Q exactive hf ms

The metabolite extract (cf. Supplement) from each biological replicate was analyzed to conduct a non-targeted metabolomics study using an ACQUITY UPLC system (Waters, USA) coupled with Q-Exactive HF MS (Thermo Fisher Scientific, USA). Details of the metabolomics analysis including sample preparation, equipment, and methods are provided in the Supporting Information.
QC samples were obtained from pooled metabolic extracts and prepared as real samples^{30 (link)}. These QC samples were analyzed every 10 injections during the entire run to monitor the robustness of the analysis.

The Q Exactive‐HF MS (Thermo Fisher Scientific, San Jose, CA, USA) was utilized for full scan MS data acquisition in the positive ion mode. The calibration solution was used to calibrate the mass axis before measurements to ensure high mass accuracy. The mass spectra were acquired across the mass range of 70–1050 with a resolution for MS1 of 60 000 at m/z 200. The MS was operated with a capillary temperature of 275 °C. The S‐Lens RF level was set to 50 and one microscan was applied. The automatic gain control (AGC) target of 1e5 and maximum injection time (IT) of 100 ms were applied for full MS scan.

The analysis was carried out using a Thermo Scientific Vanquish LC coupled to Thermo Q Exactive HF MS. An electrospray ionization interface was used as ionization source. Analysis was performed in negative and positive ionization mode. The UPLC was performed using a slightly modified version as previously described [30 ]. Peak areas were extracted using Compound Discoverer 3.1 (Thermo Fisher Scientific). Identification of compounds were performed at four levels; Level 1: identification by retention times (compared against in-house authentic standards), accurate mass (with an accepted deviation of 3 ppm), and MS/MS spectra, Level 2a: identification by retention times (compared against in-house authentic standards), accurate mass (with an accepted deviation of 3 ppm). Level 2b: identification by accurate mass (with an accepted deviation of 3 ppm), and MS/MS spectra, Level 3: identification by accurate mass alone (with an accepted deviation of 3 ppm).

Metabolomics and lipidomics were performed on an Ultimate 3000 UHPLC system coupled with a Q-Exactive HF MS (Thermo Scientific, Waltham, MA). For the aqueous phase (metabolomics), an Xbridge amide column (100 × 2.1 mm i.d., 3.5 μm; Waters Corporation, Milford, MA) was employed for compound separation at 30°C. Samples were suspended in 100 μL of acetonitrile:water (1:1, v/v), and the injection volume was 10 μL. Mobile phase A consisted of 5 mM ammonium acetate in water with 5% acetonitrile, and mobile phase B was acetonitrile. The flow rate was 0.4 mL/min with the following linear gradient: 0 min, 95% B; 3 min, 90% B; 13 min, 50% B, 14 min, 50% B; 15 min, 95% B; and 17 min, 95% B.
As for lipids, chromatographic separation was performed on a reverse-phase X-select CSH C18 column (2.1 × 100 mm, 2.5 μm; Waters) at 40°C. Two solvents, both containing 10 mM ammonium acetate and 0.1% formic acid, were used for gradient elution: (A) ACN/water (3:2, v/v) and (B) IPA/ACN (9:1, v/v). The gradient program was: 0 min, 40% B; 2 min, 43% B; 12 min, 60% B; 12.1 min, 75% B; 18 min, 99% B; 19 min, 99% B; and 20 min, 40% B. The flow rate was set to 0.4 mL/min. Samples were suspended with 100 μL of chloroform:methanol (1:1, v/v) and diluted threefold with isopropanol:acetonitrile:H₂O (2:1:1, v/v/v). The injection volume was 10 μL.