Simplifying 1H-NMR Spectra in NMR Metabolic Profiling - Applying Projections of 1H homonuclear 2D J-resolved spectra -

  • Summary

NM170014

Introduction

Spectral overlap often hampers subsequent analyses (ex. multivariate analyses) in 1H-NMR spectra of molecular mixtures such as in NMR metabolic profiling (NMR-MP). One of the most straightforward solution is using more higher field NMR system. However it is not always easy to address. In this note, the method simplifying 1H-NMR spectra of mixture is introduced by use of projections of 1H homo-nuclear 2D J-resolved spectra.

Simplifying 1H-NMR spectrum by applying projections of 1H homonuclear 2D J-resolved spectrum [1]

In the 2D J-resolved spectroscopy experiment, an evolution of J-coupling is encoded while that of chemical shift is refocused in the indirect evolution period. Therefore the proton multiplets appear at a 45 degree angle in the resultant 2D spectrum as shown in Figure 1(a). Then this 2D spectrum is tilted by 45 degrees to remove J-couplings from x-axis. After tilting, as shown in Figure 1(b), the 2D spectrum contains chemical shift and J-coupling alone in x- and y-axis, respectively. The projection of the x-axis is a pseudo pure shift spectrum (Figure 1(c): upper). You can see how spectrum goes is simplified relative to a standard single pulse experiment.

Simplifying 1H-NMR spectrum by applying projections of 1H homonuclear 2D J-resolved spectrum.
Figure1 Simplifying 1H-NMR spectrum by applying projections of 1H homonuclear 2D J-resolved spectrum. (a) The proton multiplets appear at a 45 degree angle in original 2D spectrum. (b) After tilting, the 2D spectrum contains chemical shift and J-coupling in x- and y-axis, respectively. (c) The projection of (b) is more simple than the conventional 1H single pulse spectrum.

Suppressing artifacts from strong couplings in J-resolved spectroscopy

We can record simple spectra using projections of 2D J-resolved spectra even though standard magnetic field NMR system such as 400 MHz system is used. However there is a drawback in J-resolved spectroscopy that artifacts from strong coupling can occur, especially when a lower magnetic field system is used.
As it was introduced before in our application note "Suppressing artifacts from strong couplings in 2D J-resolved spectroscopy" (NM050018), these artifacts can be suppressed when double spin-echo type of 2D J-resolved spectroscopy [2] is used.

A projection of 2D J-resolved spectrum of polar metabolites in broccoli sprout.

There are often severe spectral overlap in 1H-NMR spectra when plants are used as samples for NMR-MP because they produce various kind of metabolites including plant specialized metabolites. Here, we demonstrate that using projections of 2D J-resolved spectra is a powerful method in actual NMR-MP studies. Polar metabolites in broccoli sprouts were extracted and used as a NMR sample. All spectra were recorded with standard a NMR system (JNM-ECZ400S spectrometer with a ROYALPROBE). The double spin-echo method was used as a 2D J-resolved spectroscopy experiment.
1H spectra of polar metabolites in broccoli sprout are shown in Figure 2. Figure 2(a) and (b) show a projection of 2D J-resolved spectrum and 1H single pulse spectrum, respectively. As many metabolites were present in the sample, there is severe spectral overlap. Using a projection of 2D J-resolved spectrum, we were able to record far simpler spectra than the results of a typical 1H single pulse spectrum. For example, the insets in the spectra below highlight four different methyl protons in valine and isoleucine that were clearly resolved in the projection of 2D J-resolved spectrum (a) whereas they were ambiguous in 1H single pulse (b).
2D J-resolved spectroscopy is also useful to assign metabolites [3]. Please see detail of an actual NMR-MP study using projections of 2D J-resolved spectra in our application note "Unsupervised Multivariate Analyses for NMR-based Metabolomics" (NM170013) [4].


Figure2 A comparison between a projection of 2D J-resolved spectrum and conventional 1H single pulse spectrum of polar metabolites in broccoli sprout. (a) A projection of 2D J-resolved spectrum. The double spin-echo method was used. (b) 1H single pulse spectrum.

References

  • Ludwig; C., Viant; M., Phytochem. Anal., 21 (2010) 22-32
  • Thrippleton; M. J., Edden; R. A. E., Keeler; J., J. Magn. Reson., 174 (2005) 97
  • Kikuchi; J. et al., Anal. Chem., 88 (2016) 659
  • Application note "Unsupervised Multivariate Analyses for NMR-based Metabolomics" (NM170013).
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