Partial missing fragment ion exracted from FAIMS-DIA file by skyline

Partial missing fragment ion exracted from FAIMS-DIA file by skyline Winnie  2022-11-08

Hi skyline team,
I used skyline to extract the interested peptide precursors from the FAIMS-DIA files. The interested peptide precursors were almost extracted from files acquired from Q Exactive HF instruments or TripleTOF instruments. But there are some weird things about those peptide precursors extraction from FAIMS-DIA files. 1. Some of the peptide precursors show good MS1 extraction but without fragment ions. 2. Some of the peptide precursors could extract fragment ions from A files but could not extract from B files. The A and B files are technical replicates.
I do not know whether there are some issues with skyline settings, so I upload the skyline documents if you can check it would be better. Many thanks.

I have uploaded the file to the link.
Best regards,

Nick Shulman responded:  2022-11-08
If a molecule does not have any MS2 chromatograms, then it typically means that there were no MS2 spectra which matched the precursor, either because the isolation window did not match the precursor's m/z, or the ion mobility was not inside of the ion mobility window.

When I go to:
Settings > Transition Settings > Ion Mobility
and choose "Edit current" from the dropdown, I see that Compensation Voltage values are specified for all of your molecules, but some of the molecules also have a "High Energy Ion Mobility Offset" specified, and those are the same molecules which are missing MS2 chromatograms.

I am pretty sure that High Energy Ion Mobility Offset should never be used with Compensation Voltages (i.e. FAIMS). The high energy offset has something do to with the molecule hitting the time of flight detector at a different time because of something to do with the energy used to fragment the molecule for MS2. Compensation Voltages don't really have anything to do with the time it takes the ion to travel down a drift tube (instead it's a measure of whether a particular voltage can prevent the ion from rolling into the gutter), so the high energy offset would not apply.
I think you should change all of those High Energy Ion Mobility Offset values to zero and then extract chromatograms again (Edit > Manage Results > Reimport) and see if that fixes the problem.

If that does not fix things you should send us a couple of your raw files.

By the way, the replicates in the file that you uploaded ("") are not really named "A" or "B" or anything similar, and I am not seeing what you are describing where chromatograms might be missing from only half of the replicates.
-- Nick
Winnie responded:  2022-11-09
Hi, Nick,

I have tried to set those High Energy Ion Mobility Offset values to zero. But some of the peptide precursors still do not extract any MS2 chromatogram. I built another skyline document with consistent parameters except using the "None" option for the ion mobility spectral library. Those peptide precursors that are missing MS2 chromatograms could extract the MS2 chromatogram from the skyline document that does not set the ion mobility library. So I think some of the peptide precursors that are without MS2 chromatogram might be caused by wrong skyline settings.

I have uploaded both two skyline documents (ion mobility spectral library with zero high energy ion mobility offset and without ion mobility spectral), and two raw files that were imported into both skyline documents into this link.
I also uploaded the screenshot about some peptide precursors from the 2 skyline documents mentioned above. The left one is the ion mobility spectral library with zero high-energy ion mobility offset, and the right one is without ion mobility spectral.

Best regards.

Nick Shulman responded:  2022-11-09
Thank you for sending your raw files.
I used ProteoWizard SeeMS.exe to look at the spectra in them.
There were 20m/z wide DIA isolation windows going from 400 to 640, and all of those MS2 spectra had a compensation voltage of -68.
There were also 34m/z wide windows going from 640 to 1014 and all of those MS2 spectra had a compensation voltage of -48.

I think the problem is that any particular MS2 isolation window was either collected with a compensation voltage of -48 or -68, and there is no window where both compensation voltages were used. So, for precursors in the 400 to 640 range, you will only see MS2 chromatograms for the ions where the ion mobility library specifies that the CV is -68, and for the 640 to 1014 range, you will only see MS2 chromatograms if the CV is -48.
-- Nick
Winnie responded:  2022-11-10
Hi Nick,

You are right. I tried to modify the ion mobility spectral library to set all lower m/z precursors with -68V and all larger m/z precursors with -48V. But still, part of the files was missing MS2 spectra. I have submitted the skyline documents and raw files to this link.

Looking forward to your reply.

Nick Shulman responded:  2022-11-10
In this new set of files, can you give me an example of a peptide and replicate that is missing chromatograms?

I see that the peptide "LPTLEQPIIPSDYVAIK" is missing MS2 chromatograms in the replicate "CAC20220118ajun_ML_30min_48_68_250ng_DIA" but that is because the ion mobility library says that the CV for that peptide is -68, but the spectra that were collected for the 629.5-640.5 window used CV -48.
-- Nick