Abstract
The comprehended knowledge of the metabolic profile of the fecal matter has been recognized as an important point for understanding metabolic changes in the human systemic metabolism and it can provide precious information about host-gut microbiota interactions. However, few analytical strategies have been addressed for a broad analysis of metabolites with different chemical properties to better understand the chemical space of fecal samples. Here we report a systematic pipeline to achieve comprehensive coverage of the fecal metabolome, from high polar to nonpolar metabolites, using dog fecal samples as a proof-of-concept. This pipeline comprises a monophasic (ACN/H2O) and a biphasic extraction (methyl tert-butyl ether (MTBE)/MeOH/H2O) of the sample, followed by three liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) methods using HILIC-amide, RP-C18 and CSH-C18 columns, and a switch polarity acquisition mode in the electrospray ion source. This approach allowed the annotation of 376 metabolites from 70 different chemical classes. The chemical space analysis by molecular networking and the pathway analysis revealed the complexity of the fecal sample and the importance of combined methods to better understand biochemical pathways. This pipeline can be used as a valuable tool to comprehend the relationship between host-gut microbiota metabolites and the influence of diet, medication, or environmental changes.
Highlights
This study aimed to implement a comprehensive pipeline for the analysis of nonpolar and weakly polar metabolites to high polar metabolites in fecal samples by untargeted metabolomic and lipidomic approaches
This difference in liquid chromatographyhigh resolution mass spectrometry (LC-HRMS) chromatogram quality by methods of different scale could be due to a difficulty in sample homogenization in the microtube by the 20 mg per 200 μL proportion, especially because the solution obtained presented a high number of particulates before centrifugation, reducing the contact area between the sample and the solvent, leading to an inefficient extraction compared to the larger scale procedure
We have established a pipeline for a comprehensive analysis of the fecal metabolome using LC-HRMS/Mass spectrometry (MS)
Summary
The fecal analysis by mass spectrometry has emerged as an important field to provide insights into the relationship between the host and the gut microbiome activity and how it can affect the host homeostasis.[1,2,3] This symbiotic relationship has been shown to play a vital function in human metabolism, immunity, and reaction to diseases, such as diabetes,[4,5] coronary artery disease,[6] inflammatory disorders,[7] and for the organism health state.The metabolites produced by the gut microbiome, and other compounds that comprise the fecal matter, greatly varies and can be modified according to changes in host metabolism, such as age,[8] species,[9] diet,[10] disease,[3] or therapeutic intervention.[11]. The fecal analysis by mass spectrometry has emerged as an important field to provide insights into the relationship between the host and the gut microbiome activity and how it can affect the host homeostasis.[1,2,3] This symbiotic relationship has been shown to play a vital function in human metabolism, immunity, and reaction to diseases, such as diabetes,[4,5] coronary artery disease,[6] inflammatory disorders,[7] and for the organism health state. Mass spectrometry (MS)-based metabolomics and lipidomics have been revealed as important tools to study fecal samples. This is mostly due to the high sensitivity, specificity, and broad coverage of different classes of compounds by the mass spectrometer, especially when
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