Fast Determination of Absolute Metabolite Concentrations by Spatially Encoded 2D NMR: Application to Breast Cancer Cell Extracts

Abstract

Two-dimensional nuclear magnetic resonance (2D NMR) forms a powerful tool for the quantitative analysis of complex mixtures such as samples of metabolic relevance. However, its use for quantitative purposes is far from being trivial, not only because of the associated experiment time, but also due to its subsequent high sensitivity to hardware instabilities affecting its precision. In this paper, an alternative approach is considered to measure absolute metabolite concentrations in complex mixtures with a high precision in a reasonable time. It is based on a "multi-scan single shot" (M3S) strategy, which is derived from the ultrafast 2D NMR methodology. First, the analytical performance of this methodology is compared to the one of conventional 2D NMR. 2D correlation spectroscopy (COSY) spectra are obtained in 10 min on model metabolic mixtures, with a precision in the 1-4% range (versus 5-18% for the conventional approach). The M3S approach also shows a better linearity than its conventional counterpart. It ensures that accurate quantitative results can be obtained provided that a calibration procedure is carried out. The M3S COSY approach is then applied to measure the absolute metabolite concentration in three breast cancer cell line extracts, relying on a standard addition protocol. M3S COSY spectra of such extracts are recorded in 20 min and give access to the absolute concentration of 14 major metabolites, showing significant differences between cell lines.