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Peptide modifications not matching document settings

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Peptide modifications not matching document settings leandroxneves  2025-02-21 13:10
 

Hi,

I created a spectral library from PEAKS and when I try to add peptides in my skyline document sometimes I see an error message saying the modications don't match the document settings in spite of all the mods have been enabled in Peptide settings.

I appreciate any help in sorting out what I'm missing here

Kind regards,
Leandro
 
 
Nick Shulman responded:  2025-02-21 13:35
Unfortunately, the library in the .sky.zip file that you attached does not contain the "SWIS..." peptide in your screenshot.

I think you used the "Minimize libraries" option in the "Share Skyline Document" dialog. That removes from the library all peptides which are not in the current document, which includes the peptide you were having trouble with.

Can you send us a new .sky.zip file where "Store everything" is chosen in the Share Skyline Document dialog?
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
 
leandroxneves responded:  2025-02-21 13:47
Hi Nick

the complete sky.zip file was too large to attach in the first request but I have now uploaded it using https://skyline.ms/files.url. it is called 20250221_ESO_all_QUANT1.sky

thanks,
Leandro
 
Nick Shulman responded:  2025-02-21 14:18
Thank you for uploading that .sky.zip file.
It looks like the problem is that your settings say to use the modification "13C (R)", but that peptide has multiple arginine residues and only the one at the C-terminus is heavy. Skyline is unwilling to apply the 13C modification to the arginine at the end without also applying that same modification to all of the other R residues in the peptide.

You should go to the Modifications tab at "Settings > Peptide Settings" and change it so that the heavy modifications that you have selected all have the "Terminus" set to "C".
Unimod already provides a modification called "Label:13C(6) (C-term R)" which you can use for your 6 Dalton modification. For the 3 Dalton modifications that you have defined you will just need to select "C" for the Terminus.

By the way, I see that you have defined your 3 Dalton modifications by specifying their monoisotopic and average masses. Usually the way that you would specify that a modification involved the substitution of 3 ordinary carbons with 3 heavy carbons would be by saying the chemical formula of the modification is:
C'3-C3

C' is the symbol that Skyline uses to mean "carbon 13". Chemical formulas in Skyline are allowed to have one minus sign in them and everything to the right of the minus sign is subtracted from the stuff to the left of the minus sign.
-- Nick