Vanquish uhplc system

Ultrahigh-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) was performed on a Vanquish UHPLC system (Thermo Fisher, Germany) coupled with an Orbitrap Q-Exactive^TM HF-X mass spectrometer (Thermo Fisher). The samples were separated on a Hypesil Gold column (100 × 2.1 mm, 1.9 μm) using a 12-min linear gradient at a flow rate of 0.2 mL/min. Eluents A and B for positive-ion mode were 0.1% formic acid in water and methanol, respectively, whereas eluents A and B for negative-ion mode were 5 mM ammonium acetate (pH 9.0) and methanol, respectively. The solvent gradient was as follows: 2% B, 1.5 min; 2%–85% B, 3 min; 85%–100% B, 10 min; 100%–2% B, 10.1 min; and 2% B, 12 min. The spectrometer was operated in positive- and negative-ion mode, respectively, with a spray voltage of 3.5 kV, a capillary temperature of 320°C, a sheath gas flow rate of 35 psi, an auxiliary gas flow rate of 10 L/min, an S-lens radio frequency of 60, and an auxiliary gas temperature of 350°C.

A total of 28 LCu, SCu, LCG, and SCG Wu Ranke sheep liver samples were extracted metabolites by standard procedures [20 (link)]. The sample extracts were analyzed by a Vanquish UHPLC system coupled with an Orbitrap Q ExactiveTM HF mass spectrometer (ThermoFisher, MA, USA). The chromatographic and mass spectrometric conditions are shown in Table S5.
The raw data files generated by UHPLC-MS/MS were processed using the Compound Discoverer 3.1 (CD3.1, ThermoFisher) to perform peak alignment, peak picking, and quantitation for each metabolite. After that, peak intensities were normalized to the total spectral intensity. The normalized data were used to predict the molecular formula based on additive ions, molecular ion peaks and fragment ions. And then peaks were matched with the mzCloud, mzVault and MassList databases to obtain accurate qualitative and relative quantitative results.
Variable importance in projection (VIP) ≥ 1, |log₂ fold change|≥ 1 and p < 0.05 were identified as the significantly accumulated metabolites (DEMs) between LCu and LCG, SCu and SCG. The functions of the DEMs enriched pathways were studied using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.genome.jp/kegg/pathway.html). When the metabolic pathway p value was less than 0.05, the metabolic pathway was considered statistically significant enrichment [18 (link), 21 (link)].

Ultrahigh performance LC-MS/MS analyses were conducted using the Vanquish UHPLC system (Thermo Fisher Scientific, Karlsruhe, Germany) coupled with an Orbitrap Q ExactiveTM HF-X mass spectrometer (Thermo Fisher Scientific, Germany). Each sample was injected into a Hypersil Gold column (100 × 2.1 mm, 1.9 µm) using a 12 min linear gradient at a flow rate of 0.2 mL/min. When running the samples in the positive-polarity mode, the eluent comprised 0.1% formic acid–water (A) and methanol (B). However, in the negative-polarity mode, 5 mM ammonium acetate with a pH of 9.0 (A) and methanol (B) were used as eluents. The Orbitrap Q ExactiveTM HF-X mass spectrometer was operated in the positive/negative mode with a spray voltage of 3.5 kV, capillary temperature of 320 °C, sheath gas flow rate of 35 pounds per square inch, auxiliary gas flow rate of 10 L/min, and auxiliary gas heater temperature of 350 °C [27 (link)]. To intuitively elucidate the relationships between samples and metabolite-abundance differences between the two groups, we performed KEGG pathway enrichment analysis on the differentially abundant metabolites.

