Compound discover software

Identification analysis of metabolites in bio-samples was performed as described previously [5 (link)] using a Dionex Ultimate 3000 UHPLC system combined with a Q-Exactive Focus Orbitrap mass spectrometer (Thermo Fischer Scientific, MA, USA) fitted with a CORTECS T3 column (2.1 × 150 mm, 2.7 μm; Waters, MA, USA). An electrospray ionization (ESI) source in both negative- and positive-ionization mode was used. Data recording and processing were carried out using the Xcalibur software (version 2.2, Thermo Fischer Scientific, MA, USA) and Compound Discover software (version 3.2, Thermo Fischer Scientific, MA, USA).
A total of 100 μL plasma, bile, urine and feces homogenate samples were deproteinized with 500 μL, 500 μL, 500 μL and 900 μL of acetonitrile, respectively. After vortex-mixing and centrifugation at 14,000 rpm for 10 min twice, 5 μL of supernatant was injected into the UHPLC–HRMS system for qualitative analysis.

We used the Thermo Xcalibur software version 4.1 and Compound Discover software version 3.0 (Thermo Fisher Scientific, California, CA, USA) to process all the raw data, including full-scan MS and MS² data. We set the minimum peak intensity to 10,000 and calculated detailed chemical formula parameters from accurate masses for all the parent and fragment ions of selected peaks using a formula predictor, as follows: the maximum element counts were C30, H60, O20, and N10; the MS and MS² mass tolerances were set to 5 and 10 ppm, respectively.

Anion-exchange chromatography-mass spectrometry (IC-MS) was used for analysis of polar and ionic metabolites in the media samples as described in detail previously (46 (link)). We used compound discover software (Thermofisher Scientific) for data processing and statistical analysis. Metabolites were identified using predicted composition and searches against four global database MzCloud, Metabolika, ChemSpider, and an in-house database of authentic standards that included retention times. Matches to the in-house database were: retention time: true, RT (retention time) tolerance 2, and mass tolerance 5 ppm. Peak areas for the extracted ion chromatogram (EIC) for each identified metabolite are presented as a relative measure of abundance. To estimate the levels of itaconate and taurine, a standard curve was prepared. Standard samples were prepared in the medium used for growing the cells and all samples were measured using IC-MS. Analysis of data and calculations of EIC peak areas were performed using MestReNova software.