Coupling Targeted and Untargeted Mass Spectrometry for Metabolome-Microbiome-Wide Association Studies of Human Fecal Samples

Increasing appreciation
of the gut microbiome’s role in
health motivates understanding the molecular composition of human
feces. To analyze such complex samples, we developed a platform coupling
targeted and untargeted metabolomics. The approach is facilitated
through split flow from one UPLC, joint timing triggered by contact
closure relays, and a script to retrieve the data. It is designed
to detect specific metabolites of interest with high sensitivity,
allows for correction of targeted information, enables better quantitation
thus providing an advanced analytical tool for exploratory studies.
Procrustes analysis revealed that untargeted approach provides a better
correlation to microbiome data, associating specific metabolites with
microbes that produce or process them. With the subset of over one
hundred human fecal samples from the American Gut project, the implementation
of the described coupled workflow revealed that targeted analysis
using combination of single transition per compound with retention
time misidentifies 30% of the targeted data and could lead to incorrect
interpretations. At the same time, the targeted analysis extends detection
limits and dynamic range, depending on the compounds, by orders of
magnitude. A software application has been developed as a part of
the workflow to allows for quantitative assessments based on calibration
curves. Using this approach, we detect expected microbially modified
molecules such as secondary bile acids and unexpected microbial molecules
including Pseudomonas-associated quinolones and rhamnolipids
in feces, setting the stage for metabolome-microbiome-wide association
studies (MMWAS).