JCJC SIMI 8 - JCJC : Sciences de l'information, de la matière et de l'ingénierie : Chimie du solide, colloïdes, physicochimie

Quantitative Ultrafast Analysis by 2D NMR To Unravel Metabolic complexities – QUANTUM

At the heart of matter and life in a fraction of a second

The QUANTUM project aims at developing new, ultrafast analytical methods based on Nuclear Magnetic Resonance (NMR) for the study of biologically relevant samples, and particularly for understanding cancer cell metabolism.

New methods for the ultrafast analysis of biological samples

Understanding metabolism is a major issue, as a better understanding of biochemical reactions in biological systems opens the way towards the development of biological and medical applications, such as new diagnosis tools. One of the numerous goals of analytical chemistry is to deal with such metabolic samples. A particularly powerful technique is Nuclear Magnetic Resonance (NMR) spectroscopy, a non-invasive techniques which makes it possible to characterize the molecules present in a sample, but also to quantify them, ie. to measure their concentrations. In particular, bi-dimensional NMR (2D NMR) is a powerful and promising tool for the quantitative study of biologically relevant samples. However, it is hampered by long experiment durations (up to several hours), thus limiting its use and decreasing its performance, particularly for the study of samples with limited lifetimes. The aim of this project was to develop new NMR methods to decrease this duration to a fraction of a second, to make these methods accessible to the international scientific community, and to demonstrate their applicability on appropriate biological models.

In 2002, a promising method was proposed by Pr. Lucio Frydman, a renowned Israeli scientist, making it possible to perform, for the first time, a 2D NMR experiment in a fraction of a second. Very recently, we implemented this “ultrafast” methodology in our laboratory. We proposed several novel developments to make ultrafast 2D NMR applicable to the quantitative analysis of complex mixtures, thus enlarging the potential applications of this promising method. Based on these results, the QUANTUM project aimed at developing analytical methods to make ultrafast 2D NMR applicable for the quantitative analysis of complex metabolic mixtures. The project relies on novel and original methodological developments. These methods were fully validated to make them applicable to the quantitative study of real samples such as cancer cell extracts.

The results of the QUANTUM project have been widely spread among the international scientific community. The first quantitative applications of ultrafast 2D NMR were published in major journals. The absolute concentration of major metabolites in biological extracts (cancer cell or plant extracts) was determined with a precision that had never been reached before. The QUANTUM project also helped increasing the popularity of ultrafast NMR more popular, through numerous collaborations and projects. Finally, the PI was appointed a 5-year position at the “Institut Universitaire de France”.

While the QUANTUM project was highly successful, the results also opened new methodological barriers that will need to be broken down in order to consider future applications of ultrafast methods:

• The application of ultrafast quantitative NMR to isotopic NMR –one of our major investigation fields for the last 40 years– is the main goal of our future investigations. Promising results in enriched samples were obtained during the QUANTUM project. But the application of ultrafast NMR to natural abundance samples will require increasing the precision by one order of magnitude (from 1% to 0.1%) in order to make it compatible with the 13C natural abundance isotopic deviation range. It will require new methodological developments that will lead to new authentication and control analytical tools.
• The application of ultrafast NMR to metabolomics, whose interest was demonstrated in the QUANTUM project, is still limited by the range of accessible concentrations. In the qUANTUM project, the applications were targeting ca. 10 major metabolites, but they would highly benefit from an increase in sensitivity. A promising perspective would be to combine quantitative NMR with hyperpolarization techniques, and promising preliminary results were obtained along this research direction.

The first results of the QUANTUM project have already led to 25 publications in international peer-reviewed journals and 1 book chapter. They have also been presented in 71 communications at the international (42) and national (27) levels, including 24 invited lectures. The complete list of this scientific production is available at:
www.univ-nantes.fr/giraudeau-p

Nuclear Magnetic Resonance (NMR) is a very powerful analytical tool employed in a wide range of situations, such as elucidation of organic structures, biochemical studies, pharmaceutical analysis or in vivo spectroscopy. However, it suffers from major limitations due to strong overlap between peaks, and this is particularly true for complex metabolic mixtures. Therefore, 1D NMR offers a limited capacity for the precise quantification of interesting biomarkers in complex samples. Fortunately, two dimensional (2D) spectroscopy allows the unraveling of spectral complexity along a second dimension and thus presents a great potential to unambiguously and simultaneously measure a larger number of metabolite contributions. However, it is still rarely used for quantification, first because quantitative analysis by 2D NMR requires a calibration procedure due to the multi-impulsional nature of 2D NMR experiments, and above all because of the prohibitive experiment duration that is necessary to obtain such a calibration curve, due to the multi-scan nature of 2D NMR experiments.
Fortunately, the past few years have witnessed the emergence of a novel and promising method that makes it possible to acquire a whole 2D spectrum in a fraction of a second. This so-called “ultrafast 2D NMR” method, proposed by Pr. Lucio Frydman, is based on a single-scan approach. Very recently, we have successfully implemented this methodology at the CEISAM laboratory. We have proposed several methodological developments to make ultrafast 2D NMR suitable for quantitative analysis of mixtures, thus enlarging the application range of this promising method.
Based on this experience, the ambition of the QUANTUM project is to develop a complete methodology to set ultrafast 2D NMR as a standard tool for fast and precise quantitative analysis of complex metabolic mixtures. This project will consider original NMR developments and programming aspects, focusing on the resolution and sensitivity aspects of ultrafast methods. A complete analytical validation of these optimized methods will be carried out, in order to make them applicable to quantitative studies of real metabolic samples such as tumor cell extracts. At the end of the project, we will propose a package including the analytical strategy, the pulse sequences and the pre-acquisition and processing routines for quantitative ultrafast 2D experiments, that will be easy to implement in routine. This package will be available online on the CEISAM website, for free distribution to the academic community
Our interest in ultrafast 2D NMR is an emergent research axis in our research group, which has an international expertise in isotopic analysis applied to natural product authentication and for elucidating metabolic pathways. However, even though we started working on ultrafast 2D NMR in 2007, we became in three years the second group in the world (first in Europe) regarding the number of papers on ultrafast 2D NMR. Moreover, we are the authors of the only paper published so far reporting the application of ultrafast 2D NMR to quantitative analysis. Fortified by our experience and in order to enlarge the diffusion of ultrafast methods, we wish to make ultrafast 2D NMR accessible in routine to a large number of research groups in the world, by developing adapted programs integrated to existing commercial software, and by making these available to the scientific community.
Finally, the QUANTUM project will be an opportunity to develop fast quantitative 2D NMR methods applied to metabolomic studies, which really constitutes a new approach in the CEISAM laboratory. It should open new application perspectives that are currently out of the laboratory expertise.

Project coordinator

Monsieur Patrick GIRAUDEAU (UNIVERSITE DE NANTES) – patrick.giraudeau@univ-nantes.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CEISAM UNIVERSITE DE NANTES

Help of the ANR 240,000 euros
Beginning and duration of the scientific project: - 36 Months

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