Computational protocol: Electrospray Quadrupole Travelling Wave Ion Mobility Time-of-Flight Mass Spectrometry for the Detection of Plasma Metabolome Changes Caused by Xanthohumol in Obese Zucker (fa/fa) Rats

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Protocol publication

[…] MSE data were converted into NetCDF format (Network Common Data Form) using MassLynx software (Waters Corp, Milford, MA). Converted data sets were analyzed by the XCMS program using the R package (2.15.1) for initial data processing, including peak grouping, retention time alignment and filling in missing features []. The resulting data matrices were then imported into MetaboAnalyst software (2.0) for statistical analysis [,]. Xanthohumol and its related metabolites were excluded from the sample list prior to statistical validation [,]. Features in a data matrix were filtered based on relative standard deviation (RSD) of whole intensity values, and log transformation and auto-scaling were incorporated to ensure equal contributions from each variable to the models. Partial Least Square Discriminant Analysis (PLS-DA) was performed to identify discriminating features between control treatment groups. Data visualization using unsupervised, two-way hierarchical clustering was performed using the Ward algorithm using the Euclidian distance on the normalized metabolites. A Student t-test was performed for statistical comparison between control and high dose rat samples, and a p-value of 0.05 or less was considered significant. Significant features were selected based on the p-value (p < 0.05) and fold change threshold (FC > 1.5). MetaboSearch was used to perform m/z-based searches simultaneously against the four major metabolite databases: Human Metabolome Database (HMDB), Madison Metabolomics Consortium Database (MMCD), Metlin and LipidMaps []. Finally, the compound assignments were confirmed by matching the high energy MSE data with the reported and predicted fragmentation patterns for selected metabolites. […]

Pipeline specifications

Software tools XCMS, MetaboAnalyst, MetaboSearch
Databases HMDB METLIN MMCD
Application MS-based untargeted metabolomics
Organisms Rattus norvegicus