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

• Published in: Analytical chemistry (Washington) 2024-10, Vol.96 (42), p.16917-16925
• Main Authors: Christopher, Michael W., Ericson, Aiden C., Klug, Alexander C., Dinglasan, Rhoel R., Prentice, Boone M., Garrett, Timothy J.
• Format: Article
• Language: English
• Subjects: amino acid metabolism; Amino acids; analytical chemistry; Analyzers; Aromatic compounds; Biological properties; blood; Deuteration; Deuterium; dissociation; Energy of dissociation; enols; High resolution; Hydrogen-deuterium exchange; Indoles; Isomers; Liquid chromatography; Mass spectrometry; Mass spectroscopy; Metabolism; Metabolomics; Photodissociation; photolysis; Pyruvic acid; Resolution; Scientific imaging; tandem mass spectrometry; Tautomers
• ISSN: 0003-2700; 1520-6882; 1520-6882
• DOI: 10.1021/acs.analchem.4c03862

Tautomers are one of the many types of isomers, and differences in tautomeric structures confer altered chemical and biological properties. Using ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) ex vivo metabolomics, we investigate, in whole blood, the divergent metabolism of enol and keto forms of indole-3-pyruvate (IPyA), a tautomeric product of aromatic amino acid metabolism. Two new compounds resulting from IPyA metabolism were discovered, 3-(1H-indol-3-yl)-2,3-dioxopropanoic acid or “indole-3-oxopyruvic acid” and glutathionyl-indole pyruvate (GSHIPyA), which were characterized via ultraviolet photodissociation (UVPD) and higher-energy collisional dissociation (HCD). Computational calculations support the hypothesis that GSHIPyA forms specifically through the enol form of IPyA. GSHIPyA is also hypothesized to be tautomeric, and a hydrogen–deuterium exchange-high-resolution tandem mass spectrometry (HDX-HRMS/MS) approach is developed to prove the presence of an enol and keto tautomer. HDX of GSHIPyA labels the keto form with an additional deuterium, relative to the enol form. HRMS/MS of the labeled isomers is employed to leverage the relationship of resolving power scaling inversely with the square root of m/z, for Orbitrap mass analyzers. HRMS/MS yields a smaller-molecular-weight deuterated tautomeric product ion, reducing the analyte ion m/z and thus lowering the resolving power necessary to separate the deuterated keto tautomer product ion from the [13]C product ion.