Q exactive hf x mass spectrometer

For transition library construction, shotgun proteomics analyses were performed using an EASY-nLCTM1200 UHPLC system (Thermo Fisher, Rockford, IL, USA) coupled with an Q Exactive HF (X) mass spectrometer (Thermo Fisher, Rockford, IL, USA) operating in the data-dependent acquisition (DDA) mode. The sample was injected into a homemade C18 Nano-Trap column (2 cm × 100 μm, 3 μm).
Peptides were separated in a homemade analytical column (12 cm × 150 μm, 1.9 μm). The separated peptides were analyzed by Q Exactive HF (X) mass spectrometer (Thermo Fisher, Rockford, IL, USA), with ion source of Nanospray Flex™ (ESI), spray voltage of 2.5 kV and ion transport capillary temperature of 320 °C. The full scan range was from m/z 407 to 1500 with resolution of 60,000 (at m/z 200), the automatic gain control (AGC) target value was 3 × 10⁶ and the maximum ion injection time was 20 ms. The top 40 precursors of the highest abundance in the full scan were selected and fragmented by higher energy collisional dissociation (HCD) and analyzed in MS/MS [20 (link)], where the resolution was 15,000 (at m/z 200), the automatic gain control (AGC) target value was 5 × 10⁴, the maximum ion injection time was 45 ms, the normalized collision energy was set as 32%, the intensity threshold was 2.2 × 10⁴ and the dynamic exclusion parameter was 20 s.

The total proteins were extracted from WT, RNAi line 7 and OE line 27. The concentration of the total protein was determined by a Bradford protein quantitative kit, and the quality of the proteins was assessed by SDS-PAGE. Then, 3 μL 1 μg/μL trypsin (Promega, Madison, WI, USA) and a 500 μL 50 mM TEAB buffer (Triethyl ammonium bicarbonate) were used to digest the proteins of each sample overnight at 37 °C, and the products purified by the C18 desalination column were labeled with TMT. Next, we used an L-3000 HPLC system to gradient elute the solution containing protein powder, and 10 fractions were obtained. The chromatographic column was Waters BEH C18 (4.6 × 250 mm, 5 μm), and the column temperature was set at 50 °C. For transition library construction, shotgun proteomics analyses were performed using an EASY-nLCTM 1200 UHPLC system (Thermo Fisher, Waltham, MA, USA) coupled with an Q Exactive HF-X mass spectrometer (Thermo Fisher) operating in the data-dependent acquisition (DDA) mode. A 1-μg sample was injected into a home-made C18 Nano-Trap column (2 cm × 75 μm, 3 μm). Peptides were separated in a home-made analytical column (15 cm × 150 μm, 1.9 μm), using a linear gradient elution. The separated peptides were analyzed by a Q Exactive HF-X mass spectrometer (Thermo Fisher). The resulting MS/MS data were processed using Proteome Discoverer 2.2 (PD 2.2, Thermo).

LC-MS/MS analysis was performed by Biotree biotech Co., Ltd. (Shanghai, China) using high-performance liquid chromatography with a diode array detection system (Thermo Fisher Scientific). A UPLC BEH Amide column (2.1 × 100 mm, 1.7 μm) coupled to a Q Exactive HFX mass spectrometer (Thermo) was used for separation. The mobile phase consisted of 25 mmol/L ammonium acetate and 25 mmol/L aqueous ammonia hydroxide solution (pH 9.75) and acetonitrile. The working temperature of the automatic sampler was 4 °C, and the sample injection volume was 3 μL. A Q Exactive HFX mass spectrometer was used because of its ability to collect tandem mass spectrometry (MS/MS) spectra in information correlation acquisition mode under the control of the acquisition software (Xcalibur, Thermo). In this mode, the acquisition software continuously evaluates the full-scan MS spectrum. The electrospray ionization source conditions were 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 7500, collision energy 10/30/60 in NCE mode, and spray voltage 3.6 kV (positive) or − 3.2 kV (negative), respectively.