Trouble with Target Quantification and Integration in Skyline for PRM Data

support
Trouble with Target Quantification and Integration in Skyline for PRM Data afshari1  2024-09-13 14:44
 

Hello,

I am analyzing my PRM results using Skyline and encountering an issue with quantifying certain targets. Specifically, in the attached file, there is a peak at 69.x minutes in the bottom panel, which corresponds to one of my targets. The MS/MS data from Thermo Fisher software confirms this match. However, when I import the same RAW file into Skyline, this target is not being quantified or integrated. Could you please help me understand why this might be happening and how I can resolve this issue?

 
 
Nick Shulman responded:  2024-09-13 15:26
Can you send us your Skyline document and your .raw file?
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.
The "Share Document" dialog gives you the option to include the .raw files, or you could send those to us separately.
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
 
afshari1 responded:  2024-09-13 15:49
I just uploaded the havva_only_TLPPs.sky.zip in the file sharing folder.
Thanks
 
Nick Shulman responded:  2024-09-13 16:38
Thank you for uploading that Skyline document and the .raw files.
I think that those missing chromatograms would show up if you told Skyline to extract chromatograms again.
That is, you should go to:
Edit > Manage Results
and push the "Reimport" button.

What I think happened was that you went to:
Edit > Manage Results
and removed all of the replicates and then did "File > Import > Results" with the intention that Skyline would extract chromatograms.

Unfortunately, you need to save the Skyline document after removing all of the results and before telling Skyline to extract chromatograms again.
The act of saving the document after removing the results causes Skyline to delete the .skyd file from disk.
If you do not save the document before telling Skyline to extract chromatograms again, Skyline will see that there is already a .skyd file on disk which has chromatograms for the necessary files and so Skyline thinks that no work needs to be done.

We are going to fix it in Skyline-daily soon so that it tells you to save the document in a scenario like this.

I am not 100% sure that this is what happened to you. When I look at the Audit Log ("View > Live Reports > Audit Log") I cannot tell whether the document was saved in between the last time that you removed results and imported results.
However, when I tell Skyline to extract chromatograms from the .raw files you sent, I do see chromatograms for the things that you have missing chromatograms for, and this is the only explanation I could think of about what is going on.

-- Nick
 
afshari1 responded:  2024-09-13 17:02
Thank you so much for your response. I could solve the problem.
I have another question regarding some signs in the target list. I have marked three signs in the attached file. I would be grateful if you could explain what these signs mean.
 
Nick Shulman responded:  2024-09-13 17:25
There is a bit of an explanation of the red, green and yellow symbols here:
https://skyline.ms/announcements/home/support/thread.view?rowId=64988

The symbol next to a transition is always going to be either red, green, or missing.
If "Settings > Integrate All" is checked, the symbol next to the transition is going to be green unless the chromatogram is completely flat between the peak boundaries.
If "Settings > Integrate All" in unchecked then Skyline does a few more checks including making sure that the apex of the peak is within a quarter of a peak width of the apex of the highest chromatogram in the group, and also that the Pearson correlation between that transition's chromatogram and the tallest chromatogram is above a minimum value.

At the precursor and peptide levels, the symbol will be green if all of the transitions have a green symbol.
The symbol will be yellow if more than half of the transitions have a green symbol.
The symbol will be red if fewer than half of the transitions have a green symbol.
-- Nick