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Pipeline publication

[…] ll column eluent was transferred to the mass spectrometer and full-scan profiling data were acquired in the m/z range 100-1000 in the Orbitrap mass analyser (mass resolution 30,000; full width half maximum at m/z=400). The source and ion transfer parameters applied were as follows: source heater=200°C, sheath gas=50 (arbitrary units), aux gas=15 (arbitrary units), capillary temperature=300°C, ISpray voltage=4 kV (positive ion mode) and 3 kV (negative ion mode), s lens=65% and AGC=5×105., Raw instrument data were converted to netCDF file format with FileConverter software available in XCalibur (Thermo Fisher Scientific, Bremen, Germany). Deconvolution was performed using the freely available XCMS software as described previously (; ). Data were exported from XCMS as a .csv file for further data analysis. The quality of data was assessed by applying QC data as previously described (), with all metabolite features with an RSD >20% or with greater than 30% missing values for QC samples being removed from the dataset prior to data analysis. Metabolite annotation was performed by applying the PUTMEDID_LCMS workflow as previously described (). All metabolite annotations are reported at level 2 according to MSI reporting standards (). The processed data was analysed in R with the univariate t-test applying the Benjamini–Hochberg procedure for FDR calculation (q<0.05) after normalisation to total peak area. The fold change was calculated by applying the median response per class with 95% confidence limits. The processed data were also analysed by applying unsupervised PCA, and pairwise comparisons of classes by applying supervised PLS-DA; both of these analyses were performed in the open-access software MetaboAnalyst (). Metabolites were manually clustered into classes defining similar chemical structure or metabolic pathway to identify biologically relevant and robust metabolic changes., All ex vivo analyses were performed blinded to treatment (naïve or MCAO) and genotype (control or obese). Sample sizes were determined by power calculation (α=0.05, β=0.2) of our previous data. Statistical analyses for all non-metabolomic data were performed using GraphPad software (GraphPad Software Inc., La Jolla, CA, USA). Data was tested for equal variance using the Brown–Forsythe and Bartlett's tests, and appropriate transformations applied if necessary. Two-way ANOVA was used for comparisons by genot […]

Pipeline specifications

Software tools XCMS, PUTMEDID-LCMS, MetaboAnalyst
Organisms Mus musculus
Diseases Body Weight, Overnutrition, Body Weight, Cerebrovascular Disorders, Brain Diseases, Cerebrovascular Disorders, Brain Diseases
Chemicals Fatty Acids