Reprosil pur 120 c18 aq

Each technical replicate was analysed as a single shot on the Q Exactive HF (Thermo Fischer) mass spectrometer coupled to an EASY-nLC 1200 System (Thermo Fischer) with 15 cm long in-house pulled and packed column, filled with ReproSil-Pur 120 C18-AQ, 1.9 µm (r119.aq; Dr. Maisch). Peptides were separated out on a 77-min low pH gradient with a mobile phase of 0.1% Formic acid (1.00264.1000, VWR) and an Acetonitrile concentration gradient ranging from 0% to 80%. The mass spectrometer was operated using the ‘sensitive method’ described previously^{93 (link)}. More precisely, ions were accumulated to a target value of 1e6 and MS1 spectra (m/z 300–1750) were acquired using the Orbitrap at a resolution of 70 000 resolution (for m/z 200). The 12 most intense ions that were multiply charged were isolated with a fixed injection time of 120 ms, fragmented by higher-energy collisional dissociation (HCD) with HCD collision energy 25% and analysed in the orbitrap with a resolution of 35 000 resolution.

Extracted peptides were separated at a 300 nL/min flow rate using a reversed‐phase column (100 μm × 150 mm, 3 μm ReproSil‐Pur 120 C18‐AQ, 1.9 μm, Dr Math), followed by analysing with the EASY‐nLC nano‐UPLC (Thermo Fisher Scientific, Bremen, Germany). It performed at a 120‐min gradient elution with the UPLC mobile phase A [formic acid (FA) (0.1%) with acetonitrile (ACN) (2%)] and B [ACN (80%) with FA (0.1%)] (8%–30% B for 92 min, 30%–40% B for 20 min, 40%–100% B for 2 min, 100% B for 2 min, 100%–2% B for 2 min and finally 2% B for 2 min). Q‐Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) applying the DDA (data‐dependent acquisition) strategy which MS for the 20 most intense ions using a normalized collision energy of 27% for HCD and an isolation window of 2 m/z were used to analyse the peptides. Resolution of 70,000 for MS1 (at 200 m/z) and 17,500 for MS2 in an orbitrap analyser were processed in the following analyses. The automatic gain control target for MS1 and MS2 were set to 3.0 E⁺⁶ with max IT 50 ms and 5.0 E⁺⁴ with max IT 100 ms respectively. Thirty seconds were set for dynamic exclusion.