Ultimate 3000 nanolc ms ms system

Peptides were separated using an Ultimate 3000 nanoLC-MS/MS system (Thermo Fisher Scientific) equipped with a 50 cm × 75 μm ID Acclaim Pepmap (C18, 1.9 μm) column. After injection, peptides were trapped at 3 μl/min on a 10 mm × 75 μm ID Acclaim Pepmap trap at 2% buffer B (buffer A, 0.1% formic acid [Fisher Scientific]; buffer B, 80% ACN, 0.1% formic acid) and separated at 300 nl/min in a 10% to 40% buffer B gradient in 90 min (125 min inject-to-inject) at 35 °C. Eluting peptides were ionized at a potential of +2 kVa into a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). Intact masses were measured from m/z 350 to 1400 at resolution 120.000 (at m/z 200) in the Orbitrap using an AGC target value of 3E6 charges and a maxIT of 100 ms. The top 15 for peptide signals (charge states 2+ and higher) were submitted to MS/MS in the HCD (higher-energy collision) cell (1.4 amu isolation width, 26% normalized collision energy). MS/MS spectra were acquired at resolution 15,000 (at m/z 200) in the Orbitrap using an AGC target value of 1E6 charges, a maxIT of 64 ms, and an underfill ratio of 0.1%, resulting in an intensity threshold for MS/MS of 1.3E5. Dynamic exclusion was applied with a repeat count of one and an exclusion time of 30 s.