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Importing peptide search issue vaclavek jana  2023-11-15 05:44
 

Dear Skyline Team,
I have searched the MS data using Proteome Discoverer 3.0 and I wanted to import the peptide search data to Skyline and analyse the peptide peaks. I need to import the data including low number of synthetic heavy labelled peptides (in some cases the file should include a single peptide).
I found out that it is not possible to use the data when the PSMs are validated using Fixed Value PSM Validator because the q-value is absent. It is needed to use the Percolator. However, using of Percolator is not good for me in case of such a peptide poor data. Is it possible to solve this and use the data validated by Fixed Value PSM Validator somehow? In some cases, the synthetic peptides which I measured and I know that they are present could not be seen in Skyline when I import the data validated with Percolator but they are seen in Proteome Discoverer search validated with Fixed Value PSM Validator. In case of files which include only a single peptide I was not successful with importing the peptide search at all, see the attached printscreen.
Additionally, I have one more question regarding importing the peptide search data. I need to analyse some specific peptides which were previously identified by our shotgun proteomic analysis and should be further studied. These peptides are relatively long (20 - 25 AAs) and contain some miscleavages. In this case the Skyline showed that the shorter version without miscleavages is present and did not show the longer version with miscleavages despite the fact that the longer version with miscleavages is present for sure and the shorter should not be present at all. Is there some solution how to see the correct peptide which was synthetized for this purpose?

Thank you very much for your comments,
Jana Vaclavkova
 
 
Nick Shulman responded:  2023-11-15 08:16
What do you mean when you say that Skyline shows that the longer version of your missed cleavage peptide is not present?
Are you saying that no peak was found in the extracted chromatogram, or are you saying that the peptide did not appear in the Targets tree?

If there are peptides in your spectral library which are not appearing in the Targets tree, you can always add them by going to:
View > Spectral Libraries
and pushing the "Add All" button.

I am not sure whether there is anything that can be done about dealing with "Fixed Value PSM Validator" q-values. I am not the expert on reading Proteome Discoverer files, but if you send us all your data files we might be able to figure something out.
I am not sure which files we would need. Probably the .pdresult files and your .raw files, and you could also use the "File > Share" menu item in Skyline to create a .sky.zip file containing your Skyline document and supporting files including extracted chromatograms and spectral libraries.

You can zip everything up together and upload it here:
https://skyline.ms/files.url

-- Nick
 
vaclavek jana responded:  2023-11-16 06:57
Hi Nick,
concerning the longer version of peptides, the missed cleavage peptide is this: XXXXXXIKLEKXXXXHGK. In Skyline, I see the two versions: XXXXXXIK and XXXXXXIKLEK but not the whole XXXXXXIKLEKXXXXHGK which I was originally synthetized and which I need to see.
I have not seen it in the targets tree. In case I went to View > Spectral Libraries and pushed the "Add All" button, some "Library Peptides" appeared in the Targets tree but they are not present - the chromatogram information were unavailable.

I will zip up both the Fixed Value PSM Validator and Percolator searched file of one "one-peptide" file which reported an error as I provided. I have used the .msf files for importing peptide search, thus, I am sending the .msf files as well.

Jana

Note: Tomorrow, there is a bank holiday in the Czech Republic, so next time I will be available on Monday.
 
Nick Shulman responded:  2023-11-16 08:13
When you add new things to the Targets tree, you usually need to tell Skyline to extract chromatograms again by pushing the "Reimport" button at "Edit > Manage Results".
Newly added things will show "Chromatogram information unavailable" until you tell Skyline to extract chromatograms again.
-- Nick
 
vaclavek jana responded:  2023-11-16 08:33
Thank you for your effort. Now, I see the chromatograms for the Library peptides. However, the library peptides did not help with my problem. I still do not see what I need to see.
Jana