Advertisement

Considerations of Sample Preparation for Metabolomics Investigation

  • Teresa Whei-Mei FanEmail author
Protocol
Part of the Methods in Pharmacology and Toxicology book series (MIPT)

Abstract

Sample preparation is the gateway to metabolomic analysis, the importance of which cannot be overemphasized. There are general rules of thumb for sample preparation that help maximize sample integrity and metabolite recovery. The wide range of variations in metabolite functional groups, polarity, sizes, and stability precludes the use of a single extraction method in metabolomic studies. Common extraction methods for polar metabolites that utilize trichloroacetic acid or aqueous acetonitrile are suitable for both nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis while others that use chloroform/methanol/water partition or boiling water may not. Control of extract pH is crucial for consistent NMR assignments and chemical derivatization-linked MS analysis. Sequential polar and lipid extractions reduce sample size requirement and provide a better coverage for direct-infusion MS analysis of lipids, possibly by removing interfering salts. Cleanup of sample extracts, such as removal of fine particles or interfering cations, is often necessary but should be limited to reduce loss of metabolites.

Key words

NMR GC-MS FT-ICR-MS Trichloroacetic acid Perchloric acid Chloroform/methanol/water Acetonitrile Boiling water Mouse liver Human lung 

Notes

Acknowledgments

This work was supported in part by the National Cancer Institute grants # 1R01 CA101199-01 and 1R01CA118434-01A2, NIH Grant Number RR018733 from the National Center for Research Resources, National Science Foundation EPSCoR grant # EPS-0447479, Kentucky Challenge for Excellence, and the Brown Foundation. Dr. Zhengzhi Xie and Ms. Vennila Arumugum are acknowledged for respective assistance in sample processing and FT-ICR-MS analysis.

Glossary

BHT

Butylated hydroxytoluene

CMW

Chloroform/methanol/water partition

FT-ICR-MS

Fourier transform-ion cyclotron resonance mass spectrometry

GC-MS

Gas chromatography-mass spectrometry

GSH

Reduced glutathione

GSSG

Oxidized glutathione

HSQC

Heteronuclear single quantum coherence spectroscopy

Isotopomer

Compounds of identical chemical structure but differing in isotopic composition at individual atoms

