A Dual UHPLC-HRMS-Based Fecal Metabolomics and Lipidomics Analysis and Automated Data Processing Pipeline for Comprehen-sive Gut Phenotyping

17 January 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

In recent years, feces has surfaced as the matrix of choice for investigating the gut microbiome-health axis because of its non-invasive sampling and the unique reflection it offers of an individual’s lifestyle. In cohort studies where the number of samples required is large, but availability is scarce, a clear need exists for high-throughput analyses. Such analyses should combine a wide physicochemical range of molecules with a minimal amount of sample and resources, and downstream data processing workflows that are as automated and time efficient as possible. We present a dual fecal extraction and UHPLC-HR-Q-Orbitrap-MS-based workflow that enables widely targeted and untargeted metabolome and lipidome analy-sis. A total of 836 in-house standards were analyzed, of which 360 metabolites and 132 lipids were consequently detected in feces. Their targeted profiling was validated successfully with respect to repeatability (78% CV<20%), reproducibility (82% CV<20%) and linearity (81% R2>0.9), while also enabling holistic untargeted fingerprinting (15 319 features, CV<30%). To automate targeted processing, we optimized an R-based targeted peak extraction (TaPEx) algorithm relying on a database comprising retention time and mass-to-charge ratio (360 metabolites and 132 lipids), with batch-specific quality control curation. The latter was benchmarked towards vendor-specific targeted and untargeted software and our IPO/XCMS-based untargeted pipeline in Lifelines Deep cohort samples (n = 97). TaPEx clearly outperformed the untargeted approaches (81.3 vs. 56.7-66.0% compounds detected). Finally, our novel dual fecal metabolomics-lipidomics-TaPEx method was successful-ly applied to Flemish Gut Flora Project cohort (n = 292) samples, leading to a sample-to-result time reduction of 60%.

Keywords

UHPLC-HRMS
Metabolomics
Lipidomics
Feces
Data processing

Supplementary materials

Title
Description
Actions
Title
Supporting Information
Description
Supporting information (Figures and overview of tables) for A Dual UHPLC-HRMS-Based Fecal Metabolomics and Lipidomics Analysis and Automated Data Processing Pipeline for Comprehensive Gut Phenotyping
Actions
Title
Supplementary Tables
Description
.zip file containing all the supplementary tables described in the SI document.
Actions

Comments

Comments are not moderated before they are posted, but they can be removed by the site moderators if they are found to be in contravention of our Commenting Policy [opens in a new tab] - please read this policy before you post. Comments should be used for scholarly discussion of the content in question. You can find more information about how to use the commenting feature here [opens in a new tab] .
This site is protected by reCAPTCHA and the Google Privacy Policy [opens in a new tab] and Terms of Service [opens in a new tab] apply.