A maximum injection time of 60 ms was used for ion accumulation.
HCD collision energy was set to 27, AGC target to 5e4 and maximum injection time to 200 msec.
The resolution was set to 17,500 at 200 m/z combined with a maximum injection time of 60 ms.
MS/MS spectra were acquired at a resolution of 17,500 with a target value of 1 × 105 and a maximum injection time of 50 ms.
Peptides were loaded on an in-house packed C18-column (15 cm, 75 μm I.D., and 2-μm particle size) with a gradient of 12 32% buffer B (98% acetonitrile and 0.1% acetic acid) for 45 min followed by a 5-min wash with 95% buffer B. Full scan MS spectra were acquired at a resolution of 70,000 with an AGC target value of 3 × 106 and a maximum injection time of 100 ms.
Top 20 precursor ions were selected into the HCD chamber for MS2 fragmentation analysis, MS/MS scan resolution was 17,500, AGC was set as 5e 4, the maximum injection time was 80 ms.
Data was acquired in a top ten data dependent acquisition mode (DDA), in which a high resolution 70,000 (at m/z 200 MS survey scan (m/z 350 1800, AGC value of 1e6, maximum injection time of 100 ms) was followed by an acquisition of tandem mass spectra (35,000 (at m/z 200), target AGC value of 1e6, maximum injection time of 120 ms).
The scan range was m/z 50 1500, the maximum injection time was 50 ms, and two scan events were prescribed to run sequentially in the mass spectrometer.
The maximum injection time was 250 ms for MS1 and 200 ms for MS2 scans.
Maximum injection time was set to 100 ms for both MS and MS/MS analyses.
The MS/MS target was 1E6 with a 100 ms maximum injection time.
