Hf hybrid quadrupole orbitrap mass spectrometer

Panoramic mass spectrometric analysis was carried out on a sensitive Q Exactive HF mass spectrometer (HF Hybrid Quadrupole Orbitrap mass spectrometer, Thermo Fisher Scientific™, Rockwell, IL, USA), which allows panoramic protein search and is used in routine analysis.
The peptides for each sample were separated by high-performance liquid chromatography (HPLC, Ultimate 3000 Nano LC System, Thermo Scientific, Rockwell, IL, USA) on a 15 cm long 75 µm id C18 column (Acclaim^® pepmap™ RSLC, Thermo Fisher Scientific, Rockwell, IL, USA).
The peptides were eluted with a gradient from 5–35% buffer B (80% acetonitrile, 0.1% formic acid) over 115 min at a flow rate of 0.3 µL⁻¹ min. The total run time was 90 min to reach 99% buffer B, 10 min to wash with 99% buffer B, and 15 min to re-equilibrate with buffer A (0.1% formic acid).

Mass spectrometric measurements were carried out according to the procedure described in a previous study [43 (link)]. The analysis was performed using a Q Exactive HF mass spectrometer (HF Hybrid Quadrupole Orbitrap mass spectrometer, Thermo Fisher Scientific™, Rockwell, IL, USA). Mass spectra were obtained with a resolution of 60,000 (MS) and 15,000 (MS/MS) in the m/z range 400–1500 (MS) and 200–2000 (MS/MS).
The resulting RAW files, unprocessed with a mass spectrometer, were analyzed with the maxquant program (version 1.5.5.1, Jurgen Cox, Max Planck Institute for Biochemistry, Martinsried, Germany) [44 (link)] with the built-in Andromeda search system [45 (link)].
Protein N-terminal acetylation and methionine oxidation were variable modifications for the peptide search. A maximum mass deviation of 5 ppm was allowed for precursor identification, and 20 ppm was set as a tolerance for fragment identification. For trypsin digestion, 2 missing sites were allowed. The false discovery rate (FDR) of resulting protein identifications was 0.01.