Then, 2.5 mL were injected (time interval 0.5 minutes).
Collision energy was set to 45, maximum inject time was 250 ms and maximum ion count was 1 × 10 counts.
For MS and MS/MS spectra acquisition, the maximum ion inject time was set as 500 and 250 ms, respectively.
The maximum inject time for both precursor and fragmentation scans was 1000 and 400 ms for SIM scans.
The following scan parameters were applied during measurements: scan range, m/ z 100 to 2,000 ; resolution, 100,000; microscans, 1; AGC target, 1e6; maximum inject time, 50 ms.
It should be noted that all data were acquired using automatic gain control (AGC), which ensures that less abundant, low intensity precursor ions are accumulated in the LTQ ion trap for a proportionally longer inject time (fill time) prior to fragmentation in the HCD cell (as long as the limit set by the maximum inject time is not reached).
Both a CID scan for identification (automated gain control (AGC) target value 1 × 10, maximum inject time 400 ms, minimum signal threshold 500 counts) and a HCD scan for quantification (AGC target value 3 × 10, maximum inject time 500 ms, minimum signal threshold 500 counts) were acquired for each selected precursor (monoisotopic precursor selection on, rejection of singly charged ions).
LXQ settings were as follows: spray voltage, 1.6 kV; 1 microscan for MS scans at maximum inject time 10 ms with mass range 400-1400 m/z, 3 microscans for MS/MS at maximum inject time100 ms with automatic mass range.
Automatic gain control was set to 1,000,000 for FT-MS and 50,000 for FT-MS, full FT-MS maximum inject time was 500 ms and normalized collision energy was set to 35% with an activation time of 10 ms.
Survey MS1 scan m/z range 400 2000 profile mode, resolution 60,000 or 30,000 - 400 m/z with AGC target 1E6, and one microscan with maximum inject time 200 ms.
