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1H NMR-Based Metabolomics Methods for Chemical Genomics Experiments

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Plant Chemical Genomics

Abstract

Metabolomics and chemical genomics studies can each provide unique insights into plant biology. Although a variety of analytical techniques can be used for the interrogation of plant systems, nuclear magnetic resonance (NMR) provides unbiased characterization of abundant metabolites. An example methodology is provided for probing the metabolism of Arabidopsis thaliana in a chemical genomics experiment including methods for tissue treatment, tissue collection, metabolite extraction, and methods to minimize variance in biological and technical sample replicates. Additionally, considerations and methods for data analysis, including multivariate statistics, univariate statistics, and data interpretation are included. The process is illustrated by examining the metabolic effects of chemical treatment of Arabidopsis with Sortin 1, also known as vacuolar protein sorting inhibitor 1. Sortin 1 was applied to Arabidopsis seedlings to examine metabolic effects in a chemical genomics experiment and to demonstrate the utility of metabolomics in conjunction with other “omics” techniques.

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References

  1. Nicholson JK, Lindon JC, Holmes E (1999) ‘Metabonomics’: understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica 29:1181–1189

    Article  PubMed  CAS  Google Scholar 

  2. Fiehn O (2002) Metabolomics—the link between genotypes and phenotypes. Plant Mol Biol 48: 155–171

    Article  PubMed  CAS  Google Scholar 

  3. Robertson DG (2005) Metabonomics in toxicology: a review. Toxicol Sci 85:809–822

    Article  PubMed  CAS  Google Scholar 

  4. Robertson DG, Watkins PB, Reily MD (2011) Metabolomics in toxicology: preclinical and clinical applications. Toxicol Sci 120:S146–S170

    Article  PubMed  CAS  Google Scholar 

  5. Zouhar J, Hicks GR, Raikhel NV (2004) Sorting inhibitors (Sortins): chemical compounds to study vacuolar sorting in Arabidopsis. Proc Natl Acad Sci U S A 101:9497–9501

    Article  PubMed  CAS  Google Scholar 

  6. Fiehn O (2008) Extending the breadth of metabolite profiling by gas chromatography coupled to mass spectrometry. Trends Analyt Chem 27:261–269

    Article  PubMed  CAS  Google Scholar 

  7. Koek MM, Jellema RH, van der Greef J, Tas AC, Hankemeier T (2011) Quantitative metabolomics based on gas chromatography mass spectrometry: status and perspectives. Metabolomics 7:307–328

    Article  PubMed  CAS  Google Scholar 

  8. Lei Z, Huhman DV, Sumner LW (2011) Mass spectrometry strategies in metabolomics. J Biol Chem 286:25435–25442

    Article  PubMed  CAS  Google Scholar 

  9. Scalbert A, Brennan L, Fiehn O, Hankemeier T, Kristal BS, van Ommen B, Pujos-Guillot E, Verheij E, Wishart D, Wopereis S (2009) Mass-spectrometry-based metabolomics: limitations and recommendations for future progress with particular focus on nutrition research. Metabolomics 5:435–458

    Article  PubMed  CAS  Google Scholar 

  10. Keun HC, Ebbels TMD, Antti H, Bollard ME, Beckonert O, Schlotterbeck G, Senn H, Niederhauser U, Holmes E, Lindon JC, Nicholson JK (2002) Analytical reproducibility in H-1 NMR-based metabonomic urinalysis. Chem Res Toxicol 15:1380–1386

    Article  PubMed  CAS  Google Scholar 

  11. Sumner LW, Amberg A, Barrett D, Beale MH, Beger R, Daykin CA, Fan TWM, Fiehn O, Goodacre R, Griffin JL, Hankemeier T, Hardy N, Harnly J, Higashi R, Kopka J, Lane AN, Lindon JC, Marriott P, Nicholls AW, Reily MD, Thaden JJ, Viant MR (2007) Proposed minimum reporting standards for chemical analysis. Metabolomics 3:211–221

    Article  PubMed  CAS  Google Scholar 

  12. Sanderfoot AA, Pilgrim M, Adam L, Raikhel NV (2001) Disruption of individual members of Arabidopsis syntaxin gene families indicates each has essential functions. Plant Cell 13:659–666

