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Plant Metabolomics Using NMR Spectroscopy

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NMR-Based Metabolomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2037))

Abstract

The major goal in plant metabolomics is to study complex extracts for the purposes of metabolic exploration and natural products discovery. To achieve this goal, plant metabolomics relies on accurate and selective acquisition of all possible chemical information, which includes maximization of the number of detected metabolites and their correct molecular assignment. Nuclear magnetic resonance (NMR) spectroscopy has been recognized as a powerful platform for obtaining the metabolite profiles of plant extracts. In this chapter, we provide a workflow for targeted and untargeted metabolite profiling of plant extracts using both 1D and 2D NMR methods. The protocol includes sample preparation, instrument operation, data processing, multivariate analysis, biomarker elucidation, and metabolite quantitation. It also addresses the annotation of plant metabolite peaks considering NMR’s capabilities to cover a broad range of metabolites and elucidate structures for unknown compounds.

Denise Medeiros Selegato and Alan Cesar Pilon are co-first authors.

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References

  1. Weng J-K (2014) The evolutionary paths towards complexity: a metabolic perspective. New Phytol 201:1141–1149

    Article  Google Scholar 

  2. Hall R (2002) Plant metabolomics: the missing link in functional genomics strategies. Plant Cell 14:1437–1440

    Article  CAS  Google Scholar 

  3. Kim HK, Choi YH, Verpoorte R (2011) NMR-based plant metabolomics: where do we stand, where do we go? Trends Biotechnol 29:267–275

    Article  CAS  Google Scholar 

  4. Hong J, Yang L, Zhang D, Shi J (2016) Plant metabolomics: an indispensable system biology tool for plant science. Int J Mol Sci 17:767

    Article  Google Scholar 

  5. Ortmayr K, Causon TJ, Hann S, Koellensperger G (2016) Increasing selectivity and coverage in LC-MS based metabolome analysis. TrAC Trends Anal Chem 82:358–366

    Article  CAS  Google Scholar 

  6. Brunetti AE, Carnevale Neto F, Vera MC et al (2018) An integrative omics perspective for the analysis of chemical signals in ecological interactions. Chem Soc Rev 47:1574–1591

    Article  CAS  Google Scholar 

  7. Funari CS, Castro-Gamboa I, Cavalheiro AJ, Bolzani V da S (2013) Metabolomics, an optimized approach for the rational exploitation of Brazilian biodiversity: state of the art, new scenarios, and challenges. Quim Nov 36:1605–1609

    Article  CAS  Google Scholar 

  8. Fiehn O (2001) Combining genomics, metabolome analysis, and biochemical modelling to understand metabolic networks. Comp Funct Genomics 2:155–168

    Article  CAS  Google Scholar 

  9. Hoffmann JF, Carvalho IR, Barbieri RL et al (2017) Butia spp. (Arecaceae) LC-MS-based metabolomics for species and geographical origin discrimination. J Agric Food Chem 65:523–532

    Article  CAS  Google Scholar 

  10. Vargas LHG, Neto JCR, de Aquino Ribeiro JA et al (2016) Metabolomics analysis of oil palm (Elaeis guineensis) leaf: evaluation of sample preparation steps using UHPLC–MS/MS. Metabolomics 12: 153

    Google Scholar 

  11. Hantao LW, Aleme HG, Passador MM et al (2013) Determination of disease biomarkers in Eucalyptus by comprehensive two-dimensional gas chromatography and multivariate data analysis. J Chromatogr A 1279:86–91

    Article  CAS  Google Scholar 

  12. Uarrota VG, Moresco R, Coelho B et al (2014) Metabolomics combined with chemometric tools (PCA, HCA, PLS-DA and SVM) for screening cassava (Manihot esculenta Crantz) roots during postharvest physiological deterioration. Food Chem 161:67–78

    Article  CAS  Google Scholar 

  13. Garrett R, Schmidt EM, Pereira LFP et al (2013) Discrimination of arabica coffee cultivars by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and chemometrics. LWT - Food Sci Technol 50:496–502

    Article  CAS  Google Scholar 

  14. do Prado RM, Porto C, Nunes E et al (2018) Metabolomics and agriculture: what can be done? mSystems 3:e00156–e00117

