Abstract
Metabolite and fatty acid analysis play important roles in evaluating the metabolic state of microorganisms. To examine the growth state and metabolism response of cells to environmental stress or genetic modification, intracellular and extracellular metabolites including fatty acids are usually analyzed to help understand the cellular biochemical changes in microorganisms. In this protocol, gas chromatography-mass spectrometry based analysis was employed to investigate the fatty acids and other metabolites in yeast cells.
Keywords: Metabolite profiling, Fatty acid analysis, Saccharomyces cerevisiae
Materials and Reagents
Equipment
Software
Procedure
Note: Workflow of metabolite and fatty acid analyses of yeast cells and culture supernatants is shown in Figure 1.
Representative data
Figure 2. The representative GC-MS spectra for a S. cerevisiae fatty acid profile. Fatty acid composition and concentration may differ from those shown here as a result of altered cell culturing conditions and/or alternative genetic backgrounds of the yeast cells employed. Figure 3. A representative palmitoleic acid methyl ester standard curve. Different concentrations (0.125-0.75 mg/ml) of FAMEs standard were used for GC-MS analysis. To make individual FAME standard curves, the concentration of FAME was determined by mutiplying its percent concentration (as indicated on the cerificate of analysis from the supplier) by the prepared concentrations (0.125-0.75 mg/ml). Figure 4. A representative GC-MS spectra for L-Alanine derived from total ion chromatograms. Intracellular metabolites were extracted from the S. cerevisiae cells and metabolic profiling was conducted. The metabolite composition and concentration may differ from those shown here as a result of altered cell culturing conditions and/or alternative genetic backgrounds of the yeast cells employed.
Notes
Recipes
Acknowledgments
This protocol was adapted from previous work (Chen et al., 2014). This research is supported by a Competitive Research Programme (CRP) grant from the National Research Foundation of Singapore.
References
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