Modifications for this peptide do not match current document settings. licky lck  2022-02-28
 

Hi Skyline team,

I am having a hard time when I am trying to add some identified peptides from the spectral library explorer to my peptide list since some of my included modifications were somehow could not be interpreted by Skyline. I am quite certain that I have added these modifications manually (in the peptide setting->modifications) and their masses actually matched the ones that Skyline could not be interpreted. I selected all modifications to be variable, and I tried to play with other settings, such as max m/z, max peptide length and etc. However, the problem still remained. So I think I really need your help, thanks. :)

Bests,
Chengkang

 
 
Nick Shulman responded:  2022-02-28
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 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

After we see your Skyline document we will probably be able to figure out what is going wrong.
-- Nick
 
licky lck responded:  2022-02-28
Dear Nick,

Thank you for your response.

I think my .zip file was too large (100+MB) to be uploaded to the file sharing, plus, there are unpublished data, so can I send the data to you in an alternative way?

Best regards,
Chengkang
 
Nick Shulman responded:  2022-02-28
Thank you for uploading your Skyline document.

I see that Skyline is unable to match modifications on Arginine whose masses are:
54.04738
52.0315
72.06266

I see that you have defined some modifications at "Settings > Peptide Settings > Modifications".
The monoisotopic mass of the modifications that you have in your Skyline document do not match those masses, but the average masses do.
In order for Skyline to decide that a modification in your library is the same as the one in your Skyline document, the monoisotopic masses need to match.

I imagine that these average masses are in the library because that is what your peptide search engine used. I imagine you could fix this by doing the peptide search again and use monoisotopic masses instead of average masses.

If you really need to use peptide search results where the mass modifications are a little incorrect, there is a way to do it. Many years ago, BiblioSpec only cared about the first decimal place of modification masses. Nowadays, whenever Skyline tells BiblioSpec to create a spectral library from peptide search results, Skyline always includes the "-H" command line argument which tells BiblioSpec to remember all of the digits of modification mass precision. There is no way to tell Skyline not to use the "-H" command line argument, but if you run BlibBuild.exe yourself from the commandline, you can leave off the "-H" commandline argument.

We might be able to give you more ideas if we could see your peptide search results.
-- Nick
 
licky lck responded:  2022-02-28
Dear Nick,

Thank you for your response, but that was strange because we did use the monoisotopic masses for the search.

Bests,
Chengkang
 
Nick Shulman responded:  2022-02-28
Which peptide search engine did you use? Can you send us your peptide search results?
It is possible that BiblioSpec is misinterpreting the modification masses in your peptide search results and that we should fix that.
-- Nick
 
licky lck responded:  2022-02-28
Dear Nick,

sorry about that, I think you are right, the masses for the search were average masses. thank you so much for your inspiration. we can close this case now. :)

Best regards,
Chengkang