Skyline dropout with Astral data when using wrong window scheme in DIA

Skyline dropout with Astral data when using wrong window scheme in DIA telgierari  2023-07-13 10:41

when extracting spiked in Pepcal peptides we noticed dropout in the signal on some of the peptides, this was effecting primarily methods with small DIA widows, the Skyline document was wrongly set to " SWATH(100VW)", once the transition setting is changed to "all ions" peaks looked excellent.

Nick Shulman responded:  2023-07-13 11:03
I am not sure I understand what are saying that you did or what you were hoping to see.

When you set the Isolation Scheme to "All Ions", that tells Skyline that the chromatogram for a particular transition should be extracted from every MS2 spectrum, regardless of what the mass spectrometer said was isolated in that MS2 spectrum.
The "All Ions" setting is supposed to be used with Waters MSe data.
If you use the "All Ions" setting on data which is not MSe, then you would expect that the extracted chromatogram would be spiky like in your "Before correcting transition setting" screenshot, because some of the spectra had an actual isolation window that contained your compound of interest, and other spectra did not, and would be therefore expected to have much lower signal.

It sounds like you are saying that when you switched it to MSe, the spikiness disappeared, which is a little bit unexpected.
When you are trying to decide whether a chromatogram is spiky, you should make sure to use the menu item "View > Transform > None".
Sometimes, when "View > Transform > Interpolated" is selected, the chromatogram happens to look smooth even though the actual chromatogram that was extracted was very spiky.

Can you send us your Skyline document and some of your .raw files? We will be able to give you a better description of what is going on.

In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

If that .zip file and/or your raw files are less than 50MB you can attach them to this support request.
You can upload larger files here:

-- Nick
telgierari responded:  2023-07-13 11:45
thanks Nick for responding, yes that is correct, changing the transition setting to "all ion" did remove the dropout, I don't understand why, the raw file and zip skyline document are both uploaded.
best regards.
Nick Shulman responded:  2023-07-13 13:11
Thank you for sending that .raw file and your Skyline document.

I see that the isolation windows in the .raw file were approximately 2 Daltons wide and covered the range from 380.4228 to 980.69564.
The "SWATH(100VW)" would not be an appropriate isolation scheme setting to use with this data because the windows in that scheme are completely different from what is in the .raw file.

If you want Skyline to use whatever isolation scheme is actually in the file without you having to specify an isolation scheme, you should use the isolation scheme called "Results Only".
It turns out, however, that choosing "All Ions" will give you the same chromatograms as the "Results Only" isolation scheme would give you.
The reason for this is that Skyline only allows you to use the All Ions isolation scheme with Agilent or Waters data.
If your data is from a different brand of mass spectrometer, then Skyline treats "All Ions" the same as "Results Only".

Does that answer your question?
It sounds like you might have been saying that you might have been expecting SWATH(100VW) to work with this data, but the expected behavior is exactly what you observed.
-- Nick
telgierari responded:  2023-07-13 14:14
that answers my question, thank you for clarifying, I did not expect the SWATH setting to work, hence I referred to it as wrong setting, but I was wandering why "All Ions" worked.