Abstract
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.
Highlights
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 detect the naıve molecular localization in mammalian tissues
EGCG peaks were not observed with dihydroxy benzoic acid (DHB), cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA), or 9-AA, which are the most effective major matrices for ionizing small molecules[14]
Because ion suppressive effects are predominantly caused by competing endogenous species in tissues[14], we determined which of these matrix candidates in its corresponding optimum solvent (1,5-DAN or ferulic acid in acetone (ACE), or harmane, norharmane, or harmine in MeOH) could effectively ionize EGCG spotted on the tissue sections (Fig. 1b)
Summary
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 detect the naıve molecular localization in mammalian tissues. 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 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. Mass spectrometry imaging (MSI) is a new technology capable of determining the naıve distribution of ionizable biological molecules in tissue sections without any labeling on the basis of their specific mass-to-charge ratios This technique can theoretically detect target molecules and their metabolites simultaneously in a single analysis, and is widely used for in situ imaging of endogenous and exogenous molecules such as proteins, lipids, drugs, and their metabolites[8,9,10]. In this study, we attempted to establish an in situ label-free technique for the simultaneous imaging of a bioactive polyphenol and its metabolites in mammalian tissues following in vivo dosing
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