Hfx orbitrap ms system

LC-MS/MS experiments were performed using an Orbitrap Fusion Tribrid MS system (Thermo Scientific) as described previously (9 (link)). Subsequent analysis was also performed using an HFX Orbitrap MS system (Thermo Scientific) equipped with a Dionnex 3000 Ultimate HPLC (Thermo Fisher). Injected peptides were trapped on an Acclaim PepMap C18 column (3 µm particle size, 75 µm inner diameter x 20 mm length). After the elution the peptides were introduced into the mass spectrometer and analyzed as previously described (9 (link)). Briefly the capillary temperature was set at 275°C. Data acquisition was carried out using a top 20 based data-dependent method. MS was conducted in the range of 350–1,350 m/z at a resolution of 120,000 FWHM. The filling time was set at a maximum of 100 ms with limitation of 3 x 10⁶ ions. MSMS was acquired with a filling time maximum 300 ms with limitation of 5 x 10⁴ ions, a precursor ion isolation width of 2.0 m/z and resolution of 15,000 FWHM. The normalized collision energy was set to 28%. Only multiply charged (2+ to 5+) precursor ions were selected for MS2. The dynamic exclusion list was set to 30 s.

LC-MS/MS analysis was performed using an HFX Orbitrap MS system (Thermo Scientific, San Jose, CA, USA) equipped with a Dionnex 3000 Ultimate HPLC (Thermo Fisher, USA). Settings were used as previously described by Petruk et al.⁵⁵ with minor changes. MS range was 350–1600 m/z, the start of LC gradient at 4 min, solvent B was increased from 4% to 10% during 3 min, to 65% during 11 min, followed by 100% for 2 min. MS/MS spectra were searched with PEAKS (version 10) against UniProt Homo Sapiens (version 2020_02). The precursor tolerance was set to 10 ppm and 0.02 Da for the MSMS fragments, and no enzyme was selected, allowing unspecific cleavage. Samples from two independent digestions were analyzed.

LC-MS/MS analysis was performed using an HFX Orbitrap MS system (Thermo Scientific, San Jose, CA, USA) equipped with a Dionnex 3000 Ultimate HPLC (Thermo Fisher, USA). Settings were used as previously described by Petruk et al.⁵⁵ with minor changes. MS range was 350–1600 m/z, the start of LC gradient at 4 min, solvent B was increased from 4% to 10% during 3 min, to 65% during 11 min, followed by 100% for 2 min. MS/MS spectra were searched with PEAKS (version 10) against UniProt Homo Sapiens (version 2020_02). The precursor tolerance was set to 10 ppm and 0.02 Da for the MSMS fragments, and no enzyme was selected, allowing unspecific cleavage. Samples from two independent digestions were analyzed.