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Setting match luzjpaulo25041  2024-07-24 13:30
 

Dear Skyline Support Team,

I hope this message finds you well. I am writing to seek assistance regarding an issue I am experiencing with the Skyline program. Specifically, I am encountering difficulties importing a peptide with a PTM (Post-Translational Modification) due to a configuration mismatch with my current settings.

Here’s a brief overview of the problem: Skyline indicates that it cannot import a specific peptide, even though there is a similar peptide in the library with the same modification mass that Skyline does recognize. This discrepancy has left me unsure about which settings I need to adjust in order for Skyline to correctly recognize and import the peptides from my library.

Could you please provide guidance on what configuration settings I should review or modify to ensure that Skyline recognizes the peptides with PTMs from my library? Any advice or steps you can provide would be greatly appreciated.

Thank you very much for your assistance. I look forward to your prompt response.

Best regards,
 
 
Nick Shulman responded:  2024-07-24 13:58
Which peptide were you hoping that Skyline would recognize?

When I go to:
View > Spectral Libraries
I see the picture that I have attached "UnrecognizedModifications.png".

Do you know what those modifications are supposed to be?

Skyline does not typically allow a single amino acid to have more than one modification on it, so, if those modification masses (e.g. 140.08373 on a "K") are a combination of multiple modifications, you should go to the "Modifications" tab at:
Settings > Peptide Settings
and define a single modification which has the chemical formula of the two modifications added together.

If you would like to learn more about working with modifications in Skyline, this is the webinar that we always recommend:
http://skyline.ms/webinar10.url

By the way, if you ever need to send your Skyline document to someone else, the best way to do that is to use the menu item:
File > Share
to create a .zip file.

Skyline will make sure that the .zip file contains all necessary files including extracted chromatograms, spectral libraries and the audit log.
-- Nick
 
luzjpaulo25041 responded:  2024-07-24 14:58
Hello Nick

Thanks for the reply

I have a example of a peptide
for stance for KSAPATGGVKKPHR++ there is a peptide with the following PTMs K(+112,05243)SAPATGGVK(+70,04186)K(+56,02621)PHR++ that a cannot import even though I ve created especific modification in settings menu > Propionyl_metyl (K) (for + 70,041865 C4H6O ), 2PRO (K Nterminal) (for + 112,05243 C6H802 ) and Propionyl(K) (+ 56,026215 C3H40)
 
Nick Shulman responded:  2024-07-24 15:45
The problem is that there are some modifications at "Settings > Peptide Settings > Modifications" which have a checkmark next to them but which have not been marked as "Variable".

Usually, the only modification that should be like that is "Carbamidomethyl (C)".

Modifications with checkmarks next to them in the "Structural Modifications" list at "Settings > Peptide Settings > Modifications" are either "Implicit" or "Variable".
Skyline believes that implicit modifications must always be applied to every amino acid that they apply to.
For this reason, Skyline is unable to figure out what combination of modifications can produce the masses in your library because Skyline thinks that every Lysine must have "Propionyl_metyl (K)", and "Propionyl(K)" applied to it.

You should go to the Modifications tab at "Settings > Peptide Settings" and change it so that all of the implicit modifications are marked as "Variable".
-- Nick
 
luzjpaulo25041 responded:  2024-07-24 16:43
Hello Nick

Thank you once again for your prompt and effective support. Your guidance on adjusting the configuration settings to ensure recognition of peptides with PTMs from my library was invaluable and greatly appreciated.

Regards Joao