Divergent Metabolic Fates of Aromatic Amino Acid-Derived\nIsomers: Insights from Ex Vivo Metabolomics and HDX-HRMS/MS-Based\nResolution of Tautomers

Tautomers\nare one of the many types of isomers, and differences\nin tautomeric structures confer altered chemical and biological properties.\nUsing ultrahigh-performance liquid chromatography-high-resolution\nmass spectrometry (UHPLC-HRMS) ex vivo metabolomics, we investigate,\nin whole blood, the divergent metabolism of enol and keto forms of\nindole-3-pyruvate (IPyA), a tautomeric product of aromatic amino acid\nmetabolism. Two new compounds resulting from IPyA metabolism were\ndiscovered, 3-(1<i>H</i>-indol-3-yl)-2,3-dioxopropanoic\nacid or “indole-3-oxopyruvic acid” and glutathionyl-indole\npyruvate (GSHIPyA), which were characterized via ultraviolet photodissociation\n(UVPD) and higher-energy collisional dissociation (HCD). Computational\ncalculations support the hypothesis that GSHIPyA forms specifically\nthrough the enol form of IPyA. GSHIPyA is also hypothesized to be\ntautomeric, and a hydrogen–deuterium exchange-high-resolution\ntandem mass spectrometry (HDX-HRMS/MS) approach is developed to prove\nthe presence of an enol and keto tautomer. HDX of GSHIPyA labels the\nketo form with an additional deuterium, relative to the enol form.\nHRMS/MS of the labeled isomers is employed to leverage the relationship\nof resolving power scaling inversely with the square root of <i>m</i>/<i>z</i>, for Orbitrap mass analyzers. HRMS/MS\nyields a smaller-molecular-weight deuterated tautomeric product ion,\nreducing the analyte ion <i>m</i>/<i>z</i> and\nthus lowering the resolving power necessary to separate the deuterated\nketo tautomer product ion from the [13]C product ion.