    PubMed  CAS  Google Scholar 

  13. Lukowitz W, Mayer U, Jurgens G (1996) Cytokinesis in the Arabidopsis embryo involves the syntaxin-related KNOLLE gene product. Cell 84:61–71

    Article  PubMed  CAS  Google Scholar 

  14. Rojo E, Gillmor CS, Kovaleva V, Somerville CR, Raikhel NV (2001) VACUOLELESS1 is an essential gene required for vacuole formation and morphogenesis in Arabidopsis. Dev Cell 1:303–310

    Article  PubMed  CAS  Google Scholar 

  15. Rosado A, Hicks GR, Norambuena L, Rogachev I, Meir S, Pourcel L, Zouhar J, Brown MQ, Boirsdore MP, Puckrin RS, Cutler SR, Rojo E, Aharoni A, Raikhel NV (2011) Sortin1-hypersensitive mutants link vacuolar-trafficking defects and flavonoid metabolism in Arabidopsis vegetative tissues. Chem Biol 18:187–197

    Article  PubMed  CAS  Google Scholar 

  16. Lee DY, Fiehn O (2008) High quality metabolomic data for Chlamydomonas reinhardtii. Plant Methods 4:13

    Article  CAS  Google Scholar 

  17. Kaiser KA, Barding GA, Larive CK (2009) A comparison of metabolite extraction strategies for H-1-NMR-based metabolic profiling using mature leaf tissue from the model plant Arabidopsis thaliana. Magn Reson Chem 47:S147–S156

    Article  PubMed  CAS  Google Scholar 

  18. Sekiyama Y, Chikayama E, Kikuchi J (2011) Evaluation of a semipolar solvent system as a step toward heteronuclear multidimensional NMR-based metabolomics for C-13-labelled bacteria, plants, and animals. Anal Chem 83:719–726

    Article  PubMed  CAS  Google Scholar 

  19. Davis RA, Charlton AJ, Godward J, Jones SA, Harrison M, Wilson JC (2007) Adaptive binning: an improved binning method for metabolomics data using the undecimated wavelet transform. Chemometr Intell Lab Syst 85:144–154

    Article  CAS  Google Scholar 

  20. Holmes E, Foxall PJD, Nicholson JK, Neild GH, Brown SM, Beddell CR, Sweatman BC, Rahr E, Lindon JC, Spraul M, Neidig P (1994) Automatic data reduction and pattern recognition methods for analysis of H-1 nuclear magnetic resonance spectra of human urine from normal and pathological states. Anal Biochem 220:284–296

    Article  PubMed  CAS  Google Scholar 

  21. Craig A, Cloareo O, Holmes E, Nicholson JK, Lindon JC (2006) Scaling and normalization effects in NMR spectroscopic metabonomic data sets. Anal Chem 78:2262–2267

    Article  PubMed  CAS  Google Scholar 

  22. De Meyer T, Sinnaeve D, Van Gasse B, Tsiporkova E, Rietzschel ER, De Buyzere ML, Gillebert TC, Bekaert S, Martins JC, Van Criekinge W (2008) NMR-based characterization of metabolic alterations in hypertension using an adaptive, intelligent binning algorithm. Anal Chem 80:3783–3790

    Article  PubMed  Google Scholar 

  23. Dieterle F, Ross A, Schlotterbeck G, Senn H (2006) Probabilistic quotient normalization as robust method to account for dilution of complex biological mixtures. Application in H-1 NMR metabonomics. Anal Chem 78:4281–4290

    Article  PubMed  CAS  Google Scholar 

  24. Anderson PE, Mahle DA, Doom TE, Reo NV, DelRaso NJ, Raymer ML (2011) Dynamic adaptive binning: an improved quantification technique for NMR spectroscopic data. Metabolomics 7:179–190

    Article  CAS  Google Scholar 

  25. Broadhurst DI, Kell DB (2006) Statistical strategies for avoiding false discoveries in metabolomics and related experiments. Metabolomics 2:171–196