LC-MS

Liquid chromatography-mass spectrometry

MTBSTFA

N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide

PCA

Perchloric acid

TCA

Trichloroacetic acid

TOCSY

Total correlation spectroscopy

Supplementary material

References

  1. 1.
    De Vos RC, Moco S, Lommen A, Keurentjes JJ, Bino RJ, Hall RD. Untargeted large-scale plant metabolomics using liquid chromatography coupled to mass spectrometry. Nat Protoc. 2007;2:778–91.PubMedCrossRefGoogle Scholar
  2. 2.
    Lu X, Xu G. LC-MS metabonomics methodology in biomarker discovery. In: Wang F, editor. Biomarker methods in drug discovery and development. Totowa, NJ: Humana press; 2008. p. 291–315.CrossRefGoogle Scholar
  3. 3.
    Fan TWM, Colmer TD, Lane AN, Higashi RM. Determination of metabolites by proton NMR and GC analysis for organic osmolytes in crude tissue extracts. Anal Biochem. 1993;214:260–71.PubMedCrossRefGoogle Scholar
  4. 4.
    Sellick CA, Hansen R, Maqsood AR, et al. Effective quenching processes for physiologically valid metabolite profiling of suspension cultured mammalian cells. Anal Chem. 2009;81:174–83.PubMedCrossRefGoogle Scholar
  5. 5.
    Troy H, Chung Y-L, Mayr M, et al. Metabolic profiling of hypoxia-inducible factor-1beta-deficient and wild type Hepa-1 cells: effects of hypoxia measured by 1H magnetic resonance spectroscopy. Metabolomics. 2005;1:293–303.CrossRefGoogle Scholar
  6. 6.
    Lutz U, Lutz RW, Lutz WK. Metabolic profiling of glucuronides in human urine by LC-MS/MS and partial least-squares discriminant analysis for classification and prediction of gender. Anal Chem. 2006;78:4564–71.PubMedCrossRefGoogle Scholar
  7. 7.
    Azmi J, Connelly J, Holmes E, Nicholson JK, Shore RF, Griffin JL. Characterization of the biochemical effects of 1-nitronaphthalene in rats using global metabolic profiling by NMR spectroscopy and pattern recognition. Biomarkers. 2005;10:401–16.PubMedCrossRefGoogle Scholar
  8. 8.
    Bolten CJ, Kiefer P, Letisse F, Portais JC, Wittmann C. Sampling for metabolome analysis of microorganisms. Anal Chem. 2007; 79:3843–9.PubMedCrossRefGoogle Scholar
  9. 9.
    de Koning W, van Dam K. A method for the determination of changes of glycolytic metabolites in yeast on a subsecond time scale using extraction at neutral pH. Anal Biochem. 1992;204:118–23.PubMedCrossRefGoogle Scholar
  10. 10.
    Buchholz A, Hurlebaus J, Wandrey C, Takors R. Metabolomics: quantification of intracellular metabolite dynamics. Biomol Eng. 2002;19:5–15.PubMedCrossRefGoogle Scholar
  11. 11.
    Wittmann C, Kromer JO, Kiefer P, Binz T, Heinzle E. Impact of the cold shock phenomenon on quantification of intracellular metabolites in bacteria. Anal Biochem. 2004; 327:135–9.PubMedCrossRefGoogle Scholar
  12. 12.
    Schaub J, Schiesling C, Reuss M, Dauner M. Integrated sampling procedure for metabolome analysis. Biotechnol Prog. 2006;22:1434–42.PubMedCrossRefGoogle Scholar
  13. 13.
    Link H, Anselment B, Weuster-Botz D. Leakage of adenylates during cold methanol/glycerol quenching of Escherichia coli. Metabolomics. 2008;4:240–7.CrossRefGoogle Scholar
  14. 14.
    Villas-Buas SG, Bruheim P. Cold glycerol-saline: the promising quenching solution for accurate intracellular metabolite analysis of microbial cells. Anal Biochem. 2007;370:87–97.CrossRefGoogle Scholar
  15. 15.
    Winder CL, Dunn WB, Schuler S, et al. Global metabolic profiling of Escherichia coli cultures: an evaluation of methods for quenching and extraction of intracellular metabolites. Anal Chem. 2008;80:2939–48.PubMedCrossRefGoogle Scholar
  16. 16.
    Fan TWM, Bandura L, Higashi RM, Lane AN. Metabolomics-edited transcriptomics analysis of Se anticancer action in human lung cancer cells. Metabolomics. 2005;1:325–39.CrossRefGoogle Scholar
  17. 17.
    Villas-Boas SG, Hojer-Pedersen J, Akesson M, Smedsgaard J, Nielsen J. Global metabolite analysis of yeast: evaluation of sample preparation methods. Yeast. 2005;22:1155–69.PubMedCrossRefGoogle Scholar
  18. 18.
    Liang YS, Kim HK, Lefeber AW, Erkelens C, Choi YH, Verpoorte R. Identification of phenylpropanoids in methyl jasmonate treated Brassica rapa leaves using two-dimensional nuclear magnetic resonance spectroscopy. J Chromatogr. 2006;1112:148–55.CrossRefGoogle Scholar
  19. 19.
