LC-MS Metabolomics Data Processing

Acquired LC–MS data were processed using the Thermo Scientific TraceFinder 5.1 software. Targeted metabolites were identified by matching accurate mass and retention times to the University of Iowa Metabolomics Core Facility’s in-house library of confirmed standards (Table S2). During data analysis in TraceFinder, mass tolerance was set to 2 millimass units (0.002 Daltons). After peak area integration by TraceFinder, NOREVA software was applied for signal drift correction on a metabolite-to-metabolite basis using the pooled QC sample that had been analyzed throughout the instrument run as the normalizing reference [17 (link)]. We further accounted for technical variation in sample quantity and autosampler injection volume by normalizing each metabolite signal intensity to the sum of all metabolite signal intensities within a sample to generate a ratiometric metabolite fingerprint. NOREVA and ratiometrically normalized individual QC samples were superimposed on a PCA plot, providing evidence of high run quality and accurate data normalization (Figure S1). Internal standards were used to test for differences in extraction efficiency amongst samples, which were not observed. Batch effects were not present in this study since all samples were run in one batch on the LC–MS. The processing blank showed no evidence of carryover or contamination.