Hello and good day.
I am using a Q Exactive Plus and in the experiment pane for a Full MS / ddMS2 (TopN) there are three settings for the automatic gain control (AGC), the number of ions to be in the detector Orbitrap. They are labeled as such in the Full MS submenu, dd-MS2 / dd-SIM and dd Settings (minimum AGC target) as marked in the attachment.
The first one is for Full MS, the second I guess is the threshold that indicates if its selected for MS2 when there is not an inclusion list and the third one when there is an inclusion list, is this correct?
My goal in this case is to use an inclusion list. In my last run some targets were not acquired for MS2 despite being clearly present in MS1. Decreasing dd AGC minimum (the third setting) helped for some (1e3 down from 8e3) but not all, and in the TIC trace the missing ones are above 1e3. What can I do to force these missing ions into MS2?
Thank you!
this is what I believe is true, please correct me if I am wrong.
The first AGC target setting is indeed for MS1.
The second AGC target setting is the desired charges to be accumulated for an MS2 scan. This is not a threshold but simply the number of charges that should be collected/isolated for a MS2 scan within the time given for the maximum injection time. We usually keep this at 5e5, which will likely take "very long", for whole proteome analyses and set the maximum injection time to ensure maximum parallelization. But in your case it seems more about quality than a trade off between quality and speed.
The third AGC target setting is the minimum number of charges that need to be accumulated to trigger a data dependent MS2 event. If this limit is not reached no MS2 will be recorded. In my view this setting is not linked to the inclusion list.
Since you have inclusion and exclusion on, is there an overlap between the masses +/- isolation window? How many entries to do you have? Have you given the retention time interval for incl/ excl? It might help to increase the isolation window a bit to ensure that the MS2 threshold is reached or to further increase the maximum injection time (if you go up to 110-120 ms for MS2 max Injection Time then you can also increase the resolution to 35.000)
As Hendrik said above. Additionally if you are working with a large exclusion list your loop count/topN is 1 and you have dynamic exclusion turned off which may cause you to miss some of your targets as you may instead be repeatedly sampling more abundant ions with overlapping retention times.
Thank you for your suggestions, indeed I was missing my compounds. I have 7 compounds in my inclusion list but without much of an overlap in terms of mass but in time. Perhaps with the dynamic exclusion off what I am sampling during one time point is making difficult for the machine to simultaneously fragment the coeluting compounds. So I applied dynamic exclusion.
Also after discussing with some colleagues, this is what we believe happens: the "dd-MS2 / dd-SIM" settings are the optimal conditions in which it will try to reach an AGC target during the determined C-trap filling time. If for some reason this ion target is not reached it will try to use the dd Settings, in which only a minimum AGC target is required (this then automatically set a minimum intensity threshold value, for each AGC there has to be some internal math done by the software). So in short, with dynamic exclusion on, if idle select others -off- and AGC targets of 2e4 at 50 ms (dd-MS2 settings) or minimum 1e2 (dd settings) the missing peaks which were missing from a Top10 experiment were finally selected.
Hi Luis,
- First AGC target is for the MS1 (based on a pre-scan).
- Second AGC target is for the MS2 (based on the MS1).
- When the AGC target for MS2 cannot be reached, maximum injection time applies and AGC possible is recalculated for this time: if it is under the minimum AGC target, the MS2 event is skipped.
If you want to force MS2 for some precursors I would rather increase the maximum injection time, it may have some advantages versus reducing the minimum AGC.
And of course use an inclusion list or even better a data independent acquisition: DIA or PRM will always try to isolate your precursor in a time range of your choice.
I hope it helps
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