Q exactive hf x mass spectrometer

Samples were analysed on a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific, Rockford, IL, USA) coupled with a high-performance liquid chromatograph (EASY-nLC 1200 System, Thermo Fisher). Dried peptide samples were dissolved in solvent A (0.1% formic acid in water) and loaded onto a trap column (100 μm × 2 cm, home-made; particle size, 3 μm; pore size, 120 Å; SunChrom) with a maximum pressure of 280 bar using solvent A, and were then separated on a home-made 150 μm × 12 cm silica microcolumn (particle size, 1.9 μm; pore size, 120 Å; SunChrom) with a gradient of 5–35% mobile phase B (acetonitrile and 0.1% formic acid) at a flow rate of 600 nL/min for 75 min. MS analysis was conducted with one full scan (300–1400 m/z, R = 120,000 at 200 m/z) at an automatic gain control (AGC) target of 3e6 ions, followed by up to 20 data-dependent MS/MS scans with higher-energy collision dissociation (target 5e4 ions, max injection time 20 ms, isolation window 1.6 m/z, normalized collision energy of 27%). Detection was performed using Orbitrap (Q Exactive HF-X mass spectrometer, Thermo Fisher Scientific) and data were acquired using Xcalibur software (Thermo Fischer Scientific).

Shotgun proteomic analyses were performed using an EASY-nLC™ 1200 UHPLC system (Thermo Fisher, Waltham, MA, USA) coupled with a Q Exactive™ HF-X mass spectrometer (Thermo Fisher, Waltham, MA, USA) at Novogene Genetics, Beijing, China. Specifically, 1 µg sample was injected into a C18 Nano-Trap column (4.5 cm × 75 µm, 3 µm). Peptides were separated in an analytical column (15 cm × 150 µm, 1.9 µm) using a linear gradient elution. The separated peptides were analyzed using the Q Exactive™ HF-X mass spectrometer (Thermo Fisher, Waltham, MA, USA) combined with Nanospray Flex™ (electrospray ion source) (Thermo Fisher, Waltham, MA, USA), with a spray voltage of 2.3 kV and an ion transport capillary temperature of 320 °C. The full scan range was 350 to 1500 (m/z) with a resolution of 60,000 (at m/z 200). The automatic gain control target value was 3 × 10⁶, and the maximum ion injection time was 20 ms. The 40 most abundant precursors in the full scan were selected and fragmented by higher energy collisional dissociation for the MS/MS analysis with a 10-plex resolution of 45,000 (at m/z 200). The automatic gain control target value was 5 × 10⁴, and the maximum ion injection time was 86 ms. The normalized collision energy was set at 32%; the intensity threshold was 1.2 × 10⁵, and the dynamic exclusion parameter was 20 s.

Samples were randomized prior to the mass spectrometry (MS) procedures. For transition library construction, shotgun proteomics analyses were performed using a Q Exactive HF-X mass spectrometer (Thermo Fisher) operating in the data-dependent acquisition (DDA) mode. A sample volume containing 1 μg of total peptides from the fraction sample reconstituted in 0.1% FA was injected onto a home-made C18 Nano-Trap column (2 cm × 100 μm, 3 μm). Peptides were separated on an analytical column (25 cm × 75 μm, 100 Å) using an 80-min linear gradient from 0% to 100% of eluent B at a flow rate of 600 nL/min. There was a single full-scan mass spectrum in the Orbitrap (350–1,500 m/z, 120,000 resolution) followed by data-dependent MS/MS scans in an ion-routing multipole at 27% normalized collision energy (NCE). The single-sample was reconstituted in 0.1% FA and injected onto U3000 UHPLC system (Thermo Fisher) coupled with a Q Exactive HF-X mass spectrometer (Thermo Fisher) operating in the PRM mode. The liquid conditions were the same as above. Parameters were set as follows: MS1 and MS2 resolution 60,000, scan range 150–2,000 m/z, and an NCE of 28%.

