HK2 Metabolomics
Description
HK2 cells were incubated with interferon gamma or vehicle for 24 hours. HK2 cells were washed twice with ice-cold PBS, drained, snapped-frozen in liquid nitrogen and stored at -80°C until analyses. After addition of an extraction solution made of 50% methanol, 30% acetonitrile, and 20% water 33 (1 mL/1.106 cells or 500 l for 20 l urine), the samples were vortexed for 5 min at 4°C, and then centrifuged at 16,000 g for 15 min at 4°C. The supernatants were collected and separated by liquid chromatography–mass spectrometry using SeQuant ZIC-pHilic column (Millipore). The aqueous mobile-phase solvent was 20 mM ammonium carbonate plus 0.1% ammonium hydroxide solution and the organic mobile phase was acetonitrile. The metabolites were separated over a linear gradient from 80% organic to 80% aqueous for 15 min. The column temperature was 50°C and the flow rate was 200 μl/min. The metabolites were detected across a mass range of 75-1,000 m/z using the Q-Exactive Plus mass spectrometer at a resolution of 35,000 (at 200 m/z) with electrospray ionization and polarity switching mode. Lock masses were used to insure mass accuracy below 5 ppm. The peak areas of different metabolites were determined using Thermo TraceFinder software using the exact mass of the singly charged ion and known retention time on the HPLC column. Our metabolomics analyses are focused on small polar compounds in central carbon metabolism. We applied an established and largely referenced method for sample extraction and LC-MS analyses using pHILIC HPLC column for polar metabolites separation 33. Notably, we used the same extraction solution for cells and urine samples. As a part of the routine analytical pipeline, we apply the recommendations of the metabolomics Quality Assurance and quality Control Consortium (mQACC). The routine quality controls include regular equipment maintenance (Thermo), the use of standard operating procedures for sample extraction, storage and analyses. General practices also include weekly test runs to assure system stability and quality of runs. Regarding the QCs in relation to this study, we used (1) pooled interstudy QC, (2) process and extraction blanks, (3) system stability blanks, (4) solvents blanks, (5) long-term reference standard inter-laboratory QC mix to ensure system stability and (6) the samples were blinded and loaded in randomized order. The analyses of pooled samples QC showed no significant difference in metabolites levels between QCs.