Skip to main content
SpringerLink
Log in

Assessing the potential of quantitative 2D HSQC NMR in 13C enriched living organisms

  • Article
  • Published:
Journal of Biomolecular NMR Aims and scope Submit manuscript

Abstract

In vivo Nuclear Magnetic Resonance (NMR) spectroscopy has great potential to interpret the biochemical response of organisms to their environment, thus making it an essential tool in understanding toxic mechanisms. However, magnetic susceptibility distortions lead to 1D NMR spectra of living organisms with lines that are too broad to identify and quantify metabolites, necessitating the use of 2D 1H–13C Heteronuclear Single Quantum Coherence (HSQC) as a primary tool. While quantitative 2D HSQC is well established, to our knowledge it has yet to be applied in vivo. This study represents a simple pilot study that compares two of the most popular quantitative 2D HSQC approaches to determine if quantitative results can be directly obtained in vivo in isotopically enriched Daphnia magna (water flea). The results show the perfect-HSQC experiment performs very well in vivo, but the decoupling scheme used is critical for accurate quantitation. An improved decoupling approach derived using optimal control theory is presented here that improves the accuracy of metabolite concentrations that can be extracted in vivo down to micromolar concentrations. When combined with 2D Electronic Reference To access In vivo Concentrations (ERETIC) protocols, the protocol allows for the direct extraction of in vivo metabolite concentrations without the use of internal standards that can be detrimental to living organisms. Extracting absolute metabolic concentrations in vivo is an important first step and should, for example, be important for the parameterization as well as the validation of metabolic flux models in the future.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Akhter M et al (2016) Identification of aquatically available carbon from algae through solution-state NMR of whole 13C-labelled cells. Anal Bioanal Chem 408:4357–4370

    Google Scholar 

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

    Google Scholar 

  • Anaraki MT, Simpson M, Simpson A (2018) Reducing impacts of organism variability in metabolomics via time trajectory in vivo NMR. Magn Reson Chem. https://doi.org/10.1002/mrc.4759

    Article  Google Scholar 

  • Aoto PC, Fenwick RB, Kroon GJA, Wright PE (2014) Accurate scoring of non-uniform sampling schemes for quantitative NMR. J Magn Reson 246:31–35

    ADS  Google Scholar 

  • Bastawrous M, Jenne A, Tabatabaei Anaraki M, Simpson AJ (2018) In-vivo NMR spectroscopy: a powerful and complimentary tool for understanding environmental toxicity. Metabolites 8:1–24

    Google Scholar 

  • Beauvoit BP et al (2017) Model-assisted analysis of sugar metabolism throughout tomato fruit development reveals enzyme and carrier properties in relation to vacuole expansion model-assisted analysis of sugar metabolism througho tomato fruit development reveals enzyme and carrie. Plant Cell 2017:114

    Google Scholar 

  • Bendall MR (1995) Broadband and narrowband spin decoupling using adiabatic spin flips. J Magn Reson 112:126–129

    ADS  Google Scholar 

  • Bendall MR, Skinner TE (1998) Calibration of STUD + parameters to achieve optimally efficient broadband adiabatic decoupling in a single transient. J Magn Reson 134:331–349

    ADS  Google Scholar 

  • Bingol K, Zhang F, Bruschweiler-Li L, Brüschweiler R (2013) Quantitative analysis of metabolic mixtures by two-dimensional 13C constant-time TOCSY NMR spectroscopy. Anal Chem 85:6414–6420

    Google Scholar 

  • Bundy JG, Davey MP, Viant MR (2009) Environmental metabolomics: a critical review and future perspectives. Metabolomics 5:3–21

    Google Scholar 

  • Burton IW, Quilliam MA, Walter JA (2005) Quantitative 1H NMR with external standards: use in preparation of calibration solutions for algal toxins and other natural products. Anal Chem 77:3123–3131

    Google Scholar 

  • Castañar L, Parella T (2015) Recent advances in small molecule NMR: improved HSQC and HSQMBC experiments. Annu Rep NMR Spectrosc 84:163–232

    Google Scholar 

  • Castañar L, Sistaré E, Virgili A, Williamson RT, Parella T (2015) Suppression of phase and amplitude J(HH) modulations in HSQC experiments. Magn Reson Chem 53:115–119

