CONICET | Buscador de Institutos y Recursos Humanos

Similar to genomics and proteomics which yield vast amounts of data about the expression of genes and proteins, metabolomics refers to the whole metabolic profile of the cell. NMR spectroscopy has been used for a long time to analyze complex mixtures in the areas of organic and analytical chemistry, and has recently emerged as a key tool for monitoring metabolic processes in living systems and for the analysis of complex biofluids.1-3 The ease required for sample preparation plus the possibility of studying several compounds in a single, fast experiment, make this technique ideal for high-throughput studies. Metabolomic NMR data usually consists of spectra recorded over time, or on samples coming from different treatments. There are two ways in which this data can be analyzed quantitatively. The more general approach is aimed to process several spectra at a time in order to find similarities and differences among them, usually by the application of chemometric techniques like Principal Components Analysis. The second way to analyze metabolomic NMR data is to assign peaks of interesting compounds, calculate their absolute or relative concentrations, and then compare these concentrations along a series of samples. We have developed a computer program called Mixtures that performs both types of analyses in a windows-based interface, requiring minimum knowledge about the maths involved in the statistical procedures, signal fitting, integration and spectrum display by the operator. The program runs in MS Windows operating systems and bears all the features of standard Windows programs, providing a simple, friendly interface with no command lines. Mixtures collects all spectra from a given study inside a project. Spectra can be opened one by one in a window that allows easy visualization and basic editing. This window also provides a wizard that aids in fitting spectra from a database of known compounds to the signals in the spectrum. Fitted signals can then be integrated and the integrals exported to a file that can be loaded in any standard spreadsheet program. Mixtures also features a MultiViewer in which selected regions of the spectra are binned according to users settings, and then PCA is carried out with only a few clicks. Mixtures was used with a set of NMR spectra from tomato samples collected at different ripening stages and conditions (green, red, and red matured outside the plant) in order to follow fluctuations in metabolite concentrations during ripening and to study the effect of off-plant maturation (Figure 1, left). A rapid PCA analysis performed on the whole spectra (excluding the water region) is able to separate the samples in three classes with just 2 principal components, revealing that fruit matured outside the plant is different than fruit matured on the plant (Figure 1, right). Figure 1. Screenshots from the MultiViewer (left) and results of Principal Components Analysis. Color keys: green: green fruits, red: red fruits matured in-plant, blue: red fruits matured off-plant. Inspection of each spectrum with the Fittings Wizard loaded with a subset of the metabolite database from BioMagResBank allowed us to unambiguously assign signals from nearly 20 compounds of our interest and integrate them. Comparison of these integrals with the integral of an internal standard yielded the concentration of each substance in each sample. PCA performed on these values allowed us to discriminate more clearly between the three classes of samples, and to study the correlation between them in a loading plot. An interesting outcome is that in fruits matured off-plant the percent abundance of some metabolites is more similar to that of green fruits than that of red fruits matured in-plant. Since some of these metabolites such as Glu are responsible of exalting flavor, these results are of most importance to the field of food quality assessment (Figure 2). Figure 2. Percentage composition of the most representative amino acids and amino acid precursor in green fruits (green bars), red fruits matured in-plant (red bars) and red fruits matured off-plant (orange bars). Two metabolites related to flavor are highlighted. Mixtures proved useful for the simple analysis of metabolomic NMR data using the two most common approaches in a quick and versatile way. Its application to studies of tomato ripening revealed differences between in-plant and off-plant maturation. REFERENCES 1-Eisenreich W, Bacher A., Phytochemistry 2008 Nov-Dec;68(22-24):2799-815. 2-Ellis DI, Dunn WB, Griffin JL, Allwood JW, Goodacre R. Pharmacogenomics 2007 Sep;8(9):1243-66. 3- Lindon JC, Holmes E, Nicholson JK. FEBS J. 2007 Mar;274(5):1140-51.