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

Abstract

New sulfatase-based assay for the selective identification of sulfate esters in human urine and fecal samples.

Coupled Enzymatic Treatment and Mass Spectrometric Analysis for Identification of Glucuronidated Metabolites in Human Samples

“…The complexity of metabolomics analysis and challenges to overcome metabolite structure identification as well as absolute validation require the development of advanced chemical biology and interdisciplinary methodologies . We have recently reported a new strategy for the identification of O‐sulfated metabolites by using a combination of sulfatase treatment, chemical synthesis, and bioinformatic metabolomics analysis to selectively identify sulfated metabolites . This enzyme‐driven metabolomics discovery approach identified three times as many sulfated metabolites as are reported in the entire Human Metabolome Database .…”

“…We have recently reported a new strategy for the identification of O‐sulfated metabolites by using a combination of sulfatase treatment, chemical synthesis, and bioinformatic metabolomics analysis to selectively identify sulfated metabolites . This enzyme‐driven metabolomics discovery approach identified three times as many sulfated metabolites as are reported in the entire Human Metabolome Database . This new metabolomics tool opens up new opportunities for the discovery of unknown bioactive molecules.…”

“…A highly purified glucuronidase was essential to the success of the analysis, as unselective reactions could create artefacts and lead to erroneous identification of glucuronides and increased data complexity . Although glucuronidases overexpressed or extracted from different organisms can be purchased, significant quantities of active enzyme cannot be obtained at low cost .…”

“…Although glucuronidases overexpressed or extracted from different organisms can be purchased, significant quantities of active enzyme cannot be obtained at low cost . Recently, we reported an untagged purification of an arylsulfatase from a commercial crude extract from the snail Helix pomatia . While the extract was marketed as an arylsulfatase, characterization of the enzymatic activities in the crude mixture revealed glucuronidase activity approximately ten times higher than the sulfatase activity.…”

“…In metabolomics, the highest level of confidence for the identification of metabolite structures requires a synthesized or commercially available standard compound . Co‐injection experiments of the natural and the standard metabolite must result in the same retention time as well as a perfectly matching fragmentation pattern to validate the structure of the natural compound . As one example for validation of the absolute metabolite structure, we synthesized p ‐cresyl glucuronide ( 5 ) to confirm the metabolite (see the Supporting Information).…”