Difficulties reading SWATH results

Difficulties reading SWATH results laura corveleyn  2021-09-27


I am having some troubles when importing SWATH data as results. I want to compare histone peptidoform expression across several conditions (cell lines) using peak areas. To do this, I made a QC sample (mixture of all conditions) as a baseline. However, when importing the SWATH raw data of these QC samples into Skyline, it seems like something goes wrong when reading it. The XICs look a bit strange. I tried reimporting results several times and made a new Skyline project, but nothing helps. You can find a screenshot of some XICs in the attachment (the QC samples are in the bottom left). Hope you can help me out :)

Thanks in advance!

Kind regards,


Nick Shulman responded:  2021-09-27

The chromatograms in the lower left corner of your screenshots look like what you get when the mass spectrometer is doing DDA. With DDA, a particular precursor does not get sampled at a regular frequency, so the MS2 chromatograms look like that, with long straight lines connecting the places where a matching precursor happened to be chosen by the mass spectrometer for fragmentation.

If you send us your Skyline document and a few of your raw files we can probably give you more definitive answer about what is happening.

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 your raw files are less than 50MB you can attach them to this support request. Otherwise, you can upload them here:

-- Nick
laura corveleyn responded:  2021-09-27
Hi Nick,

Thanks for the quick answer. You can find the files in the attachment. The QC samples were run throughout the entire batch, so normally they are all acquired in SWATH mode.

laura corveleyn responded:  2021-09-27
Oh, now I see the files were indeed too big to attach here. I have uploaded them on the link you sent me:

Skyline project: "TALL_SWATH_DDA_SL.sky.zip"
Raw files: "QC_RAW_SWATH.zip"
Nick Shulman responded:  2021-09-27

Can you also send the .wiff.scan files? We cannot look at a .wiff files unless we also have the .wiff.scan file that goes with it.
Which peptide were you looking at in your screenshots?
-- Nick
laura corveleyn responded:  2021-09-28
Hi Nick,

Sorry I added them to the link ("QC_RAW_SWATH_ALL.zip"). The peptides from the screenshots are:
1: G[Poy]K[Ac]QGGK[Poy]AR from H2A1A
2: K[Me3Prop]STGGK[Poy]APR from H31
3: E[Poy]AQD[Dea]FK[Byr]TDLR from H31

However, there are many more examples that look weird in the QC samples.

Nick Shulman responded:  2021-09-28
Thanks for sending those .wiff.scan files.
The files that you sent me definitely look like they are DDA.
When I look at the files using ProteoWizard SeeMS.exe, I see that any particular precursor m/z is only getting selected once, with a 1 m/z wide isolation window.
-- Nick