    Article  Google Scholar 

  15. Cardoso S, Maraschin M, Peruch LAM et al (2017) A chemometrics approach for nuclear magnetic resonance data to characterize the partial metabolome banana peels from southern Brazil. J Integr Bioinform 14:1–10

    Google Scholar 

  16. Roessner U (2001) Metabolic profiling allows comprehensive phenotyping of genetically or environmentally modified plant systems. Plant Cell 13:11–29

    Article  CAS  Google Scholar 

  17. Padilla-González GF, Diazgranados M, da Costa FB (2017) Biogeography shaped the metabolome of the genus Espeletia: a phytochemical perspective on an andean adaptive radiation. Sci Rep 7:1–11

    Google Scholar 

  18. Liang YS, Choi YH, Kim HK et al (2006) Metabolomic analysis of methyl jasmonate treated Brassica rapa leaves by 2-dimensional NMR spectroscopy. Phytochemistry 67:2503–2511

    Article  CAS  Google Scholar 

  19. Choi YH, Choi H-K, Hazekamp A et al (2003) Quantitative analysis of bilobalide and ginkgolides from Ginkgo biloba leaves and Ginkgo products using 1H-NMR. Chem Pharm Bull (Tokyo) 51:158–161

    Article  CAS  Google Scholar 

  20. Sedio BE, Boya PCA, Rojas Echeverri JC (2018) A protocol for high-throughput, untargeted forest community metabolomics using mass spectrometry molecular networks. Appl Plant Sci 6:1–13

    Article  Google Scholar 

  21. Pavarini DP, Da Silva DB, Carollo CA et al (2012) Application of MALDI-MS analysis of rainforest chemodiversity: a keystone for biodiversity conservation and sustainable use. J Mass Spectrom 47:1482–1485

    Article  CAS  Google Scholar 

  22. Pauli GF, Gödecke T, Jaki BU, Lankin DC (2012) Quantitative1H NMR. Development and potential of an analytical method: an update. J Nat Prod 75:834–851

    Article  CAS  Google Scholar 

  23. Markley JL, Brüschweiler R, Edison AS et al (2017) The future of NMR-based metabolomics. Curr Opin Biotechnol 43:34–40

    Article  CAS  Google Scholar 

  24. Dagnino D, Schripsema J (2005) 1H NMR quantification in very dilute toxin solutions: application to anatoxin-a analysis. Toxicon 46:236–240

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  26. Szasz PL (2009) Thought suppression, depressive rumination and depression: a mediation analysis. J Cogn Behav Psychother 9:199–209

    Google Scholar 

  27. Mompeán M, Sánchez-Donoso RM, de la Hoz A et al (2018) Pushing nuclear magnetic resonance sensitivity limits with microfluidics and photo-chemically induced dynamic nuclear polarization. Nat Commun 9:1–8

    Google Scholar 

  28. Simmler C, Napolitano JG, McAlpine JB et al (2014) Universal quantitative NMR analysis of complex natural samples. Curr Opin Biotechnol 25:51–59

    Article  CAS  Google Scholar 

  29. Phansalkar RS, Simmler C, Bisson J et al (2017) Evolution of quantitative measures in NMR: quantum mechanical qHNMR advances chemical standardization of a red clover (Trifolium pratense) extract. J Nat Prod 80:634–647

    Article  CAS  Google Scholar 

  30. Wei S, Zhang J, Liu L et al (2011) Ratio analysis nuclear magnetic resonance spectroscopy for selective metabolite identification in complex samples. Anal Chem 83:7616–7623

    Article  CAS  Google Scholar 

  31. Defernez M, Colquhoun IJ (2003) Factors affecting the robustness of metabolite fingerprinting using 1H NMR spectra. Phytochemistry 62:1009–1017

    Article  CAS  Google Scholar 

  32. Kim HK, Verpoorte R (2010) Sample preparation for plant metabolomics. Phytochem Anal 21:4–13

    Article  CAS  Google Scholar 

  33. Kim HK, Choi YH, Verpoorte R (2010) NMR-based metabolomic analysis of plants. Nat Protoc 5:536–549

    Article  CAS  Google Scholar 

  34. Schripsema J (2010) Application of NMR in plant metabolomics: techniques, problems and prospects. Phytochem Anal 21:14–21

