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Missing Peaks viktoria thomanek  2025-02-12 03:30
 

Hi there,

I have some problems with the Skyline software regarding small molecule analysis. The details with screenshots are added as a Word file. In short, I optimized the measuring for optimized retention times with Skyline, but after method optimization one Peak is missing. Why is the peak gone and what can I do to improve my measurements?

Thanks for your help!
VT
 
 
Nick Shulman responded:  2025-02-12 06:59
It sounds like you might have wanted to attach a Word document to this support request, but no files have been attached.

It might also be helpful if you could send us your Skyline document.
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.

Files which are less than 50MB can be attached to these support requests. You can always upload larger files here:
https://skyline.ms/files.url
-- Nick
 
viktoria thomanek responded:  2025-02-12 22:12
 
viktoria thomanek responded:  2025-02-12 22:13
I hope the upload of the worked this time and you can utilize them.
 
Nick Shulman responded:  2025-02-12 22:30
Can you send me your .wiff files and the .wiff.scan files that go with them?
That would be:
Data241216_VT_Ashgo_std.wiff
Data241216_VT_Ashgo_std.wiff.scan
Data241213_VT_Ashgo_std.wiff
Data241213_VT_Ashgo_std.wiff.scan
Data250203_AshgoSTD_WD.wiff
Data250203_AshgoSTD_WD.wiff.scan

It sounds like you are saying that some of the chromatograms from your .wiff file are not being imported into your Skyline document.
You have already seen the message that Skyline gives you when it discards a chromatogram because it does not overlap with the Explicit Retention Time.

The Q1 and Q3 values of the chromatogram in the .wiff file need to be within the "Method match tolerance m/z" value from the "Instrument" tab at "Settings > Transition Settings". This value is set to 0.2 which is a reasonable number.

After I see your .wiff files I will probably be able to figure out what is going wrong.
-- Nick
 
viktoria thomanek responded:  2025-02-12 23:27
Hi Nick,

thanks for your help.

I attached the files for you.
 
Nick Shulman responded:  2025-02-12 23:55
Thank you for sending those files.
I used ProteoWizard SeeMS.exe to look at the chromatograms in "Data241216_VT_Ashgo_std.wiff"
I see that you have two different chromatograms which both have Q1=854.6 Q3=347.
One of those chromatograms goes from 7.4 minutes to 9.4 minutes, and that is the one that you were hoping that Skyline would use for Beta-hydroxybutyryl-CoA.
However, Skyline is choosing the other chromatogram. It looks like that chromatogram was supposed to only have data in it from 2.6 minutes to 4.6 minutes, but it actually has data for the entire range from 0 minutes to 15 minutes (see attached screenshot).

Skyline only pays attention to the Q1, Q3 and the time range over which the chromatogram has data so Skyline has no way of knowing that you would like to use the other chromatogram with Beta-hydroxybutyryl-CoA.
There might be a way to get this to work if you used MSConvert to convert the .wiff file to .mzML and also removed the chromatograms that you do not want Skyline to use.
If you are able to run your experiment again, there might be a way for you to change your instrument method so that the chromatogram only has data from 2.6 minutes to 4.6 minutes instead of the entire range that it seems to have.
-- Nick