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EBSI TEAM

Elucidation of Biosynthesis by isotopic spectrometry

 

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methodological developments in quantitative NMR

Fast and Ultrafast 2D NMR : methodological developments and quantitative applications

Research staff: Patrick Giraudeau (PI), Serge Akoka, Benoît Charrier, Estelle Martineau
PhD students: Laetitia Rouger (2014-17), Boris Gouilleux (2014-17), Tangi Jézéquel (2015-18), Jérémy Marchand (2015-2018)
Former PhD students: Adrien Le Guennec (2012-15), Meerakhan Pathan (2011-13), Estelle Martineau (2008-11)

General description

Measuring the precise concentration of analytes in chemical or biochemical samples is, together with the identification of these components, one of the main goals of analytical chemistry. Nuclear Magnetic Resonance (NMR) is a widely used tool towards quantitative purposes. However, 1D proton NMR suffers from large overlap between peaks that restrain its quantitative use, particularly when complex biological mixtures are studied. In order to overvcome these drawbacks, we are developing new quantitative 2D NMR approaches , as multidimensional NMR offers a better discrimination between resonances in complex mixtures. Still, conventional 2D NMR experiments are affected by long experiment durations that, above evident timetable constraints, limit their use for quantitative analysis (long experiments are highly sensitive to spectrometer instabilities) or for studying fast-occuring processes (kinteics, dynamics, etc.). For this reason, we focus on obtaining fast and precise 2D NMR experiments and we apply them to a variety of quantitative studies

 

Fast quantitative 2D NMR

A first approach consists in carefully optimizing conventional 2D NMR experiments for quantitative analysis.

 

A first approach consists in carefully optimizing conventional 2D NMR experiments for quantitative analysis. The pulse sequences, as well as the acquisition and processing parameters, are carefully optimized in order to obtain precise results in the shortest time possible. We have decreased to a few minutes the experiment duration of several 2D NMR experiments ( J -resolved, DQF-COSY, 1 H INADEQUATE, etc.) while obtaining a significant improvement in terms of precision. Absolute concentrations can be precisely measured provided that a calibration procedure is carried out, which is now possible in a reasonable duration. These methodologies have been successfully applied to the metabolic discrimination of breast cancer cell lines (PhD of Estelle Martineau, 2008-2011). This research axis is still under continuous investigation through several projects and collaborations. Very recently, we reached a ca . 0.1% precision, paving the way towards the application of these methods to isotopic analysis at natural abundance.

Analytical workflow illustrating the quantitative method capable of determining absolute metabolite concentrations in biological extracts
Photo © U. Salvagnin, copyright-free picture downloaded from this link .

 

Ultrafast 2D NMR and hybrid methods

 

 

In the last twenty years, NMR spectroscopists have developed a number of approaches to reduce the duration of multi-dimensional NMR experiments. The most efficient one is probably ultrafast 2D NMR, recently proposed by L. Frydman and co-workers. This method allows the acquisition of a 2D spectrum in a single scan, and thus in a fraction of a second.

In 2007, we have successfully implemented this methodology on the spectrometers of the CEISAM laboratory. In the last 8 years, we have brought a number of methodological improvements to ultrafast 2D NMR, which have highly improved its analytical performances, in terms of resolution, lineshape, sensitivity and accessible spectral width. We also developed new tools to make ultrafast NMR accessible to non-specialists.

 

Thanks to these new developments, we have been able to apply our methods to a broad range of situations, such as (non-exhaustive list):

  • targeted and untargeted metabolomics of biological samples
  • the determination of site-specific isotopic enrichments in fluxomics
  • the real-time study of reaction mechanisms
  • the in situ monitoring of electrochemical reactions
This projects have been funded by the ANR (QUANTUM project, 2011-2014). New methodological developments and applications are currently under investigation, thanks to funding from the Institut Universitaire de France, the Région Pays de Loire (RésoNantes Grant, 2014-2017), and to our industrial partners.
Quantitative Ultrafast 2D NMR COSY spectrum of a 50 mM metabolic mixture, recorded in 0.2 s on our 500 MHz spectrometer with a cryoprobe. Thanks to the methodological improvements we brought to the ultrafast methodology, the spectrum provides the same information as its conventional 2D NMR counterpart.

The acquisition and processing routines can be obtained on demand by contacting P. Giraudeau .

 

Collaborations

 

Selected publications

An updated list of our publications can be downloaded here .