Q exactive orbitrap ms

LC-MS/MS analyses were performed using a HPLC system with a HSS T3 column coupled to Q Exactive (Orbitrap MS, Thermo). The mobile phase A was 0.1% formic acid in water and the mobile phase B was acetonitrile. The elution gradient was set as follows: 0 min, 2% B; 1 min, 2% B; 18 min, 100% B; 22 min, 100% B; 25 min, 2% B. The flow rate was 0.3 mL/min. The injection volume was 2 μL. The QE mass spectrometer was used for its ability to acquire MS/MS spectra on an information-dependent basis (IDA) during an LC/MS experiment. ESI source conditions were set as following: Aux gas flow rate as 16 Arb, Full ms resolution as 70,000, Collision energy as 25 eV in NCE model, MS/MS resolution as 17,500, and spray voltage as −3.0 kV (negative) or 3.6 kV (positive), respectively.

For the HPLC-MS/MS analysis, the peptides were resolved in 5 µL 30% formic acid and diluted with 40 µL of mobile phase A (97.9% H₂O, 2% acetonitrile, 0.1% formic acid). Then, 1 µL for the supernatant samples and 10 µL of cytoplasmic and nuclear samples were injected into the Dionex UltiMate 3000 RSLCnano LC system, coupled to the QExactive Orbitrap MS (all Thermo Fisher Scientific, Vienna, Austria). Peptides were trapped on a C18 2 cm × 100 μm precolumn, and LC separation was performed on a 50 cm × 75 μm Pepmap100 analytical column (both Thermo Fisher Scientific, Austria). Following this, 1 µL of sample was injected. The 85 min HPLC method included a 43 min gradient from 7% to 40% mobile phase B (79.9% acetonitrile, 20% H₂O, 0.1% formic acid) at a flow rate of 300 nL/min. The resolution on the MS1 level was set to 70,000 (at m/z = 200), with a scan range from 400 to 1400 m/z. The top eight abundant peptide ions were chosen for fragmentation at 30% normalized collision energy and resulting fragments were analyzed in the Orbitrap at a resolution of 17,500 (at m/z = 200).

A 2 μL stock solution (10 mg/mL) was loaded using a disposable glass Pasteur pipette (Volac; Poulten & Graf Ltd., Barking, UK) onto the center of a chromatographic paper tip (Whatman 1 Chr., Kent, UK). The positive-ion mode of the Q-Exactive orbitrap MS (Thermo Fisher Scientific, Inc., Rockford, IL, USA) was used to collect the data over the range of m/z 50–600. To make a sharp tip, the chromatographic paper was cut into dimensions of 6 mm base and 14 mm height. A syringe pump (Fusion 100T; Chemyx, Stafford, TX, USA) was used to load the ethanol solvent onto the sample-loaded paper at a flow rate of 15 μL/min. A spray voltage of 4.5 kV was directly applied to the paper tip for the ionization of the sample. The other parameters for the PSI experiment were as follows: capillary temperature 300, S-lens RF level 50, mass resolution 140,000 (full-width at half-maximum), and maximum injection time 150 ms. The automatic gain control was set to 1 × 10⁶.
To perform the MS/MS experiments, three different stepped normalized collision energies (10, 30, and 50) were used with the same instrument. The instrument was operated in the positive-ion mode. The other operative parameters for the MS/MS experiments were as follows: sheath and auxiliary gas flow rate 10 and 0 (arbitrary units), respectively; spray voltage 3.60 kV; capillary temperature 300; and S-lens RF level 50.