MSACL 2025 Abstract

Chemical Biology Tools for the Selective Metabolomics Investigation of Gut Microbiota-Derived and Dietary Metabolites

Ioanna Tsiara (1), Belén Hervás Povo (1), Fan Yang (1), Daniel Globisch (1)
(1) Department of Chemistry, Uppsala University, Uppsala/Sweden

Daniel Globisch, PhD (Presenter) Uppsala University

Presenter Bio: Daniel Globisch studied Chemistry at the Technical University of Kaiserslautern (Germany) and the University of Southern Denmark, Odense (Denmark). He received his Ph.D. from the Ludwig-Maximilians-University Munich (Germany) with Professor Thomas Carell in 2011 and joined the laboratory of Professor Kim D. Janda at The Scripps Research Institute (CA, USA) for his postdoctoral studies. He started his independent career in 2015 at Uppsala University (Sweden) as a Science For Life Laboratory Fellow and was appointed as Associate Professor in 2017. He received tenure in December 2020 and was promoted to Full Professor in November 2023. Daniel has been elected as a board member of the Nordic Metabolomics Society. The interdisciplinary nature of the research projects is focused on the elucidation of the metabolic interaction between the gut microbiota and their human host. His laboratory develops new Chemical Biology tools to extend the scope of metabolomics research for discovery of unknown biomarkers and bioactive metabolites.

Relevant Financial Disclosures (within past 24 months, reported on Mar 19, 2025) No relevant financial relationship(s) to disclose.

INTRODUCTION:
Metabolites produced by the gut microbiome play a crucial and diverse role in host physiology, which are detectable in a wide range of biological samples including feces, plasma, urine, and cerebrospinal fluid. Microbiota dysbiosis has been associated with the development of diseases, however, the metabolic link has yet to be detected. The detailed and targeted analysis of these metabolites is important for the discovery of biomarkers and unknown bioactive molecules. Mass spectrometric metabolomics is the method of choice for identification and quantification of these metabolites.

OBJECTIVES:
We have developed a series of new methodologies to overcome limitations in the selective mass spectrometric analysis. We are developing and utilizing techniques at the interface of chemistry and biology to allow for an advanced targeted qualitative and quantitative metabolomics analysis for the discovery of disease-specific biomarkers.

METHODS:
We have developed a series of chemical biology tools by combining selective enzymatic metabolite conversion with metabolomics data analysis. We are selectively converting sulfated [1-4] and glucuronidated [1,3] metabolites in different human sample types for subsequent UPLC-MS/MS analysis using recombinant sulfatases and glucuronidases, respectively. The obtained raw data were then processed with R using the XCMS metabolomics framework to selectively identify metabolites with a sulfate and glucuronide moiety. We have performed MS/MS fragmentation for all identified features in order to validate the chemical structure. Recently, we have applied this methodology to dietary intervention studies [4]. To enhance the scope of our method, we have now applied two recombinant enzymes, ASPC and B-One, in order to increase the number of sulfated and glucuronidated metabolites and enhance the analysis for identification of additional scaffolds.

RESULTS:
Through this combined metabolomics and enzymatic analysis, we detected more than 200 sulfated and more than 150 glucuronidated metabolites [1-3], which is a phase II metabolite coverage of more than double compared to other methodologies. We have built up a library of metabolites identified at the highest two confidence levels that we can screen in different sample types. In one of our applications, we investigated metabolic phase II product changes of a (poly)phenol-rich diet [2]. In a large-scale analysis, we discovered six polyphenol-derived metabolites as potential biomarkers for the dietary intake [1]. Due to the stability of the enzymes, we immobilized them to magnetic beads for the separate mass spectrometric quantitative investigation of sulfated and glucuronidated metabolites on a large scale [4].

CONCLUSION:
We have succeeded in identifying numerous metabolites that are correlated with microbiome host co-metabolism using two recombinant enzymes demonstrating the broad applicability of our analytical tools for discovery of dietary markers and microbiome-derived metabolites.

REFERENCES
(1) I. Tsiara ... D. Globisch J. Agric. Food Chem. 2025, accepted.
(2) M.S.P. Correia … D. Globisch Free Radic. Biol. Med. 2020, 160, 745.
(3) C. Ballet … D. Globisch Chem. Sci. 2018, 9, 6233.
(4) I. Tsiara … D. Globisch Anal. Chem. 2023, 95, 12565.