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Cannot see product ion chromatogram in Surequant run

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Cannot see product ion chromatogram in Surequant run yufeicui  2023-05-31 13:54
 

Hello,

I did a Surequant run using synthetic heavy isotope-labeled peptides as triggers and for one peptide (HLVEALYL[+7]V), the product ion chromatogram was not found for the light peptide (attached) although there is a trace for the precursor. However, I could see the light peptide MS2 spectra at the correct retention time when I searched the Surequant data (attached). It seems like somehow this spectra information was not extracted by Skyline. I am using Skyline-daily 22.2.1.501
 
 
Nick Shulman responded:  2023-05-31 13:56
Can you send us your Skyline document and one of your .raw files?
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 file are less than 50MB you can attach them to this support request.
You can upload larger files here:
https://skyline.ms/files.url
-- Nick
 
yufeicui responded:  2023-05-31 14:13
The .raw is uploaded with the name 20230531_yufeicui.raw and the skyline document is attached.

Thanks!
 
Nick Shulman responded:  2023-05-31 15:24
Thank you for sending those files.
When I look at your raw file using ProteoWizard SeeMS.exe, I see something strange going on with the values in the "Precursor Info" and "Isolation Windows" column.
For spectrum #25098, the value in the Precursor Info is 528.80833 which probably means that the spectrum is supposed to match the light version of HLVEALYLV (528.8080).
However, when Skyline is deciding which precursors should get a chromatogram point from a particular spectrum, Skyline actually looks at the isolation window of the spectrum. The isolation window is 529.79321 - 529.79321, which, notably, does not actually contain 528.808.

My understanding is that the precursor is always supposed to be inside of the isolation window so this either means that there is a bug in the way that ProteoWizard is interpreting the data in your .raw file, or there is something very strange going on with the .raw file.

I will ask my coworkers and see if anyone can figure out what is going on.
-- Nick
 
yufeicui responded:  2023-06-02 10:30
Thanks for the reply. It is a weird thing that I never noticed. I also wonder if this would make the fragment ion chromatogram for the peptide completely not extractable despite there being other MS2 scans that correspond to the same precursor (scan 25221, 25371, 25358 etc)?
 
yufeicui responded:  2023-06-02 10:44
I wonder if ProteoWizard only shows the precursor monoisotopic mass. When I look at the raw data on the qual browser, the precursor mass for scan 25098 is 530.2932, which is within the isolation window. It seems like this is an isotopic precursor of the peptide (see the comparison to scan 25221)?