Qe plus

The alterations that occurred to the mouse physiology after exposure were investigated using metabolic techniques on the basis of high-resolution mass spectroscopy (QE plus, Thermo Fisher, Waltham, MA, USA), and the samples involved the serum, feces, and liver. The involved methods are described in Supplementary File S1 (Methods for Metabolic Profiling Analysis).

To confirm FAHFA species as endogenous metabolites rather than artifacts of metabolite extraction or data acquisition, we added 50 μM hydroxy-palmitic acid (HPA). HPA was first conjugated to FA-free BSA to mimic serum conditions as previously described (28 (link), 29 (link)). Briefly, Krebs-Henseleit bicarbonate solution (118 mM NaCl, 25 mM NAHCO₃, 4.7 mM KCl, 1.2 mM KH₂PO₄, 2.5 mM CaCl₂, 1.2 mM MgSO₄, pH 7.4) was heated to 40–44°C. FA-free BSA was dissolved in Krebs for final concentration of 0.13 mM. About 20 mM HPA was dissolved in ethanol and water. Dissolved HPA was slowly added to BSA and then dialyzed against Krebs-Henseleit solution with a 7 kDa cassette overnight. HPA-BSA was spiked into human serum and extracted for untargeted metabolomics, and data were acquired on HILIC-ESI(−)-MS/MS as described previously.
Direct infusion experiment was performed using MS parameters described previously. About 50 μM [U-¹³C₁₈] 18:1 and 50 μM HPA in AcN:H₂O (1:1) were infused into Thermo QE Plus at a flow rate of 5 μl/min. FS data were acquired in a mass window of m/z 200–700 for 5 min. Dimer m/z were predicted based on chemical formula and targeted for MS/MS (±0.5 m/z) at normalized collision energy = 35, including m/z = 543.5, 553.5, 571.5, and 599.6. Fragmentation data were acquired for 3 min.

Metabolomics of collected fecal samples was performed by LC-MS/MS. Taken 200 mg of lyophilized feces and added to 4 mL of ultrapure water, then vortexed well for 30 s. Added 1 mL of ice-cold MeOH and H₂O (80:20) mixture, vortexed for 1 min, centrifuged the mixture at 13,000 g for 10 min. The supernatant was collected and passed through a polyamide filter (25 mm, pore size 0.45 μm), diluted with water (1:3) and transferred to a glass vial for LC-MS/MS detection. Pooled 10 µL aliquots from each sample to create a composite quality control sample for metabolomic analysis.
An ultra-high-performance liquid chromatography system (Ultimate 3000, Thermo Fisher Scientific, USA) was employed, and the analysis was carried out with a high-resolution tandem electrostatic field Orbitrap mass spectrometer (QE Plus, Thermo Fisher Scientific). The Compound Discoverer 3.2 software facilitated the chromatographic peak identification, alignment, and normalization processes, yielding files that included m/z values, retention times (Rt), and peak areas for subsequent analysis. PCA of the collected data, differential metabolite screening and pathway enrichment were done by metaboanalyst 6.0. Differential metabolites between groups were screened using VIP > 1.0 and p < 0.05 as threshold.

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.

Each sample was collected by electrospray ionization (ESI) in positive (+) and negative (−) modes. The samples were separated by UPLC and analyzed by mass spectrometry using a QE Plus mass spectrometer (Thermo Scientific, Waltham, MA, USA). The ionization conditions were as follows: spray voltage: 3.8 kv (+) and 3.2 kv (−); capillary temperature: 320 (±); spray voltage: 3.8 kv (+) and 3.2 kv (−); capillary temperature: 320 (±); sheath gas: 30 (±); aux gas: 5 (±); probe heater temp: 350 (±); S-lens RF level: 50.

An ACQUITY UPLC I-Class plus (Waters Corporation, Milford, CT, USA) coupled with a QE plus (Thermo Fisher Scientific, Shanghai, China) high-resolution tandem mass spectrometer was used to analyze the metabolic profiling in both ESI positive and ESI negative ion modes. An ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 × 100 mm) was employed in both positive and negative modes. The binary gradient elution system consisted of (A) water (containing 0.1% formic acid, v/v) and (B) acetonitrile (containing 0.1% formic acid, v/v) and separation was achieved using the following gradients: 0–2 min, 95% A, 5% B; 2–14 min, 95% to 0% A, 5% to 100% B; 14 min, 100% B; 15 min, 100% B; and 15.1–16 min, 95% A, 5% B. Formic acid and acetonitrile were provided by Thermo Fisher Scientific. The flow rate was 0.35 mL/min and the column temperature was 45 °C. All the samples were kept at 10 °C during the analysis. The injection volume was 3 μL.
The mass range was from m/z 100 to 1000. The resolution was set at 70,000 for the full MS scans and 17,500 for HCD MS/MS scans. The Collision energy was set at 10, 20 and 40 eV. The mass spectrometer operated as follows: spray voltage, 3800 V (+) and 3000 V (−); sheath gas flow rate, 35 arbitrary units; auxiliary gas flow rate, 8 arbitrary units; capillary temperature, 320 °C; aux gas heater temperature, 350 °C; s-lens RF level, 50.

Reversed-phase microcapillary/tandem mass spectrometry (LC/MS/MS) was performed using an Easy-nLC nanoflow HPLC (Proxeon Biosciences) with a self-packed 75 μm × 15 cm C18 column connected to a QE-Plus (Thermo Scientific) in data-dependent acquisition and positive ion mode at 300 nL/min. Passing MS/MS spectra were manually inspected to ensure that all b-and y-fragment ions aligned with the assigned sequence and modification sites. A 25 ul reaction mixture contained 2 µM SETD3 or SETD3 mutants (final concentration) and 20 µM peptide (final concentration) in a buffer containing 10 mM Tris-HCl, (pH 7.5), 20 mM NaCl and 10 µM AdoMet. The reaction was incubated at 37°C for 2 hr before being quenched (at 70°C for 10–15 mins). Then, reactions were analyzed by LC/MS/MS and Proteomics Browser software, with the relative abundances of substrate and product reflecting the methylation activities of proteins.