Optimized Fast Filtration-Based Sampling and Extraction Enables Precise and Absolute Quantification of the Escherichia coli Central Carbon Metabolome

Precise and accurate quantification is a prerequisite for interpretation of targeted metabolomics data, but this task is challenged by the inherent instability of the analytes. The sampling, quenching, extraction, and sample purification conditions required to recover and stabilize metabolites in re...

Full description

Saved in:
Bibliographic Details
Published in:Metabolites 2023-01, Vol.13 (2), p.150
Main Authors: Thorfinnsdottir, Lilja Brekke, García-Calvo, Laura, Bø, Gaute Hovde, Bruheim, Per, Røst, Lisa Marie
Format: Article
Language:English
Subjects:
Accuracy
Acetonitrile
Biotechnology
Carbon
central carbon metabolism
Chromatography
Cold
E coli
Escherichia coli
fast filtration sampling
Filtration
mass spectrometry
Metabolism
metabolite extraction
Metabolites
Metabolomics
Protocol
Sampling
targeted metabolite profiling
Trace elements
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Precise and accurate quantification is a prerequisite for interpretation of targeted metabolomics data, but this task is challenged by the inherent instability of the analytes. The sampling, quenching, extraction, and sample purification conditions required to recover and stabilize metabolites in representative extracts have also been proven highly dependent on species-specific properties. For , unspecific leakage has been demonstrated for conventional microbial metabolomics sampling protocols. We herein present a fast filtration-based sampling protocol for this widely applied model organism, focusing on pitfalls such as inefficient filtration, selective loss of biomass, matrix contamination, and membrane permeabilization and leakage. We evaluate the effect of and need for removal of extracellular components and demonstrate how residual salts can challenge analytical accuracy of hyphenated mass spectrometric analyses, even when sophisticated correction strategies are applied. Laborious extraction procedures are bypassed by direct extraction in cold acetonitrile:water:methanol (3:5:2, %), ensuring compatibility with sample concentration and thus, any downstream analysis. By applying this protocol, we achieve and demonstrate high precision and low metabolite turnover, and, followingly, minimal perturbation of the inherent metabolic state. This allows us to herein report absolute intracellular concentrations in and explore its central carbon metabolome at several commonly applied cultivation conditions.
ISSN:2218-1989
2218-1989
DOI:10.3390/metabo13020150