Question on LC/MS/MS DDA acquisition and mass inclusion list

[prk018]

Hi,
I was running some samples on the Orbitrap and set the acquisition mode as DDA, and put a mass inclusion list (~50 rows in Excel).
Let's say one of the compound in the inclusion list had a mz of 495.53 (for the purpose of this question). When I pulled out the EIC, I set the mass range to 495.50-495.55 and found a peak eluting at 10min. However, I couldn't find any MS2s with a precursor ion of 495.5 . Why isn't the mass spec fragmenting and measuring this compound when it's included in the inclusion list and is clearly there (based on the EIC)?

[Gaetan Glauser]

I can see three possible explanations why the mass spec did not select the precusor for MS/MS although the peak was present in the EIC trace of the full scan:

1. Problem of mass tolerance for the inclusion list ions? Is the peak detected in the EIC really 495.53? If you set a mass tolerance of a few mDa, but a much higher EIC window (50 mDa), it is possible that the peak detected in the EIC does not have exactly the same mass as the precusor specified in the inclusion list and thus not selected for MS/MS.

2. Problem of threshold? I do not know for the Orbitrap, but on our Q-TOF, the threshold set for MS/MS applies to all peaks, including those of the inclusion list. If the peak at 495.53 has a lower intensity than that set for MS/MS switch, it would not be selected.

3. Very unlikely for only 50 ions in your inclusion list, but if you have e.g. 5 MS/MS max and 5 other ions of the inclusion list have a higher intensity than m/z 495.53, the latter would not be chosen for MS/MS. The peaks set in the inclusion list have a higher priority than all other peaks but within the inclusion list, stronger signals are still preferentially selected.

Let us know if one of these three options is plausible in your case.

[Gaetan Glauser]

And a fourth possibility:

in the dynamic exclusion list (if activated), you must select a time during which the instrument will not switch to MS/MS after the acquisition of a precursor ion. If this time is set too long and you had an isomer eluting before your peak at 10 min, your peak will not be selected.