    Google Scholar 

  • Colombié S et al (2015) Modelling central metabolic fluxes by constraint-based optimization reveals metabolic reprogramming of developing Solanum lycopersicum (tomato) fruit. Plant J 81:24–39

    Google Scholar 

  • Di Gialleonardo V et al (2016) High-throughput indirect quantitation of 13C enriched metabolites using 1 H NMR. Anal Chem 88:11147–11153

    Google Scholar 

  • Farjon J, Milande C, Martineau E, Akoka S, Giraudeau P (2018) The FAQUIRE approach: fast, quantitative, highly resolved and sensitivity enhanced 1H, 13C data. Anal Chem 90:1845–1851

    Google Scholar 

  • Farooq H et al (2013) HR-MAS NMR spectroscopy: a practical guide for natural samples. Curr Org Chem 17:3013–3031

    Google Scholar 

  • Fu R, Bodenhausen G (1995a) Ultra-broadband decoupling. J Magn Reson Ser A 117:324–325

    ADS  Google Scholar 

  • Fu R, Bodenhausen G (1995b) Broadband decoupling in NMR with frequency-modulated ‘chirp’ pulses. Chem Phys Lett 245:415–420

    ADS  Google Scholar 

  • Fu R, Bodenhausen G (1996) Evaluation of adiabatic frequency-modulated schemes for broadband decoupling in isotropic liquids. J Magn Reson Ser A 119:129–133

    ADS  Google Scholar 

  • Fugariu I, Bermel W, Lane D, Soong R, Simpson AJ (2017) In-phase ultra high-resolution in vivo NMR. Angew Chem Int Ed 56:6324–6328

    Google Scholar 

  • Giraudeau P, Akoka S (2013) Fast and ultrafast quantitative 2D NMR. Vital tools for efficient metabolomic approaches. In: Advances in botanical research, Elsevier, Amsterdam

    Google Scholar 

  • Gronwald W et al (2008) Urinary metabolite quantification employing 2D NMR spectroscopy. Anal Chem 80:9288–9297

    Google Scholar 

  • Heikkinen S, Toikka MM, Karhunen PT, Kilpeläinen IA (2003) Quantitative 2D HSQC (Q-HSQC) via suppression of J-dependence of polarization transfer in NMR spectroscopy: application to wood lignin. J Am Chem Soc 125:4362–4367

    Google Scholar 

  • Hertkorn N et al (2007) High-precision frequency measurements: indispensable tools at the core of the molecular-level analysis of complex systems. Anal Bioanal Chem 389:1311–1327

    Google Scholar 

  • Hu K, Westler WM, Markley JL (2011) Simultaneous quantification and identification of individual chemicals in metabolite mixtures by two-dimensional extrapolated time-zero 1H-13C HSQC (HSQC 0). J Am Chem Soc 133:1662–1665

    Google Scholar 

  • Jézéquel T et al (2015) Absolute quantification of metabolites in tomato fruit extracts by fast 2D NMR. Metabolomics 11:1231–1242

    Google Scholar 

  • Koskela H (2009) Chapter 1 quantitative 2D NMR studies. In: Annual reports on NMR spectroscopy, Elsevier, Amsterdam

    Google Scholar 

  • Koskela H, Kilpeläinen I, Heikkinen S (2005) Some aspects of quantitative 2D NMR. J Magn Reson 174:237–244

    ADS  Google Scholar 

  • Koskela H, Heikkilä O, Kilpeläinen I, Heikkinen S (2010) Quantitative two-dimensional HSQC experiment for high magnetic field NMR spectrometers. J Magn Reson 202:24–33

    ADS  Google Scholar 

  • Kupče Ē, Freeman R (1995) Adiabatic pulses for wideband inversion and broadband decoupling. J Magn Reson Ser A 115:273–276

    ADS  Google Scholar 

  • Lam B, Simpson AJ (2008) Direct 1H NMR spectroscopy of dissolved organic matter in natural waters. Analyst 133:263–269

    ADS  Google Scholar 

  • Lewis IA et al (2007) Method for determining molar concentrations of metabolites in method for determining molar concentrations of metabolites in complex solutions from two-dimensional 1H-13C NMR spectra. 79:9385–9390

  • Mahrous EA, Farag MA (2015) Two dimensional NMR spectroscopic approaches for exploring plant metabolome: a review. J Adv Res 6:3–15

