Waters m class uplc system

Dissolved samples were injected by a Waters M‐class UPLC system (Waters AG) and separated on a C18 reverse‐phase column (HSS T3 1.8 μm, 75 μm × 250 mm, Waters AG). The column was equilibrated with 99% solvent A (0.1% formic acid (FA) in water) and 2% solvent B (0.1% FA in ACN). Peptides were eluted using the following gradient: 2–25% B in 50 min; 25–35% B in 10 min; 35–98% B in 5 min. The flow rate was constant, 0.3 μl/min. High accuracy mass spectra were acquired with a Q‐Exactive HF mass spectrometer (Thermo Scientific) that was operated in data‐dependent acquisition mode. A survey scan was followed by up to 12 MS2 scans. The survey scan was recorded using quadrupole transmission in the mass range of 350–1,500 m/z with an AGC target of 3E6, a resolution of 120,000 at 200 m/z, and a maximum injection time of 50 ms. All fragment mass spectra were recorded with a resolution of 30,000 at 200 m/z, an AGC target value of 1E5, and a maximum injection time of 50 ms. The normalized collision energy was set to 28%. Protein identification and reporting from MS‐raw data was performed with Mascot and Scaffold, respectively. Only proteins with a minimum of four unique spectral counts were utilized for the final dataset and contaminants were manually removed.

The extracted digested peptides were injected and separated with a Waters M-class UPLC system (Waters Corp., Milford, MA, USA) online connected to a Qexactive^PLUS mass spectrometer (Thermo Fisher Scientific, Palo Alto, USA). Peptides were first collected on a trap column (20 × 15 mm; PepSepC18 Trap, PepSep, Denmark) and subsequently separated on an analytical C18 column (100 mm × 75 μm, PepSep, Denmark) with a 30-min gradient of 4 to 16% and 15-min gradient to 25% acetonitrile in 0.1% FA followed by a column cleanup step (isocratic 80% acetonitrile in 0.1% FA) and an equilibration step (isocratic 2% acetonitrile in 0.1% FA), all at a flow rate of 200 nl min⁻¹. The eluted peptides were electrosprayed into the mass spectrometer using a Flex-Ion nanoESI Source at +2.4 kV. MS and MSMS spectra were collected in top-10 DDA mode selecting charge 2, 3, or 4 ions within mass/charge ratio (m/z) range of 400 to 1500 for MS spectra and auto-range for MSMS spectra.