Metabolomics Analysis of Viral Infection

Full scan LC/MS (m/z range 85–2000) was performed essentially as previously described [8] . Cell extracts or supernatants were treated with acetonitrile (2∶1, v/v) and centrifuged at 14,000× g for 5 min at 4°C to remove proteins. Samples were maintained at 4°C in an autosampler until injection. A Thermo Orbitrap-Velos mass spectrometer (Thermo Fisher, San Diego, CA) coupled with anion exchange chromatography was used for data collection, via positive-ion electrospray ionization (ESI). Metabolites of interest were identified by tandem mass spectrometry on a LTQ-FTMS, where the biological sample, biological sample spiked in with authentic chemical and authentic chemical reference were run sequentially. The and were done in the ion trap of the LTQ-FTMS, with an isolation width of 1 amu and a normalized collision energy of 35 eV.
The LC/MS data were processed with apLCMS program [25] (link) for feature extraction and quantification. Significant features were also verified by inspecting the raw data (Figure S5). Features were removed if their intensity is below 10,000 in every sample class. Missing intensity values were imputed to 1000. The intensities were log2 transformed. Low quality features were further filtered out if their averaged in-class coefficient of variation was greater than 0.2. Averaged ion intensity over three machine replicates was used for subsequent analysis. These 7,995 features constituted the reference list . No normalization was used because total ion counts in all samples were very similar. Student's t-test was used to compare infected samples (at 6 hr) versus mock infections (at 6 hr), and infected samples (at 6 hr) versus baseline controls (at 0 hr). Features with in both tests were included in the significant list . The feature table, , and predictions are given in Dataset S1.