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Problems with MS1 extraction frederik tellkamp  2023-06-28 07:02
 

Dear Skyline support team and community,

I am trying to set up a PRM measurement. To this end I wanted to use some DDA runs to extract chromatograms and generate a transition list for PRM. One of the peptides I am particulary interested in is an N-terminal phosphopeptide.

Unfortunately, after MS1 extraction this very phosphopeptide did not appear in Skyline, whereas the unphosphorylated one did. I was puzzled and did some troubleshooting (I am new to this software I should mention here):

  1. Adjusting the filter criteria in the "peptide settings..." and "transition settings...". Namely: possible charge states, number of modifications per peptide, number of missed cleavages
  2. I switched to a single protein FASTA to make all peptides automatically unique.
  3. I tried other filter criteria ("Pick peptides matching:").
  4. I have re-run the MQ analysis with a rather late version to make sure there is no obsolete phrasing in the output that prevents parsing.
  5. Along with point 4. I have re-run the MQ analysis using p(ST) instead of p(STY) because I realize that p(STY) is not a modification in Skyline, even when the "modifications.local.xml" file is in the correct location.

I noticed other prominent phosphopeptides in the data that have been extracted without problems.
My question: What do I wrong here?

I used MQ 2.0.1.0 and Skyline 22.2.0.527. I guess you may need some files, i attached the msms.txt, the mqpar.xml and the modifications.xml.
I am interested in the phosphorylated peptide with the Peptide ID 436 (all sites basically).

Thanks in advance your your help!
Frederik
 
 
Nick Shulman responded:  2023-06-28 07:12
I am not sure I understand your question.

Can you 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 spectral libraries and extracted chromatograms.
If that .zip file is less than 50MB you can attach it to this support request.
You can upload larger files here:
https://skyline.ms/files.url

When you import a peptide search into Skyline, Skyline creates a spectral library (.blib file) containing the peptides from the peptide search. You can use the Skyline menu item "View > Spectral Libraries" to see which peptides are in the spectral library.
If the peptide you want is in the spectral library, but, for some reason, was not added to your document, you can use the "Add" or "Add All" buttons in the Spectral Library Explorer to add the missing peptides to your document.

If the peptides that you want are in the document, but they do not have the correct modifications on them, you can right-click on the peptide in the Targets tree and choose "Modify" and add modifications to the peptide.

If you would like to learn more about modifications in Skyline, I recommend the "Working with Modifications in Skyline" webinar:
https://skyline.ms/webinar10.url
-- Nick
 
Nick Shulman responded:  2023-06-28 07:58
Thank you for uploading your Skyline document.

Which peptide are you interested in? We do not have any MaxQuant software, so I do not know what you mean by "Peptide ID 436", but if you tell me the amino acid sequence of that peptide I will be able to figure out which one it is.
-- Nick
 
frederik tellkamp responded:  2023-06-28 07:59
Dear Nick,

thanks for your quick response. So my problem is mainly that there is not chromatogram or any other kind of information extracted for that specific peptide. I can find it in the library, I can add it or I can add the modification. But there is no retention time, no ID or any other information for any sample visible. The graphs stay empty. It appears that the peptide has not been measured.
I uploaded the file "230628_PRM_phosphopeptide.sky.zip" in the file share folder. I am specifically interessted in the peptide "-.MASQRHSGPSSYK.V [1, 13]". This peptide gets its methionine cleaved off and then acetylated at A2 and phosphorylated at S3.

Regarding the webinars: yes I have started to watch them, quite a lot, they are very helpful!
Thanks,
Frederik
 
Nick Shulman responded:  2023-06-28 08:12
I think the problem is that when you remove the Methionine from the beginning of that peptide sequence, you no longer have a fully tryptic peptide.
There are a couple of ways to deal with this.
One way would be to go to:
Settings > Peptide Settings > Digestion
and maybe choose "Edit Current" from the "Enzyme" dropdown and check the checkbox that says "Allow semi-cleavage".

Probably the better way to deal with this would be to go to:
View > Spectral Libraries
and push the "Add All" button.

All of the peptides that are currently missing from your document will be added to a peptide list called "Library Peptides" and you will be able to find "ASQRHSGPSSYK" in that list.

If you want "ASQRHSGPSSYK" to get moved into the appropriate Protein, you can use the menu item "Refine > Associate Proteins".
-- Nick
 
frederik tellkamp responded:  2023-06-28 08:32
Dear Nick,

The checkbox "Allow semi-cleavage" did it! Thanks for this great and super fast support!

Frederik