    Article  CAS  Google Scholar 

  35. Kruger NJ, Troncoso-Ponce MA, Ratcliffe RG (2008) 1H NMR metabolite fingerprinting and metabolomic analysis of perchloric acid extracts from plant tissues. Nat Protoc 3:1001–1012

    Article  CAS  Google Scholar 

  36. Bernstein MA, Sýkora S, Peng C et al (2013) Optimization and automation of quantitative NMR data extraction. Anal Chem 85:5778–5786

    Article  CAS  Google Scholar 

  37. Saude EJ, Slupsky CM, Sykes BD (2006) Optimization of NMR analysis of biological fluids for quantitative accuracy. Metabolomics 2:113–123

    Article  CAS  Google Scholar 

  38. Goudarzi H, Mirsamadi ES, Ghalavand Z et al (2015) Molecular detection of metallo-beta-lactamase genes in clinical isolates of Acinetobacter baumannii. J Pure Appl Microbiol 9:145–151

    Google Scholar 

  39. Bharti SK, Sinha N, Joshi BS et al (2008) Improved quantification from1H-NMR spectra using reduced repetition times. Metabolomics 4:367–376

    Article  CAS  Google Scholar 

  40. Pauli GF, Chen SN, Simmler C et al (2014) Importance of purity evaluation and the potential of quantitative1H NMR as a purity assay. J Med Chem 57:9220–9231

    Article  CAS  Google Scholar 

  41. Malz F, Jancke H (2005) Validation of quantitative NMR. J Pharm Biomed Anal 38:813–823

    Article  CAS  Google Scholar 

  42. Akoka S, Barantin L, Trierweiler M (1999) Concentration measurement by proton NMR using the ERETIC method. Anal Chem 71:2554–2557

    Article  CAS  Google Scholar 

  43. Pilon AC, Carnevale Neto F, Freire RT et al (2016) Partial least squares model and design of experiments towards the analysis of the metabolome of Jatropha gossypifolia leaves: extraction and chromatographic fingerprint optimization. J Sep Sci 39:1023–1030

    Article  CAS  Google Scholar 

  44. Hendrawati O, Yao Q, Kim HK et al (2006) Metabolic differentiation of Arabidopsis treated with methyl jasmonate using nuclear magnetic resonance spectroscopy. Plant Sci 170:1118–1124

    Article  CAS  Google Scholar 

  45. Widarto HT, van der Meijden E, Lefeber AWM et al (2006) Metabolomic differentiation of Brassica rapa following herbivory by different insect instars using two-dimensional nuclear magnetic resonance spectroscopy. J Chem Ecol 32:2417–2428

    Article  CAS  Google Scholar 

  46. Sánchez-Sampedro A, Kim HK, Choi YH et al (2007) Metabolomic alterations in elicitor treated Silybum marianum suspension cultures monitored by nuclear magnetic resonance spectroscopy. J Biotechnol 130:133–142

    Article  Google Scholar 

  47. Simoh S, Quintana N, Kim HK et al (2009) Metabolic changes in Agrobacterium tumefaciens-infected Brassica rapa. J Plant Physiol 166:1005–1014

    Article  CAS  Google Scholar 

  48. Leiss KA, Choi YH, Abdel-Farid IB et al (2009) NMR metabolomics of thrips (Frankliniella occidentalis) resistance in senecio hybrids. J Chem Ecol 35:219–229

    Article  CAS  Google Scholar 

  49. Aranı’bar N, Ott K-H, Roongta V, Mueller L (2006) Metabolomic analysis using optimized NMR and statistical methods. Anal Biochem 355:62–70

    Article  Google Scholar 

  50. Pereira GE, Gaudillere JP, Leeuwen CV et al (2006) 1H NMR metabolite fingerprints of grape berry: comparison of vintage and soil effects in Bordeaux grapevine growing areas. Anal Chim Acta 563:346–352

    Article  CAS  Google Scholar 

  51. Schripsema J, Dagnino D (2018) Two-phase extraction for comprehensive analysis of the plant metabolome by NMR. In: Theodoridis GA, Gika HG, Wilson ID (eds) Metabolic profiling: methods and protocols. Springer, New York, NY, pp 195–202

