Optimization of Metabolite Extraction Protocols for the Identification and Profiling of Small Molecule Metabolites from Planktonic and Biofilm Pseudomonas aeruginosa Cultures
Methods: Three cell pellet wash solutions were compared for the potential to influence intracellular metabolite leakage of P. aeruginosa. We also compared four different extraction methods using (i) methanol:chloroform (2:1); (ii) 50% methanol; (iii) 100% methanol; or (iv) 100% water to extract intracellular metabolites from P. aeruginosa planktonic and biofilm cultures.
Results: Intracellular metabolite extraction efficiency was found to be dependent on the extraction method and varies between microbial modes of growth. Methods using the 60% methanol wash produced the greatest amount of intracellular material leakage. Quantification of intracellular metabolites via 1H NMR showed that extraction protocols using 100% water or 50% methanol achieved the greatest extraction efficiencies, while addition of sonication to facilitate cell lysis to the 50% methanol extraction method resulted in at least a two-fold increase in signal intensities for approximately half of the metabolites identified. Phosphate buffered saline (PBS) was determined to be the most appropriate wash solution, yielding little intracellular metabolite leakage from cells.
Conclusion: We determined that washing in 1X PBS and extracting intracellular metabolites with 50% methanol is the most appropriate metabolite extraction protocol because (a) leakage is minimal; (b) a broad range of metabolites present at sufficiently high concentrations is detectable by NMR; and (c) this method proved suitable for metabolite extraction of both planktonic and biofilm P. aeruginosa cultures.
Keywords: 1H NMR; Biofilm; P. aeruginosa; metabolite extraction; planktonic cell cultures; untargeted metabolomics
Document Type: Research Article
Publication date: 01 August 2016
Current Metabolomics publishes timely reviews, original research articles, thematic issues, and technical notes covering all aspects of the recent advancements and applications of metabolomics technology to systems biology, disease diagnosis, personalized medicine, and drug discovery. Original research articles and reviews in the following areas of metabolomics are of special interest to the readers of this journal:
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Metabolic profiling and directed metabolite analysis
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Sample preparation, extraction techniques and chromatography
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