Q exactive hf orbitrap mass spectrometer

Ultra high performance liquid chromatography tandem-mass spectrometry (UHPLC-MS/MS) analysis was performed on the Vanquish UHPLC system (Thermo Fisher Scientific, Massachusetts, USA) coupled with an Orbitrap Q Exactive™ HF mass spectrometer (Thermo Fisher Scientific, Massachusetts, USA). In detail, chromatographic separation was conducted in Vanquish UHPLC system equipped with a Hypersil GOLD columns (100 mm × 2.1 mm, 1.9 μm) using a 17-min linear gradient at a flow rate of 0.2 mL/min. The eluents for the positive polarity mode were eluent A (0.1% FA in water) and eluent B (methanol). The eluents for the negative polarity mode were eluent A (5 mM ammonium acetate, pH 9.0) and eluent B (methanol). The solvent linear gradient was set as follows: 2% B, 1.5 min; 2%−100% B, 12.0 min; 100% B, 14.0 min; 100%−2% B, 14.1 min; 2% B, 17 min. The ESI-MSn experiments were executed on a Q ExactiveTM HF mass spectrometer in positive/negative polarity mode with spray voltage of 3.2 kV. Sheath gas and auxiliary gas flow e were set at 40 and 10 arbitrary units, respectively, and the capillary temperature was 320°C. The analyzer scanned over a mass range of 100 m/z–15,000 m/z for full scan at a mass resolution of 70,000. Finally, data-dependent acquisition MS/MS experiments were performed.

In Novogene Co., Ltd. (Beijing, China), UHPLC–MS/MS analyzes were carried out utilizing a Vanquish UHPLC system (Thermo Fisher, Germany) paired with an Orbitrap Q Exactive™HF mass spectrometer (Thermo Fisher, Germany). A 17-min linear gradient was used to inject samples onto a HypesilGoldcolumn (100 × 2.1 mm, 1.9 ) at a flow rate of 0.2 mL/min. Eluents A (0.1% formic acid (FA) in water) and B (methanol) were used in the positive polarity mode. Eluents A (5 mM ammonium acetate, pH 9.0) and B (Methanol) were used in the negative polarity mode. The following settings were made for the solvent gradient: 2% B, 1.5 min; 2–85% B, 3 min; 85–100% B, 10 min; 100–2% B, 10.1 min; 2% B, 12 min. 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, the QExactive^™HF mass spectrometer was operated in positive/negative polarity mode.

LC-MS/SM analysis of digested peptides was performed on an Orbitrap Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Bremen) coupled to an EASY-nLC 1200 (Thermo Fisher Scientific). Peptides were loaded and separated at 250 nl/min on a home-made C18 50 cm capillary column picotip silica emitter tip (75 μm diameter filled with 1.9 μm Reprosil-Pur Basic C18-HD resin, (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany)) equilibrated in solvent A (2% ACN, 0.1% FA). Peptides were eluted using a gradient of solvent B (80% ACN, 0.1% FA) from 3% to 6% in 5 min, 6% to 29% in 130 min, 29% to 56% in 26 min, 56% to 90% in 5 min (total length of the chromatographic run was 180 min including high ACN level steps and column regeneration). Mass spectra were acquired in data-independent acquisition mode with the XCalibur 4.1.31.9 software (Thermo Fisher Scientific, Bremen).
Each cycle was built up as follows: one full MS scan at resolution 30 000 (scan range between 400 and 1200 m/z), AGC was set at 3*10⁶, ion trap was set at 50 ms. All MS1 was followed by 40 isolation windows of 20 m/z, covering the MS1 range from 400 m/z to 1200 m/z. The AGC target was 2*10⁵, and NCE was set to 27. All acquisitions were done in positive and profile mode.

