We propose a novel strategy for determining the elemental composition of organic compounds using the peak ratio of isotopic fine structure observed by high-magnetic field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Using 3'-phosphoadenosine 5'-phosphosulfate and CTU guanamine as standard organic compounds, isotopic peaks derived from (15)N-, (34)S-, and (18)O-substituted forms were separated from (13)C-substituted species. Furthermore, these isotopic peaks were quantitatively detected and closely matched the natural abundance of each element. These data successfully led us to determine the one elemental composition in a standard independent manner. The approach should be particularly amenable to the metabolomics research field.

Although understanding the high-resolution spatial distribution of bioactive small molecules is indispensable for elucidating their biological or pharmacological effects, there has been no analytical technique that can easily detect the naïve molecular localization in mammalian tissues. We herein present a novel in situ label-free imaging technique for visualizing bioactive small molecules, using a polyphenol. We established a 1,5-diaminonaphthalene (1,5-DAN)-based matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) technique for visualizing epigallocatechin-3-O-gallate (EGCG), the major bioactive green tea polyphenol, within mammalian tissue micro-regions after oral dosing. Furthermore, the combination of this label-free MALDI-MSI method and a standard-independent metabolite identification method, an isotopic fine structure analysis using ultrahigh-resolution mass spectrometer, allows for the visualization of spatially-resolved biotransformation based on simultaneous mapping of EGCG and its phase II metabolites. Although this approach has limitations of the detection sensitivity, it will overcome the drawbacks associated with conventional molecular imaging techniques, and could contribute to biological discovery.

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In the present study, a high-throughput analytical method for intracellular metabolites using MALDI-MS has been developed. As an analytical tool, the quantitative performance and dynamic range of MALDI-TOF-MS was confirmed to be suitable for characterizing the trends of intracellular metabolism. The technique was tested by investigating the intracellular metabolism of Escherichia coli by analyzing whole cell samples taken consecutively before and after a perturbation of the environmental carbon source. As the result, dramatic changes of metabolite concentrations responding to the perturbation were observed. The whole analysis process (i.e., sample preparation and MALDI-MS analysis for 24 time points in triplicate) was completed within 4 hours. MALDI-FTICR-MS was used to identify the elemental compositions of detected metabolites to support the reliability of the MALDI-MS-based analysis. The MALDI-MS-based analytical method developed herein should be suitable for high-throughput analysis of dynamic intracellular metabolism events.