    Google Scholar 

  • Majumdar RD et al (2017) In vivo solution-state NMR-based environmental metabolomics. Magn Res 6:133–148

    Google Scholar 

  • Marchand J, Martineau E, Guitton Y, Dervilly-Pinel G, Giraudeau P (2017) Multidimensional NMR approaches towards highly resolved, sensitive and high-throughput quantitative metabolomics. Curr Opin Biotechnol 43:49–55

    Google Scholar 

  • Martineau E, Tea I, Akoka S, Giraudeau P (2012) Absolute quantification of metabolites in breast cancer cell extracts by quantitative 2D 1H inadequate NMR. NMR Biomed 25:985–992

    Google Scholar 

  • Martineau E, Akoka S, Boisseau R, Delanoue B, Giraudeau P (2013) Fast quantitative 1H-13C two-dimensional NMR with very high precision. Anal Chem 85:4777–4783

    Google Scholar 

  • Mauve C, Khlifi S, Gilard F, Mouille G, Farjon J (2016) Sensitive, highly resolved, and quantitative 1H-13 C NMR data in one go for tracking metabolites in vegetal extracts. Chem Commun 52:6142–6145

    Google Scholar 

  • Michel N, Akoka S, Michel N, Akoka S (2004) The application of the ERETIC method to 2D- NMR. J Magn Reson. https://doi.org/10.1016/j.jmr.2004.02.006

    Article  Google Scholar 

  • Mobarhan YL et al (2016) Comprehensive multiphase NMR applied to a living organism. Chem Sci 7:4856–4866

    Google Scholar 

  • Morris GA, Freeman R (1979) Enhancement of nuclear magnetic resonance signals by polarization transfer. J Am Chem Soc 101:760–762

    Google Scholar 

  • Nagato EG et al (2013) 1H NMR-based metabolomics investigation of daphnia magna responses to sub-lethal exposure to arsenic, copper and lithium. Chemosphere 93:331–337

    ADS  Google Scholar 

  • Neves JL, Heitmann B, Khaneja N, Glaser SJ (2009) Heteronuclear decoupling by optimal tracking. J Magn Reson 201:7–17

    ADS  Google Scholar 

  • Nielsen J, Oliver S (2005) The next wave in metabolome analysis. Trends Biotechnol 23:544–546

    Google Scholar 

  • Pathan M, Akoka S, Tea I, Charrier B, Giraudeau P (2011) ‘Multi-scan single shot’ quantitative 2D NMR: a valuable alternative to fast conventional quantitative 2D NMR. Analyst 136:3157–3163

    ADS  Google Scholar 

  • Pauli GF, Gödecke T, Jaki BU, Lankin DC (2012) Quantitative 1 H NMR. development and potential of an analytical method: an update. J Nat Prod 75:834–851

    Google Scholar 

  • Peterson DJ, Loening NM (2007) QQ-HSQC: a quick, quantitative heteronuclear correlation experiment for NMR spectroscopy. Magn Reson Chem 45:937–941

    Google Scholar 

  • Schilling F et al (2014) Next-generation heteronuclear decoupling for high-field biomolecular NMR spectroscopy. Angew Chem Int Ed 53:4475–4479

    Google Scholar 

  • Shaka AJ, Barker PB, Freeman R (1985) Computer-optimized decoupling scheme for wideband applications and low-level operation. J Magn Reson 64:547–552

    ADS  Google Scholar 

  • Sharma R, Gogna N, Singh H, Dorai K (2017) Fast profiling of metabolite mixtures using chemometric analysis of a speeded-up 2D heteronuclear correlation NMR experiment. RSC Adv 7:29860–29870

    Google Scholar 

  • Simpson AJ, Liaghati Y, Fortier-McGill B, Soong R, Akhter M (2015) Perspective: in vivo NMR—a potentially powerful tool for environmental research. Magn Reson Chem 53:686–690

    Google Scholar 

  • Skinner TE, Reiss TO, Luy B, Khaneja N, Glaser SJ (2003) Application of optimal control theory to the design of broadband excitation pulses for high-resolution NMR. J Magn Reson 163:8–15

    ADS  Google Scholar 

  • Skinner TE et al (2006) Optimal control design of constant amplitude phase-modulated pulses: application to calibration-free broadband excitation. J Magn Reson 179:241–249

