC/MS results obtained from a labeling experiment with 13C-labeled glucose. The developed method was used to shed light on the complexity of the yeast Saccharomyces cerevisiae carbon–nitrogen co-metabolism by administering both 13C-labeled glucose and 15N-labeled alanine. The results indicate that not only glutamate but also alanine acts as an amino donor during alanine and valine synthesis. Metabolic studies using FTICR-MS can exploit new possibilities by the use of multiple-labeled elemental isotopes.