Hi,
we have measured tryptic peptides using both DDA and DIA method on QExactive. In DDA replicates i saw unusual intensity drops occurring at the same sections of chromatograms in DDA replicates (attached picture). I am wondering why this is happening ? as both methods worked fine for us previously.
cheers
Arslan
Hi,
It seems you are experiencing some instability in your spray.
What was the order in which the runs you show were performed?
What was the flow rate and gradient composition around the retention times at which you observe the gaps?
It's not necessarily occuring in the same retention time windows and also seems to happen in your DIA runs, although to a lesser extent.
Are the source settings the same for both the DDA and the DIA analyses?
Spray instability could be caused by several things, including clogging of the spray needle.
Hi Eef Dirksen,
many thanks for the answer. I also thought that it might be spray instability, but why is it consistent in the replicates? shouldn't it be random?
The runs were in the order as shown in picture separated by column wash.
Source settings were same for both DDA and DIA and the chromatography is generic: flow 300nL/min 2%B at 3 min to 30%B at 43 min followed by 6 min of 95%B (A=2% ACN 0.1%FA and B=80% ACN 0.1% FA).
Do you see the same view in MS1 TICs? Do these chromatograms belong to MS2 only? It seems like a sensitivity threshold filter has been incorporated in DDA mode and when the signal is low for precursors, ms/ms was not triggered, hence the obtained ms2 spectrum appeared partially null. This can be confirmed by investigating DIA plots since this acquisition mode subjects every MS1 to the fragmentation cycle and you always get a signal whether it is meaningful or not. Secondly, you may share the LC pump pressure plots to interrogate if any air pulse has been experienced during high organic solvent composition trying to be delivered by binary pumps.
Dear İsmail Emir Akyildiz,
Many thanks for the answer. The pic represent TIC (both MS1 and 2) and i also checked, the pump pressure are also fine.
warm regards
Arslan
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