For transition library construction, shotgun proteomics analyses were performed using an EASY-nLCTM 1200 UHPLC system (Thermo Fisher, Waltham, MA, USA) coupled with a Q Exactive HF-X mass spectrometer (Thermo Fisher, Waltham, MA, USA) operating in the data-dependent acquisition (DDA) mode. A total of 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 as listed in Table S2. The separated peptides were analyzed by Q Exactive HF-X mass spectrometer (Thermo Fisher, Waltham, MA, USA), with ion source of Nanospray Flex™ (ESI), spray voltage of 2.3 kV and ion transport capillary temperature of 320 °C. Full scan ranges from m/z 350 to 1500 with resolution of 60,000 (at m/z 200), an automatic gain control (AGC) target value was 3 × 106 and a maximum ion injection time was 20 ms. The top 40 precursors of the highest abundant in the full scan were selected and fragmented by higher energy collisional dissociation (HCD) and analyzed in MS/MS, where resolution was 45,000 (at m/z 200) for 10 plex, the AGC target value was 5 × 104 the maximum ion injection time was 86 ms, a normalized collision energy was set as 32%, an intensity threshold was 1.2 × 105 and the dynamic exclusion parameter was 20 s.

SDS/PAGE was used to identify the integrity of the proteins prior to follow‐up experiments. A total of 20 μg of the protein sample was separated by 12% SDS/PAGE and stained. The proteins were collected according to the following protocol: digestion with trypsin; desalting by gel filtration; thrice‐washing with PBS; elution; and lyophilization, as previously described [11]. Then, the iTRAQ reagent (Beyotime, Shanghai, China) was used to label the proteins according to the manufacturer’s protocol.
The labeled peptides were graded using the Rigol L3000 HPLC system (Dalian, China) and a Waters BEH C18 column (4.6 × 250 mm, 5 μm; (Dalian, China). The details of the elution gradient are shown in Table S1. Proteomics analyses were performed using an EASY‐nLCTM 1200 UHPLC system (Thermo Fisher, Waltham, MA, USA) coupled with a Q Exactive HF‐X mass spectrometer (Thermo Fisher) using a linear gradient elution, as listed in Table S2. The separated peptides were analyzed using a Q Exactive HF‐X mass spectrometer (Thermo Fisher).

Proteomics analysis was conducted using an EASY-nLC^TM 1200 UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) combined with a Q-Exactive^TM HF-X mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Lyophilized peptides were redissolved with 0.1% formic acid, followed by injection of 1 μg of solution which was injected into a self-made analytical column (15 cm × 150 μm, 1.9 μm). Mobile phases A (2% acetonitrile, 0.1% formic acid) and B (98% acetonitrile, 0.1% formic acid) were used to develop a gradient elution (Table S2). The fractionated peptides were analyzed with a Q-Exactive^TM HF-X mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with a Nanospray Flex^TM ion source. The spray voltage and transport capillary temperature were set at 2.1 kV and 320 °C, respectively. The m/z scan range was of 350–1500 for the full scan mode. The intact peptides were detected via Orbitrap MS at a resolution of 60,000. Peptides were then selected for MS/MS mode using an NCE (28). The fragments were detected at a resolution of 30,000. The data-dependent acquisition mode was used after 1 full scan, followed by 20 MS/MS scans. The dynamic exclusion was 20 s. The automatic gain control target value was set at 3 × 10⁶, and a maximum ion injection time was set at 20 ms.

For transition library construction, shotgun proteomics analyses were performed using an EASY-nLCTM 1200 UHPLC system (Thermo Fisher, Waltham, MA, USA) coupled with a Q ExactiveTM HF-X mass spectrometer (Thermo Fisher, Waltham, MA, USA) operating in the data-dependent acquisition (DDA) mode. A total of 1 µg sample was injected into a homemade C18 Nano-Trap column (4.5 cm × 75 µm, 3 µm). The peptides were separated in a homemade analytical column (15 cm × 150 µm, 1.9 µm), using a linear gradient elution as listed in Table S3. The separated peptides were analyzed by Q ExactiveTM HF-X mass spectrometer (Thermo Fisher, Waltham, MA, USA), with ion source of Nanospray Flex™ (ESI), spray voltage of 2.3 kV, and ion transport capillary temperature of 320 °C. Full scan ranges from m/z 350 to 1500 with the resolution of 60,000 (at m/z 200), an automatic gain control (AGC) target value was 3 × 10⁶, and a maximum ion injection time was 20 ms. The top 40 precursors of the highest abundant in the full scan were selected and fragmented by higher-energy collisional dissociation (HCD) and analyzed in MS/MS, where the resolution was 45,000 (at m/z 200) for 10 plex, the automatic gain control (AGC) target value was 5 × 104 the maximum ion injection time was 86 ms, the normalized collision energy was set as 32%, the intensity threshold was 1.2 × 10⁵, and the dynamic exclusion parameter was 20 s.