    Article  CAS  Google Scholar 

  26. Kaiser KA, Merrywell CE, Fang F, Larive CK (2008) In: Holzgrabe U, Wawer I, Diehl B (eds) NMR spectroscopy in pharmaceutical analysis. Elsevier, Oxford, pp 233–267

    Google Scholar 

  27. Barding GA Jr, Salditos R, Larive CK (2012) Quantitative NMR for bioanalysis and metabolomics. Anal. Bioanal. Chem. 404:1165-1179

    Google Scholar 

  28. Fiehn O, Kopka J, Trethewey R, Willmitzer L (2000) Identification of uncommon plant metabolites based on calculation of elemental compositions using gas chromatography and quadruple mass spectrometry. Anal Chem 72:3573–3580

    Article  PubMed  CAS  Google Scholar 

  29. Glasoe PK, Long FA (1960) Use of glass electrodes to measure acidities in deuterium oxide. J Phys Chem 64:188–190

    Article  CAS  Google Scholar 

  30. Larive CK, Jayawickrama D, Orfi L (1997) Quantitative analysis of peptides with NMR spectroscopy. Appl Spectrosc 51:1531–1536

    Article  CAS  Google Scholar 

  31. Smallcombe SH, Patt SL, Keifer PA (1995) WET solvent suppression and its applications to LC NMR and high-resolution NMR spectroscopy. J Magn Reson A 117:295–303

    Article  CAS  Google Scholar 

  32. Liu ML, Mao XA, Ye CH, Huang H, Nicholson JK, Lindon JC (1998) Improved WATERGATE pulse sequences for solvent suppression in NMR spectroscopy. J Magn Reson 132:125–129

    Article  CAS  Google Scholar 

  33. Cui Q, Lewis IA, Hegeman AD, Anderson ME, Li J, Schulte CF, Westler WM, Eghbalnia HR, Sussman MR, Markley JL (2008) Metabolite identification via the Madison Metabolomics Consortium Database. Nat Biotechnol 26:162–164

    Article  PubMed  CAS  Google Scholar 

  34. Wishart DS, Knox C, Guo AC, Eisner R, Young N, Gautam B, Hau DD, Psychogios N, Dong E, Bouatra S, Mandal R, Sinelnikov I, Xia J, Jia L, Cruz JA, Lim E, Sobsey CA, Shrivastava S, Huang P, Liu P, Fang L, Peng J, Fradette R, Cheng D, Tzur D, Clements M, Lewis A, De Souza A, Zuniga A, Dawe M, Xiong Y, Clive D, Greiner R, Nazyrova A, Shaykhutdinov R, Li L, Vogel HJ, Forsythe I (2009) HMDB: a knowledgebase for the human metabolome. Nucleic Acids Res 37:D603–D610

    Article  PubMed  CAS  Google Scholar 

  35. Braunschweiler L, Ernst RR (1983) Coherence transfer by isotropic mixing: application to proton correlation spectroscopy. J Magn Reson 53:521–528

    CAS  Google Scholar 

  36. Bodenhausen G, Ruben DJ (1980) Natural abundance N-15 NMR by enhanced heteronuclear spectroscopy. Chem Phys Lett 69:185–189

    Article  CAS  Google Scholar 

  37. Cobas C, Seoane F, Dominguez S, Sykora S, Davies A (2011) A new approach to improving automated analysis of proton NMR spectra through Global Spectral Deconvolution 8GSD. Spectrosc Eur 23:26–30

    CAS  Google Scholar 

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Acknowledgements

DJO, GAB, and CEM gratefully acknowledge support by the National Science Foundation Integrative Graduate Education Research and Training Program grant DGE-0504249. CKL and DJO acknowledge additional support from POM Wonderful, LLC.

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Orr, D.J., Barding, G.A., Tolley, C.E., Hicks, G.R., Raikhel, N.V., Larive, C.K. (2014). 1H NMR-Based Metabolomics Methods for Chemical Genomics Experiments. In: Hicks, G., Robert, S. (eds) Plant Chemical Genomics. Methods in Molecular Biology, vol 1056. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-592-7_21

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  • DOI: https://doi.org/10.1007/978-1-62703-592-7_21

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-591-0

  • Online ISBN: 978-1-62703-592-7

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