Dionex u3000 uhplc high resolution mass spectrometer

We accurately weighed 15 mg of the tissue samples into 1.5-ml of the EP tube and added the inner standard (FMOC-L-2-Chlorophe, 0.3 mg/ml; Lyso PC17: 0, 0.01 mg/ml, all configured with methanol) of 20 μl and added 400 μl of methanol–water (v/v = 4:1). After grinding, centrifugation, supernatant absorption, filtration, and transfer to the LC sample vial, the solution was stored at −80°C until LC-MS analysis.
For data processing, the metabolic profiling in positive and negative electrospray ionization (ESI) modes was analyzed by using the liquid-mass spectrometry system consisting of the Dionex U3000 UHPLC High-Resolution Mass Spectrometer and the QE plus (Thermo Fisher Scientific, Waltham, MA, USA). The determination was performed on the ACQUITY UPLC HSS T3 (100 × 2.1 mm, 1.8 μm) with a mobile phase consisting of A-water (containing 0.1% formic acid, v/v) and B-acetonitrile (containing 0.1% formic acid, v/v). The flowrate was set to 0.35 ml/min, and the column temperature was 45°C. The injection volume was 2 µl. Data acquisition was performed in the full-scan mode (m/z ranges from 70 to 1,000) combined with the IDA mode.

For metabolomics, N = 8 biological replicates were sequenced in MDFs grown at 2 kPa and N = 6 biological replicates were sequenced in MDFs grown at 50 kPa. The culture medium was completely removed and the cells were immediately placed on liquid nitrogen. Cells were scraped and lysed with ice-cold 80% methanol in water (pre-cooled in a −80 °C freezer). All samples were transferred to tubes and centrifuged at 20,000 × g for 10 min at 4 °C. The supernatant was collected and dried. Metabolites were rehydrated in 100 µl of 0.03% formic acid in liquid chromatography-mass spectrometry (LC-MS)-grade water, vortexed to remove debris, and centrifuged. The supernatant was transferred to a high-performance liquid chromatography (HPLC) vial and metabolite profiling was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS/MS was performed using a liquid mass spectrometry system consisting of a Dionex U3000 UHPLC high-resolution mass spectrometer and QE plus (Thermo Fisher Scientific). Progenesis Qi V2.3 software (Nonlinear Dynamics, Newcastle, UK) was used to process raw metabolic data after acquisition by Unifi 1.8.1. Differential metabolites were selected according to VIP values and p-values obtained from two-tailed Student’s t-test of normalised peak areas. Metabolites with VIP values > 1.0 and p < 0.05 were considered to be differential metabolites.