    Chapter  Google Scholar 

  52. Huang Y, Zhang Z, Chen H et al (2015) A high-resolution 2D J-resolved NMR detection technique for metabolite analyses of biological samples. Sci Rep 5:1–9

    Google Scholar 

  53. Zhang ZM, Chen S, Liang YZ (2010) Baseline correction using adaptive iteratively reweighted penalized least squares. Analyst 135:1138–1146

    Article  CAS  Google Scholar 

  54. Cobas C, Seoane F, Sýkora S (2008) Global spectral deconvolution (GSD) of D-NMR spectra. Poster SMASH Conf 15706

    Google Scholar 

  55. Savorani F, Tomasi G, Engelsen SB (2010) Icoshift: a versatile tool for the rapid alignment of 1D NMR spectra. J Magn Reson 202:190–202

    Article  CAS  Google Scholar 

  56. Selegato DM, Freire RT, Tannús A, Castro-Gamboa I (2016) New dereplication method applied to NMR-based metabolomics on different Fusarium species isolated from rhizosphere of Senna spectabilis. J Braz Chem Soc 27:1421–1431

    Google Scholar 

  57. Mortazavi-Tabatabaei SA, Fathi F, Ektefa F et al (2013) Investigation of metabonomics technique by analyze of NMR data, which method is better? Mean center or auto scale? J Paramed Sci 4:2–9

    Google Scholar 

  58. Pilon AC, Valli M, Dametto AC et al (2017) NuBBEDB: an updated database to uncover chemical and biological information from Brazilian biodiversity. Sci Rep 7:7215

    Article  Google Scholar 

  59. Choi HK, Choi YH, Verberne M et al (2004) Metabolic fingerprinting of wild type and transgenic tobacco plants by1H NMR and multivariate analysis technique. Phytochemistry 65:857–864

    Article  CAS  Google Scholar 

  60. Weljie AM, Newton J, Mercier P et al (2006) Targeted profiling: quantitative analysis of 1H NMR metabolomics data. Anal Chem 78:4430–4442

    Article  CAS  Google Scholar 

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Acknowledgements

The authors acknowledge support from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP fellowship 2014/05935-0 and 2017/06466-2 to DMS, and 2016/13292-8 to ACP), and the University of Washington (to FCN).

Author information

Authors and Affiliations

  1. Núcleo de Bioensaios, Biossíntese e Ecofisiologia de Produtos Naturais (NuBBE), Departamento de Química Orgânica, Instituto de Química, Universidade Estadual Paulista (UNESP), Araraquara, São Paulo, Brazil

    Denise Medeiros Selegato

  2. Núcleo de Pesquisa em Produtos Naturais e Sintéticos (NPPNS), Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil

    Alan Cesar Pilon

  3. Department of Anesthesiology and Pain Medicine, Northwest Metabolomics Research Center, University of Washington, Seattle, WA, USA

    Fausto Carnevale Neto

Authors
  1. Denise Medeiros Selegato
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  2. Alan Cesar Pilon
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  3. Fausto Carnevale Neto
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Corresponding author

Correspondence to Fausto Carnevale Neto .

Editor information

Editors and Affiliations

  1. Northwest Metabolomics Research Center, Mitochondria and Metabolism Center, Department of Anesthesiology and Pain Medicine, University of Washington, Seattle, WA, USA

    G. A. Nagana Gowda

  2. Northwest Metabolomics Research Center, Mitochondria and Metabolism Center, Department of Anesthesiology and Pain Medicine, University of Washington, Fred Hutch Cancer Research Center, Seattle, WA, USA

    Daniel Raftery

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Selegato, D.M., Pilon, A.C., Carnevale Neto, F. (2019). Plant Metabolomics Using NMR Spectroscopy. In: Gowda, G., Raftery, D. (eds) NMR-Based Metabolomics. Methods in Molecular Biology, vol 2037. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9690-2_19

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  • DOI: https://doi.org/10.1007/978-1-4939-9690-2_19

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9689-6

  • Online ISBN: 978-1-4939-9690-2

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