New enzymatic and mass spectrometric methodology for the selective investigation of gut microbiota-derived metabolites

Abstract

Gut microbiota significantly impact human physiology through metabolic interaction. Selective investigation of the co-metabolism of bacteria and their human host is a challenging task and methods for their analysis are limited. One class of metabolites associated with this co-metabolism are O-sulfated compounds. Herein, we describe the development of a new enzymatic assay for the selective mass spectrometric investigation of this phase II modification class. Analysis of human urine and fecal samples resulted in the detection of 206 sulfated metabolites, which is three times more than reported in the Human Metabolome Database. We confirmed the chemical structure of 36 sulfated metabolites including unknown and commonly reported microbiota-derived sulfated metabolites using synthesized internal standards and mass spectrometric fragmentation experiments. Our findings demonstrate that enzymatic sample pre-treatment combined with state-of-the-art metabolomics analysis represents a new and efficient strategy for the discovery of unknown microbiota-derived metabolites in human samples. Our described approach can be adapted for the targeted investigation of other metabolite classes as well as the discovery of biomarkers for diseases affected by microbiota.

Scheme 1. Workflow of the targeted metabolomics-driven identification and mass spectrometric analysis of sulfated metabolites including the chemical structure of four representative gut microbiota-derived metabolites 1–4 identified in human samples.

Fig. 1. (A) Structures of the substrates used in the sulfatase/glucuronidase comparative assay: 4-methylumbelliferyl sulfate (4) and N-acetyltyramine-O,β-glucuronide (5), and in the units calculation assay: phenolphthalein (6) and phenolphthalein β-d-glucuronide (7); (B) SDS-PAGE of the crude H. pomatia lysate and the purified arylsulfatase (ArS); (C) glucuronidase activity calculated for the H. pomatia crude mixture (Sigma-Aldrich) and the purified arylsulfatase fraction.

Fig. 2. (A) Hydrolysis experiments of selected sulfated substrates using purified sulfatase in duplicate (error bars: SEM); (B) sulfated metabolites tested as substrates in the sulfatase assay. Microbiota-derived metabolites are highlighted in red.

Scheme 2. Workflow for our described sulfatase-based mass spectrometric strategy for the analysis of human samples. Structure validation of ferulic acid 4-sulfate (3) is presented as an example. (Sulfatase crystal structure15).

Fig. 3. Example of sulfate ester validation in urine with p-cresyl sulfate (2). (A) UPLC-MS chromatogram comparison of urine sample vs. the synthesized standard molecule; (B) MS/MS fragmentation pattern of 2 in urine sample vs. the synthesized standard molecule at a collision energy at 30 V; (C) assignment of MS/MS fragments of 2.