Abstract
Salmonella enterica serovar typhimurium (S. typhimurium) causes food poisoning in human and animals. Its infection rate is the highest among all salmonella serotypes. Metabolomics is a potential way to study the pathogenesis of S. typhimurium via analysis of various small molecular substances. Due to the lack of a uniform protocol for the extraction of metabolites, we evaluated five commonly used extraction methods including cold methanol (CM), hot ethanol (HE), chloroform–methanol cocktail (CMC), perchloric acid (PCA), and alkali (AL) for their efficacy in extracting the intracellular metabolites of S. typhimurium. Samples were quenched in 60% methanol at − 40 °C, and then the five methods were used to extract the metabolites. After derivatization, all samples were analyzed on a gas chromatography–triple quadrupole mass spectrometry (GC–MS/MS). Our results suggest that CM and HE extraction methods provide the best compromise allowing identification of 98 and 95 metabolites in a single analysis. For targeted metabolome analysis, the optimal extraction method for alcohols and organic acids is HE. CMC preferentially extracted lipid metabolites. PCA is suitable for extraction of small molecular carbohydrates. The optimal extraction method for macromolecular carbohydrates is the CM method.
Similar content being viewed by others
References
Bern C, Martines J, de Zoysa I, Glass RI (1992) The magnitude of the global problem of diarrhoeal disease: a ten-year update. Bull World Health Organization 70(6):705–714
Hendriksen RS, Vieira AR, Karlsmose S, Wong DMALF, Jensen AB, Wegener HC, Aarestrup FM (2011) Global monitoring of salmonella serovar distribution from the world health organization global foodborne infections network country data bank: results of quality assured laboratories from 2001 to 2007. Foodborne Pathog Dis 8(8):887–900
Raamsdonk LM et al (2001) A functional genomics strategy that uses metabolome data to reveal the phenotype of silent mutations. Nat Biotechnol 19:45–50
Gika H, Theodoridis GA, Plumb RS, Wilson ID (2014) Current practice of liquid chromatography-mass spectrometry in metabolomics and metabonomics. J Pharm Biomed Anal 87:12–25
Ramana P, Adams E, Augustijns P, Van Schepdael A (2015) Metabonomics and drug development vol 1277. Methods in Molecular Biology, 2015/02/14 edn
Peng B, Li H, Peng XX (2015) Functional metabolomics: from biomarker discovery to metabolome reprogramming. Protein Cell 6:628–637
Ogbaga CC, Stepien P, Dyson BC, Rattray NJ, Ellis DI, Goodacre R, Johnson GN (2016) Biochemical analyses of sorghum varieties reveal differential responses to drought. PLoS ONE 11:e0154423
Wang X, Xie Y, Gao P, Zhang S, Tan H, Yang F, Lian R, Tian J, Xu G (2014) A metabolomics-based method for studying the effect of yfcC gene in Escherichia coli on metabolism. Anal Biochem 451:48–55
Kato M, Lin SJ (2014) Regulation of NAD+ metabolism, signaling and compartmentalization in the yeast Saccharomyces cerevisiae. DNA Repair 23:49–58
Sapcariu SC, Kanashova T, Weindl D, Ghelfi J, Dittmar G, Hiller K (2014) Simultaneous extraction of proteins and metabolites from cells in culture. MethodsX 1:74–80
Mashego MR, Rumbold K, De Mey M, Vandamme E, Soetaert W, Heijnen JJ (2007) Microbial metabolomics: past, present and future methodologies. Biotechnol Lett 29:1–16
Maharjan RP, Ferenci T (2003) Global metabolite analysis: the influence of extraction methodology on metabolome profiles of Escherichia coli. Anal Biochem 313:145–154
Tanga T, Gaoa Q, Barrow P, Wanga M, Chenga A, Jiaa R, Zhu D, Chena S, Liua M, Sun K, Yang Q, Chen X (2015) Development and evaluation of live attenuated Salmonella vaccines in newly hatched duckings. Vaccine 33:5564–5571
Yamamoto H, Fujimori T, Sato H, Ishikawa G, Kami K, Ohashi Y (2014) Statistical hypothesis testing of factor loading in principal component analysis and its application to metabolite set enrichment analysis. BMC Bioinform 15:51
Sevin DC, Kuehne A, Zamboni N, Sauer U (2015) Biological insights through nontargeted metabolomics. Curr Opin Biotechnol 34:1–8
Park C, Yun S, Lee SY, Park K, Lee J (2012) Metabolic profiling of Klebsiella oxytoca: evaluation of methods for extraction of intracellular metabolites using UPLC/Q-TOF-MS. Appl Biochem Biotechnol 167:425–438
Duportet X, Aggio RBM, Carneiro S, Villas-Bôas SGJM (2012) The biological interpretation of metabolomic data can be misled by the extraction method used. Metabolomics 8:410–421
Cohn EJ, McMeekin TL, Edsall JT, Weare JH (1934) Studies in the physical chemistry of amino acids, peptides and related substances. II. The solubility of α-amino acids in water and in alcohol—water mixtures. J Am Chem Soc 56(11):2270–2282
Vanyushkina AA, Fisunov GY, Gorbachev AY, Kamashev DE, Govorun VM (2014) Metabolomic analysis of three Mollicute species. PLoS ONE 9:e89312
Buchholz A, Hurlebaus J, Wandrey C, Takors R (2002) Metabolomics: quantification of intracellular metabolite dynamics. Biomol Eng 19:5–15
Buchholz A, Takors R, Wandrey C (2001) Quantification of intracellular metabolites in Escherichia coli K12 using liquid chromatographic-electrospray ionization tandem mass spectrometric techniques. Anal Biochem 295:129–137
Shryock JC, Rubio R, Berne RM (1986) Extraction of adenine nucleotides from cultured endothelial cells. Anal Biochem 159:73–81
Stipetic LH, Dalby MJ, Davies RL, Morton FR, Ramage G, Burgess KE (2016) A novel metabolomic approach used for the comparison of Staphylococcus aureus planktonic cells and biofilm samples. Metabolomics 12:75
Varik V, Oliveira SRA, Hauryliuk V, Tenson T (2017) HPLC-based quantification of bacterial housekeeping nucleotides and alarmone messengers ppGpp and pppGpp. Sci Rep 7:11022
Dietmair S, Timmins NE, Gray PP, Nielsen LK, Kromer JO (2010) Towards quantitative metabolomics of mammalian cells: development of a metabolite extraction protocol. Anal Biochem 404:155–164
Acknowledgements
This study is supported by the China Postdoctoral Science Foundation (Grant No. 2016M602691) and Sichuan Science & Technology Department Foundation (Grant No. 2017JY0240).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Tian, S., Wang, C., Yang, L. et al. Comparison of Five Extraction Methods for Intracellular Metabolites of Salmonella typhimurium. Curr Microbiol 76, 1247–1255 (2019). https://doi.org/10.1007/s00284-019-01750-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00284-019-01750-4