DOI: 10.25345/c5j679740
title: MassIVE MSV000094355 - Effect of itaconate and 4-octylitaconate on the metabolome
title: Metabolites extraction. Metabolites were extracted from cells on ice using 800 ul 100% methanol for viral inactivation, followed by 200 ul of ice cold HPLC-grade water. The cell solutions were vortexed for 10 seconds and incubated at -20 degrees C for 2 h. Then, the cell solutions were centrifuged at 4 degrees C, 16 000 g for 20 min, and the supernatants were transferred to 1.5 ml Eppendorf tubes. The supernatants were dried down in a speedvac (Labconco Centrivap) at 8oC. The dried metabolite extracts were resuspended in 100 ul of acetonitrile-water (1:1, v/v) and sonicated for 10 min in ice water. The solution was centrifuged at 4 degrees C, 16 000 g for 20 min. The supernatants were transferred to a 96-well plate (Greiner) and were subjected to targeted metabolomics LC-MS/MS analysis using a list of known itaconate-related metabolites. A quality control sample was prepared by pooling together 5ul of all samples and was used to observe the instrument performance during the run. Targeted metabolomics analysis and mass spectrometry. Targeted metabolomic analysis was performed on a triple quadrupole (QQQ) mass spectrometer (Agilent Triple Quadrupole 6495C, San Diego, CA), coupled to an ultra-high pressure liquid chromatography system (UPLC) system (1290 Infinity, Agilent Technologies) as previously described 81. Data were acquired with Agilent MassHunter Workstation Data Acquisition (version 10.1). A CSH Phenyl-hexyl column (1.7 um, 1.0 x 100 mm) (Waters, Taastrup, Denmark) was used for metabolites separation. Collision energies and product ions (MS2 or quantifier and qualifier ion transitions) were optimized. Electrospray ionisation source conditions were set as follows: gas temperature, 200 degrees C; gas flow, 15 L/min; Nebulizer, 25 psi; sheath gas temperature, 325 degrees C; cap voltage, 3000 V; and nozzle voltage, 500 V. For the liquid chromatography, the following parameters were used. The gradient consisted of buffer A, and buffer B. Buffer A was 99.9% H2O and 0.1% formic acid. Buffer B was 99.9% acetonitrile and 0.1% formic acid. The gradient with A/B ratios were as follows: T0, 99:1; T2.5, 99:1; T6, 86.9:13.10; T7, 1:99; T8.5, 1:99; T9, 99:1; T10, 99:1. The flow rate was 150ul/min. Three microliters of sample were injected. Multi reaction monitoring (MRM) was used. A standard curve was recorded and integrated using the mass hunter platform (Agilent). The transitions used for 4-octyl-itaconic acid were 241.14 -&gt; 111 (quantifier, collision energy - 12 V), 241.14 -&gt; 67.1 (qualifier, collision energy - 24 V). The transitions for itaconic acid were the following: 129.02 -&gt; 85.1 (quantifier, collision energy - 8 V), 129.02 -&gt; 41.2 (qualifier, collision energy - 12 V). Retention time for 4-octyl itaconic acid was 8.2 min, and 2.0 min for itaconic acid. Targeted metabolomics data analysis. Transition lists, retention time and raw data were loaded into Skyline (version 23.1) 82. Then, peaks were evaluated and integrated, and intensities were exported. The area under the curve of the quantifiers was used for further analysis. Data were plotted using python v3.9 83 , and the packages matplotlib v3.5.1, numpy v1.22.2 83, pandas v1.4.1, seaborn v0.12.0, statannotations v0.4.4. T-test independent was used for statistical analysis.