As described in our previous study [13 (link)], the untargeted metabolomics was used to determine the metabolomic profiles of all 22 quail serum samples. A mixture of 400 µL precooled methanol and 100 µL quail serum was vortexed. LC-MS/MS analysis was then performed using a Vanquish UHPLC system (Thermo Fisher) coupled to an Orbitrap Q Exactive series mass spectrometer (Thermo Fisher Scientific). Raw data files generated by UHPLC-MS/MS were processed using Compound Finder 3.0 (CD3.0, Thermo Fisher), to determined lignment, peak pickup, and quantification of each metabolite. R (R version R-3.4.3), Python (Python 2.7.6 version), and CentOS (CentOS release 6.6) software was used to perform subsequent statistical analysis.

Metabolite analyses were carried out on a Vanquish UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) coupled in tandem to a Q Exactive™ HF-X mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) at Novogene Co., Ltd. (Beijing, China). Chromatographic separation was performed on a Hypersil Gold column (2.1 × 100 mm, 1.9 μm) at 40 °C. The injection volume was 1 μL, and the flow rate was 0.2 mL/min. Eluent A for positive and negative ionization modes was 0.1% formic acid solution and 5 mol/L ammonium acetate solution, respectively. Eluent B for positive and negative ionization modes was methanol. The solvent gradient was set as follows: 2% B, 1.5 min; 2–100% B, 3 min; 100% B, 10 min; 100–2% B, 10.1 min; and 2% B, 12 min.
Mass spectrometry analysis was performed in positive/negative ionization mode with a spray voltage of 3.5 kV, capillary temperature of 320 °C, sheath gas flow rate of 35 psi, aux gas flow rate of 10 L/min, S-lens RF level of 60, and aux gas heater temperature of 350 °C.

LC/MS was performed to detect 15N-labeled isotopes of metabolites in the de novo pyrimidine synthesis pathway. LC separation was achieved using a Vanquish UHPLC system (Thermo Fisher Scientific) and an Xbridge BEH Amide column (2.1 mm × 150 mm × 2.5 mm particle size, 130 A °pore size; Waters, Milford, MA), column temperature 25°C. Solvent A is 95:5 water:acetonitrile with 20 mM ammonium acetate and 20 mM ammonium hydroxide at pH 9.4, and solvent B is acetonitrile. Flow rate was 150 mL/min. The LC gradient was 0 min, 85% B; 2 min, 85% B; 3 min, 80% B; 5 min, 80% B; 6 min, 75% B; 7 min, 75% B; 8 min, 70% B; 9 min, 70% B; 10 min, 50% B; 12 min, 50% B; 13 min, 25% B; 16 min, 25% B; 18 min, 0% B; 23 min, 0% B; 24 min, 85% B. 25 min, stop run. Injection volume was 5 µL. The Q-Exactive Plus mass spectrometer was operated in negative ion mode scanning from m/z 70-1000 with a resolution at 140,000. Data were analyzed by using El-Maven.

A quadrupole-orbitrap mass spectrometer (Q Exactive, Thermo Fisher Scientific, San Jose, CA) operating in a positive ion mode was coupled to vanquish UHPLC system (ThermoFisher Scientific, San Jose, CA) with electrospray ionization and used to scan from m/z 70 to 1000 at 1 Hz and 75,000 resolution. The LC separation was achieved on a XBridge BEH Amide column (2.1 mm × 150 mm, 2.5 μm particle size, 130 Å pore size; Waters, Milford, MA) using a gradient of solvent A (95:5 water: acetonitrile with 20 mM ammonium acetate and 20 mM ammonium hydroxide, pH 9.45) and solvent B (acetonitrile). Flow rate was 150 μL/min. The LC gradient was: 0 min, 85% B; 2 min, 85% B; 3 min, 80% B; 5 min, 80% B; 6 min, 75% B; 7 min, 75% B; 8 min, 70% B; 9 min, 70% B; 10 min, 50% B; 12 min, 50% B; 13 min, 25% B; 16 min, 25% B; 18 min, 0% B; 23 min, 0% B; 24 min, 85% B; 30 min, 85% B. Autosampler temperature was 5°C, and injection volume was 3 μL. Data were analyzed using the MAVEN software.