    Barsch A, Patschkowski T, Niehaus K. Comprehensive metabolite profiling of Sinorhizobium meliloti using gas chromatography–mass spectrometry. Funct Integr Genomics. 2004; 4:219–30.PubMedCrossRefGoogle Scholar
  20. 20.
    Bolling C, Fiehn O. Metabolite profiling of Chlamydomonas reinhardtii under nutrient deprivation. Plant Physiol. 2005;139:1995–2005.PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Fan TW-M, Lane AN. Structure-based profiling of metabolites and isotopomers by NMR. Prog NMR Spectr. 2008;52:69–117.CrossRefGoogle Scholar
  22. 22.
    Lane AN, Fan TWM, Higashi RM. Isotopomer-based metabolomic analysis by NMR and mass spectrometry. Methods Cell Biol. 2008; 84:541–88.PubMedCrossRefGoogle Scholar
  23. 23.
    Fan TWM, Kucia M, Jankowski K, et al. Rhabdomyosarcoma cells show an energy producing anabolic metabolic phenotype compared with primary myocytes. Mol Cancer. 2008;7:79.PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Fiehn O, Kopka J, Dormann P, Altmann T, Trethewey RN, Willmitzer L. Metabolite profiling for plant functional genomics. Nat Biotechnol. 2000;18:1157–61.PubMedCrossRefGoogle Scholar
  25. 25.
    Gradwell MJ, Fan TWM, Lane AN. Analysis of phosphorylated metabolites in crayfish extracts by two-dimensional 1H-31P NMR heteronuclear total correlation spectroscopy (hetero TOCSY). Anal Biochem. 1998;263:139–49.PubMedCrossRefGoogle Scholar
  26. 26.
    Fan TWM, Lane AN, Higashi RM. Selenium biotransformations by a euryhaline microalga isolated from a saline evaporation pond. Environ Sci Technol. 1997;31:569–76.CrossRefGoogle Scholar
  27. 27.
    Fan TWM, Higashi RM, Macdonald JM. Emergence and recovery response of phosphate metabolites and intracellular pH in intact Mytilus edulis as examined in situ by in vivo phosphorus-31 NMR. Biochim Biophys Acta. 1991;1092:39–48.PubMedCrossRefGoogle Scholar
  28. 28.
    Fan TWM, Higashi RM, Lane AN, Jardetzky O. Combined use of proton NMR and gas chromatography-mass spectra for metabolite monitoring and in vivo proton NMR assignments. Biochim Biophys Acta. 1986;882:154–67.PubMedCrossRefGoogle Scholar
  29. 29.
    Fan TWM, Lane AN, Shenker M, Bartley JP, Crowley D, Higashi RM. Comprehensive chemical profiling of gramineous plant root exudates using high-resolution NMR and MS. Phytochemistry (Oxford). 2001;57:209–21.CrossRefGoogle Scholar
  30. 30.
    Lewis IA, Schommer SC, Hodis B, Robb KA, Tonelli M, Westler WM, Suissman MR, Markley JL. Method for determining molar concentrations of metabolites in complex solutions from two-dimensional H-1-C-13 NMR spectra. Anal Chem. 2007;79:9385–90.PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Fiehn O, Kopka J, Trethewey RN, Willmitzer L. Identification of uncommon plant metabolites based on calculation of elemental compositions using gas chromatography and quadrupole mass spectrometry. Anal Chem. 2000;72:3573–80.PubMedCrossRefGoogle Scholar
  32. 32.
    Fan TW-M, Lane AN. Structure-based profiling of metabolites and isotopomers by NMR. Prog NMR Spectrosc. 2008;52:69–117.CrossRefGoogle Scholar
  33. 33.
    Lane AN, Fan TW-M, Higashi RM. Isotopomer-based metabolomic analysis by NMR and mass spectrometry. Methods Cell Biol. 2008;84:541–88.PubMedCrossRefGoogle Scholar
  34. 34.
    Lane AN, Fan TW-M. Quantification and identification of isotopomer distributions of metabolites in crude cell extracts using 1H TOCSY. Metabolomics. 2007;3:79–86.CrossRefGoogle Scholar
  35. 35.
    Rujoi M, Jin J, Borchman D, Tang D, Yappert MC. Isolation and lipid characterization of cholesterol-enriched fractions in cortical and nuclear human lens fibers. Invest Ophthalmol Vis Sci. 2003;44:1634–42.PubMedCrossRefGoogle Scholar
  36. 36.
    Yappert MC, Rujoi M, Borchman D, Vorobyov I, Estrada R. Glycero- versus sphingo-phospholipids: correlations with human and non-human mammalian lens growth. Exp Eye Res. 2003;76:725–34.PubMedCrossRefGoogle Scholar
  37. 37.
    Schwudke D, Oegema J, Burton L, et al. Lipid profiling by multiple precursor and neutral loss scanning driven by the data-dependent acquisition. Anal Chem. 2006;78:585–95.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  1. 1.Department of Chemistry, Center for Regulatory and Environmental Analytical Metabolomics (CREAM), and James Graham Brown Cancer CenterUniversity of LouisvilleLouisvilleUSA

Personalised recommendations