The cecal samples were prepared and the supernatant was injected into a liquid-mass spectrometry (LC–MS/MS) system for analysis (Want et al., 2012 (link); Yuan et al., 2012 (link)). Furthermore, the ultra-high-performance liquid Chromatography (UHPLC)-MS/MS analyses were performed using a Vanquish UHPLC system (Thermo Fisher, Germany) coupled with an Orbitrap Q Exactive™ HF mass spectrometer (Thermo Fisher, Germany) at Novogene Co. Ltd. (Beijing, China). The cecal samples were then injected onto a Hypersil Gold column (100 × 2.1 mm, 1.9 μm) using a 17 min linear gradient at a flow rate of 0.2 ml/min. The eluents for the positive polarity mode were eluent A (0.1% FA in Water) and eluent B (Methanol). The eluents for the negative polarity mode were eluent A (5 mM ammonium acetate, pH 9.0) and eluent B (Methanol), and the solvent gradient was set as follows: 2% B, 1.5 min; 2–100% B, 12.0 min; 100% B, 14.0 min; 100–2% B, 14.1 min; and 2% B, 17 min. Then, the Q ExactiveTM HF mass spectrometer was operated in positive/negative polarity mode with spray voltage of 3.2 kV, capillary temperature of 320°C, sheath gas flow rate of 40 arb, and aux gasflow rate of 10 arb.
The raw data files generated by UHPLC–MS/MS were processed using Compound Discoverer 3.1 (CD3.1, Thermo Fisher) to perform peak alignment, peak picking, and quantitation for each metabolite.

All DESI-MS analyses were performed using a laboratory-built DESI sprayer fitted to a Thermo Orbitrap Q-Exactive HF mass spectrometer at a resolving power of 60,000 in the negative ion mode in the mass range m/z 100 to 1500. Tissue sections were thawed and dried in a chemical fume hood 10 min prior to DESI-MS analysis. DESI-MS imaging of tissue sections was performed at a spatial resolution of 150 μm using a spray flow rate of 1.5 μl min⁻¹ with a histologically compatible solvent system, acetonitrile:dimethylformamide 3:1 (v/v). The pressure of the nebulizing N₂ gas was set to 180 psi. DESI-MS ion images were constructed using FireFly (Thermo Scientific) and BioMAP (Novartis) software. Ion identifications were made with high mass accuracy measurements (≤5 ppm mass error) and/or tandem MS experiments (Figs. S4 and S9) using high-energy collision dissociation. METASPACE and LIPID MAPS databases were used to aid in molecular identification.

All analyses were performed on a Vanquish UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Exactive HF mass spectrometer (Thermo Fisher). The raw data generated by UHPLC-MS/MS were processed using the Compound Discoverer (Thermo Fisher) to perform peak alignment, peak picking, and quantitation for each metabolite. After that, peak intensities were normalized to the total spectral intensity, and the normalized data was further used to predict the molecular formula based on additive ions, molecular ion peaks, and fragment ions. Then, peaks were matched with the Lipidmaps and Lipidblast database to obtain accurate qualitative and relative quantitative results. We applied univariate analysis (t test) to calculate the statistical significance (P value). The metabolites with VIP > 1 and P value < 0.05 and fold change ≥ 2 or ≤ 0.5 were considered to be differential metabolites between LT and LW.

All the hub proteins determined above and the proteins of the top 25 combinations with the smallest root mean squared error (RMSE) values and AUC value of 1 were validated by PRM in independent samples.
First, the proteins were extracted, digested and mixed samples were prepared, and the full spectrum was scanned by the “label-free” method using the EASY-nLC1200 connected to the Orbitrap Q-Exactive HF mass spectrometer (Thermo, Scientific, USA). Second, the Proteome Discoverer 2.2 software was used to search the library. The search results were imported into Skyline(version 20.1.0.155) software [33 ] to obtain the target protein peptide information. Then, the PRM method can be established, and the obtained data were imported into Skyline software for quantification. The parameters of PRM were set as follows: the primary resolution was 12,000 (at 300–1400 m/z) with an automatic gain control (AGC) target value of 3e6, a maximum injection time of 80 ms, and a Normalized Collision Energy (NCE) of 27%; the secondary resolution was 15,000 with an AGC target value of 2e4, a maximum injection time of 19 ms. The mass tolerances for precursor and fragment ions were also set at 10 ppm and 0.02 Da, respectively.
The ROC curve analyses of the validated combinations were examined for PDR, and the AUC of each ROC curve was calculated.