Abstract
Untargeted metabolomics aims to provide a global picture of the metabolites present in the system under study. To this end, making a careful choice of sample preparation is mandatory to obtain reliable and reproducible biological information. In this study, eight different sample preparation techniques were evaluated using Caulobacter crescentus as a model for Gram-negative bacteria. Two cell retrieval systems, two quenching and extraction solvents, and two cell disruption procedures were combined in a full factorial experimental design. To fully exploit the multivariate structure of the generated data, the ANOVA multiblock orthogonal partial least squares (AMOPLS) algorithm was employed to decompose the contribution of each factor studied and their potential interactions for a set of annotated metabolites. All main effects of the factors studied were found to have a significant contribution on the total observed variability. Cell retrieval, quenching and extraction solvent, and cell disrupting mechanism accounted respectively for 27.6%, 8.4%, and 7.0% of the total variability. The reproducibility and metabolome coverage of the sample preparation procedures were then compared and evaluated in terms of relative standard deviation (RSD) on the area for the detected metabolites. The protocol showing the best performance in terms of recovery, versatility, and variability was centrifugation for cell retrieval, using MeOH:H2O (8:2) as quenching and extraction solvent, and freeze-thaw cycles as the cell disrupting mechanism.
Highlights
Untargeted metabolomic approaches aim to analyze the metabolic composition of biological samples in a holistic manner, with the goal of retrieving and storing as much chemical information as possible
Medium filtering and centrifugation were compared for cell retrieval, cold MeOH:H2 O 8:2 and cold MeOH:H2 O:CHCl3 7:3:1 + ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA) 1 mM were compared as two quenching and extraction solvents and, two cell disruption mechanisms were tested
Metabolomic analyses were performed in hydrophilic liquid interaction chromatography (HILIC) mode coupled to a Vion TWIMS-quadrupole time-of-flight instruments (QTOF) operating in negative ESI polarity
Summary
Untargeted metabolomic approaches aim to analyze the metabolic composition of biological samples in a holistic manner, with the goal of retrieving and storing as much chemical information as possible. To this end, adequate sample preparation is one of the most critical steps to obtain reliable biological insights [1]. Several groups have recently demonstrated that different HILIC stationary phases enable the analysis of many chemical classes of metabolites [12,13,14]. It has been shown that polymeric columns with zwitterionic stationary phases are interesting for the analysis of compounds related to the tricarboxylic acid cycle (TCA) [14]
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