Q exactive hfx mass spectrometer orbitrap ms

The detailed protocol for untargeted metabolomics analysis was described in our previous report [20 (link)]. Briefly, a mixture of 25 mg of heart tissue and 500 μL of extract solution (acetonitrile–methanol–water (2:2:1) with isotopically-labeled internal standard mixture) was homogenized and sonicated for 3 cycles and then kept at −40 °C for 1 h. After centrifugation at 12,000 rpm for 15 min at 4 °C, the supernatant was transferred to a fresh glass vial for subsequent LC-MS/MS analyses, which were performed on a UHPLC system (Vanquish, Thermo Fisher Scientific, Cambridge, MA) coupled to a Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo Fisher Scientific, Cambridge, MA, USA).

Metabolites were extracted with 500 μL of extraction solution (methanol: acetonitrile: water = 2:2:1 (V/V), containing isotope-labeled internal standard mixture), and the supernatant was used for LC-MS/MS analysis. LC-MS/MS analyses were conducted using a UHPLC system (Vanquish, Thermo Fisher Scientific, Waltham, MA, USA) with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm) connected to a Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo Fisher). The mobile phase consisted of 25 mmol/L ammonium acetate and 25 ammonia hydroxides in water, with a pH of 9.75. The autosampler was set at a temperature of 4 °C, and the injection volume was 2 μL. For the acquisition of MS/MS spectra, the QE HFX mass spectrometer was used in information-dependent acquisition mode, controlled by the Xcalibur software from Thermo Fisher. In this mode, the software consistently assesses the full-scan MS spectrum. The raw data were converted to the mzXML format using ProteoWizard. Subsequently, an in-house program, developed with R and based on XCMS, was used for peak detection, extraction, alignment, and integration. Metabolite annotation was performed using an in-house MS2 database called BiotreeDB. The annotation cutoff was set at 0.3.

Briefly, 25 mg of cecal content was added to an EP tube with 500 μL of extract solution. Firstly, the sample was ground for 4 min at 35 Hz and sonicated for 5 min in an ice water bath. This step was repeated three times. Next, we allowed it to stand at −40 °C for an hour, then centrifuged it at 4 °C for 15 min at 12,000 rpm. Finally, we transferred the resulting supernatant into a vial for assaying. The UHPLC procedure was carried out utilizing the Vanquish instrument from Thermo Fisher Scientific. This instrument has the capability to detect a relatively wide range of substances. This system was coupled with a Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo Fisher Scientific, Waltham, MA, USA). Qualitative and quantitative analyses of peaks were carried out using XCMS, self-designed R packages, and proprietary secondary mass spectrometry databases.

In total, 1g of rumen from each sample was transferred to an EP tube. After the addition of 400 µL of extract solution (acetonitrile: methanol = 1: 1, containing isotopically-labelled internal standard mixture), the samples were vortexed for 30 s, sonicated for 10 min in ice-water bath, and incubated for 1 h to precipitate proteins. Then the sample was centrifuged at 12,000r/min for 15 min. The supernatant was transferred to a fresh glass vial for analysis. The quality control (QC) sample was prepared by mixing an equal aliquot of the supernatants from all of the samples (19 (link)).
LC-MS/MS analyses were performed using an UHPLC system (Vanquish, Thermo Fisher Scientific) with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm) coupled to Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo). The mobile phase consisted of 25 mmol/L ammonium acetate and 25 ammonia hydroxides in water (pH = 9.75) (A) and acetonitrile (B). The auto-sampler temperature was 4°C, and the injection volume was 3 μL (20 (link)). The raw data were converted to the mzXML format using ProteoWizard and processed with an in-house program, which was developed using R and based on XCMS, for peak detection, extraction, alignment, and integration. Then an in-house MS2 database (BiotreeDB) was applied in metabolite annotation. The cutoff for annotation was set at 0.3 (21 (link)).

Liquid chromatography–mass spectrometry (LC-MS) were applied to identify the differentiated metabolites in fibrotic livers of monkeys. LC-MS analyses were performed using an UHPLC system (Vanquish, Thermo Fisher Scientific) with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm) coupled to Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo) by Biotree company (Shanghai, China). All data obtained by metabolomics profiling were analyzed using MetaboAnalyst v.4.0, and pathway mapping was performed on the basis of annotated metabolic pathways in KEGG. A univariate statistical analysis was used to identify significant differences in the abundances of metabolites between fibrotic and HC groups.

We used a UHPLC system (Vanquish; Thermo Fisher Scientific, Waltham, MA, USA) with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm) combined with Q Exactive HFX mass spectrometer (Orbitrap MS; Thermo, Waltham, MA, USA) to perform LC-MS/MS analysis. The mobile phase A was formed with 25 mmol/L ammonium acetate and 25 mmol/L ammonia hydroxide in water (pH = 9.75), and the mobile phase B was acetonitrile. The temperature of the auto-sampler was 4 °C, and the injection volume was 3 μL. We acquired MS/MS spectra using the QE HFX mass spectrometer on the information-dependent acquisition (IDA) mode under the control of the acquisition software (Xcalibur; Thermo, Waltham, MA, USA). The detailed steps and parameter settings of LC-MS/MS analysis and data preprocessing and annotation were described in the Supplemental Materials.