    ADS  Google Scholar 

  • Soong R et al (2015) In vivo NMR spectroscopy: toward real time monitoring of environmental stress. Magn Reson Chem 53:774–779

    Google Scholar 

  • Starčuk Z, Bartušek K, Starčuk Z (1994) Heteronuclear broadband spin-flip decoupling with adiabatic pulses. J Magn Reson Ser A 107:24–31

    ADS  Google Scholar 

  • Tabatabaei Anaraki M et al (2018) Development and application of a low-volume flow system for solution-state in vivo NMR. Anal Chem 90:7912–7921

    Google Scholar 

  • Tredwell GD, Behrends V, Geier FM, Liebeke M, Bundy JG (2011) Between-person comparison of metabolite fitting for NMR-based quantitative metabolomics. Anal Chem 83:8683–8687

    Google Scholar 

  • Vuister W, Bax AD (1991) Letters to the editor. Tempo 435:69

    Google Scholar 

  • Watanabe R et al (2016) Quantitative nuclear magnetic resonance spectroscopy based on PULCON methodology: application to quantification of invaluable marine toxin, okadaic acid. Toxins 8:1–9

    Google Scholar 

  • Weljie AM, Newton J, Mercier P, Carlson E, Slupsky CM (2006) Targeted profiling: quantitative analysis of 1H NMR metabolomics data. Anal Chem 78:4430–4442

    Google Scholar 

  • Wheeler HL et al (2015) Comprehensive multiphase NMR: a promising technology to study plants in their native state. Magn Reson Chem 53:735–744

    Google Scholar 

  • Wider G, Dreier L (2006) Measuring protein concentrations by NMR spectroscopy. J Am Chem Soc 128:2571–2576

    Google Scholar 

  • Yuk J, McKelvie JR, Simpson MJ, Spraul M, Simpson AJ (2010) Comparison of 1-D and 2-D NMR techniques for screening earthworm responses to sub-lethal endosulfan exposure. Environ Chem 7:524–536

    Google Scholar 

  • Yuk J, Simpson MJ, Simpson AJ (2013) 1-D and 2-D NMR-based metabolomics of earthworms exposed to endosulfan and endosulfan sulfate in soil. Environ Pollut 175:35–44

    Google Scholar 

Download references

Acknowledgements

We would like to thank the Natural Sciences and Engineering Research Council of Canada (NSERC) Strategic (STPGP 494273-16) and Discovery Programs (RGPIN-2014-05423), the Canada Foundation for Innovation (CFI), the Ontario Ministry of Research and Innovation (MRI), and the Krembil Foundation for providing funding. A. S. would like to thank the Government of Ontario for an Early Researcher Award. TES gratefully acknowledges support from the National Science Foundation under Grant No. CHE-1214006.

Author information

Authors and Affiliations

  1. Environmental NMR Centre, University of Toronto Scarborough, 1265 Military Trail, Toronto, ON, M1C 1A4, Canada

    Daniel Lane, Ronald Soong, Rudraksha Dutta Majumdar, Yalda Liaghati Mobarhan, Myrna J. Simpson & André J. Simpson

  2. Department of Physics, Wright State University, Dayton, OH, 45735, USA

    Thomas E. Skinner & Naum I. Gershenzon

  3. Bruker BioSpin GmbH, Silberstreifen 4, Rheinstetten, Germany

    Wolfgang Bermel

  4. Silantes GmbH, Munich, Germany

    Sebastian Schmidt & Hermann Heumann

  5. Bruker Ltd., 2800 Highpoint Drive, Milton, ON, L9T 6P4, Canada

    Rudraksha Dutta Majumdar & Martine Monette

Authors
  1. Daniel Lane
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thomas E. Skinner
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Naum I. Gershenzon
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wolfgang Bermel
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ronald Soong
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Rudraksha Dutta Majumdar
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yalda Liaghati Mobarhan
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sebastian Schmidt
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hermann Heumann
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Martine Monette
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Myrna J. Simpson
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. André J. Simpson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to André J. Simpson.

Ethics declarations

Conflict of interest

The authors declare no conflicts of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 319 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lane, D., Skinner, T.E., Gershenzon, N.I. et al. Assessing the potential of quantitative 2D HSQC NMR in 13C enriched living organisms. J Biomol NMR 73, 31–42 (2019). https://doi.org/10.1007/s10858-018-0221-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-018-0221-2

Keywords

Search

Navigation