DIA-MS analysis: Replicates not showing up

DIA-MS analysis: Replicates not showing up nbaig  2023-02-20 09:33


I am performing DIA data analysis. I notice that only one replicate is showing up while the others are not visible. How do I resolve this issue?

Nick Shulman responded:  2023-02-20 17:22
I do not understand your question but if you send us your Skyline document we will probably be able to tell you 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 is less than 50MB you can attach it to this support request. You can upload larger files here:

It would probably also be helpful if you could send us a screenshot of what you are looking at.
-- Nick
nbaig responded:  2023-02-20 18:20
Hello Nick,

So I have 6 DIA runs from 6 different samples. However only one file is showing up. The other replicates are not visible. I tried uploading both mzml and raw formats and the problem still persists.
Nick Shulman responded:  2023-02-21 03:36
I see that in your Peak Area Replicate Comparison graph, the "A1" replicate has some nice peak areas, but the other 5 replicates have zero peak area.

Whenever you see something suspicious in the Peak Area graph the usual place to look next would be the chromatogram graphs.
You can tell Skyline to show you the chromatogram graph for a particular replicate using the menu item:
View > Chromatograms > <replicate name>

It is possible that the chromatogram graphs for the replicates with zero peak area will be completely flat. If you are looking at a completely flat chromatogram in Skyline, one thing that you can do is click on any point along the flat line and Skyline will show you the spectrum which contributed to the point on the extracted ion chromatogram.
Sometimes the spectrum that Skyline shows you really has no signal in it at all, and sometimes there is some signal there, but it is just outside of the m/z window that Skyline was summing across and so it did not contributed to the intensity value in the extracted ion chromatogram.
If you want to widen the m/z window that Skyline is summing across you can change the settings at:
Settings > Transition Settings > Full Scan

I cannot say for sure what is going on with your data, but if you send me your Skyline document and a few of your raw files, I could probably tell you.

The way to send someone your Skyline document is to use the Skyline menu item:
File > Share
That menu item allows you to create a .zip file which contains the Skyline document and supporting files including spectral libraries and extracted ion chromatograms.

That .zip file and your .raw files will probably be too large to attach to this support request, so you can upload them here:

-- Nick
nbaig responded:  2023-02-21 07:33
Hello Nick,

I uploaded my skyline file along with a couple of replicate data sets under the name: NBAIG-UMN-Skyline. Please let me know if you need additional details.
Nick Shulman responded:  2023-02-21 17:16
Thank you for uploading that .zip file.

I see that the .zip file contained three .raw files and a file whose filename extension is ".skyd".
A .skyd file contains extracted chromatograms associated with a Skyline document, but it is not possible to understand the contents of a .skyd file without having the rest of the Skyline document files.

When you use the menu item:
File > Share
Skyline will create a .sky.zip file which contains the Skyline document and its other necessary files.

By default, Microsoft Windows tries very hard to conceal from you the actual extensions on file names.

If you are having trouble finding the .sky.zip file which you just created, I would recommend turning off an option in Windows called "Hide extensions for known file types".

-- Nick
nbaig responded:  2023-02-21 17:43
Hello Nick,

I uploaded the zip folder again under the same name. I hope this works. I appreciate your help and patience. Thank you!
Nick Shulman responded:  2023-02-22 04:08
I did not see any new file uploaded to the file sharing folder.
Can you try again?
-- Nick
nbaig responded:  2023-02-22 06:39
Hi Nick. I tried again. The folder is labeled as NBAIG-UMN-skyline-22
Nick Shulman responded:  2023-02-22 13:59
Thank you for sending that new .zip file.
The spectral library file that you are using, "BLIBfilefinal.blib" has information in it which is telling Skyline where to put the peak boundaries.
In the cases where Skyline is not giving you peak areas for some of the replicates, I believe what is happening is that the spectral library does not have peak boundary information for that file.

One thing that you can do is tell Skyline to ignore the peak boundary information in the .blib file.
To do that, you would go to:
Settings > Peptide Settings > Library
and then push "Edit List"
and then select the library and choose "Edit" and then uncheck the box that says "Use explicit peak bounds"

After you do that, you should tell Skyline to choose peaks again by going to:
Edit > Manage Results
and push the "Rescore" button.

This will cause Skyline to do its own peak finding instead of using the peak boundary information in your spectral library.

I think there also might have been a bug in the way that Skyline version 22.2 handled libraries that provided peak boundary information for some but not all of the replicates. In cases where the peak was supposed to be missing from a particular replicate, Skyline would set the peak boundaries to very strange numbers. I will look into this a little bit more and I might be able to provide more information about what is happening if you do not want to uncheck the "Use explicit peak bounds" checkbox. You might also find that things work better if you use "Skyline-daily" instead of Skyline 22.2.
-- Nick