Metabolomics data for crude protein content in diets for Huangjiang mini-pigs

The metabolite contents in the jejunum and ileum of Huanjiang mini-pigs were determined using a non-targeted metabolomics approach with the UPLC-HDMS. The metabolomics procedures included sample preparation, metabolite separation and detection, data preprocessing, and statistical analysis.For metabolite identification, approximately 25 mg of each sample was weighed into a 2-mL EP tube and then added 500 mL extract solution (acetonitrile: methanol: water = 2:2:1 (v/v), with the isotopically-labeled internal standard mixture) to the EP tube. After 30 s of vortexing, the mixed samples were homogenized at 35 Hz for 4 min and sonicated in an ice-water bath for 5 min. The homogenization and sonication cycles were repeated three times. Then the samples were incubated for 1 h at -40 °C and centrifuged at 12,000 ´ g for 15 min at 4 °C. The resulting supernatants were filtered through a 0.22-µm membrane and transferred to fresh glass vials for further analysis. The quality control (QC) sample was obtained by mixing an equal aliquot of the supernatants from all samples.An ultra-performance liquid chromatography (UPLC) system (Vanquish, Thermo Fisher Scientific, Waltham, MA, USA) with a UPLC BEH Amide column (2.10 × 100 mm, 1.70 mm) coupled with Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo Fisher Scientific, Waltham, MA, USA) was used to perform LC-MS/MS analyses. The mobile phase A contained 25 mmol/L ammonium acetate and 25 mmol/L ammonia hydroxide in water, and the mobile phase B contained acetonitrile. The injection volume was 3 mL, and the temperature of the auto-sampler was set at 4 °C. To acquire MS/MS spectra on an information-dependent acquisition (IDA) mode, the QE HFX mass spectrometer was used for its ability in the control of the acquisition software (Xcalibur, Thermo Fisher Scientific, Waltham, MA, USA). In this mode, the acquisition software continuously evaluated the full scan of the MS spectrum. The conditions for ESI source were set as follows: sheath gas flow rate 30 Arb, Aux gas flow rate 25 Arb, capillary temperature 350 °C, full MS resolution 60,000, MS/MS resolution a7500, collision energy 10/30/60 in NCE mode, and spray voltage 3.60 kV (positive ion mode) or -3.20 kV (negative ion mode), respectively.For peak detection, extraction, alignment, and integration, obtained raw data were converted into mzXML format by ProteoWizard and then processed with an in-house program, which was developed using R and based on XCMS. The metabolites were annotated using an in-house MS2 (secondary mass spectrometry) database (BiotreeDB v2.1). The value of the cutoff was 0.3. The PCA and orthogonal partial least squares discriminant analysis (OPLS-DA) were established by the SIMCA software v.16.0.2 (Sartorious Stedim Data Analytics AB, Umea, Sweden) to visualize the distinction and detect differential metabolites among different CP content groups. Moreover, the Kyoto Encyclopedia of Genes and Genomes (KEGG) and MetaboAnalyst 5.0 were used for pathway analysis.