The supernatant was extracted from 200 mg of faeces and transferred to sample vials[19 (link)]. Two microlitres of a sample was separated with an HSS T3 column and used for LC-MS/MS analysis. Mass spectrometric data were collected using a UHPLC-Q Exactive system (Thermo Fisher Scientific, Waltham, MA, United States) with an electrospray ionization source operating in the positive- and negative-ion modes. Data acquisition was performed in the data-dependent acquisition mode.
The raw LC-MS/MS data were preprocessed using Progenesis QI (Waters Corporation, Milford, MA, United States) software. Internal standard peaks and false-positive peaks were removed from the data matrix, redundant signals were removed, and the peaks were pooled. In addition, the metabolites were searched and identified in the HMDB, KEGG and Metlin databases.
Metabolites detected in at least 80% of any set of samples were retained[20 (link)]. After filtering, the metabolite response intensity of the mass spectrum peaks was normalized using the sum-normalization method. Moreover, variables with a relative standard deviation > 30% relative to the quality control samples were removed, and log10 logarithmization was performed to obtain the final data matrix for subsequent analysis.
The raw LC-MS/MS data were preprocessed using Progenesis QI (Waters Corporation, Milford, MA, United States) software. Internal standard peaks and false-positive peaks were removed from the data matrix, redundant signals were removed, and the peaks were pooled. In addition, the metabolites were searched and identified in the HMDB, KEGG and Metlin databases.
Metabolites detected in at least 80% of any set of samples were retained[20 (link)]. After filtering, the metabolite response intensity of the mass spectrum peaks was normalized using the sum-normalization method. Moreover, variables with a relative standard deviation > 30% relative to the quality control samples were removed, and log10 logarithmization was performed to obtain the final data matrix for subsequent analysis.
