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Global peptide, can't see the options described in the previous answer

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Global peptide, can't see the options described in the previous answer yurubi  2025-04-08 13:07
 

Hello there, I found an answer that described what I am trying to do, set one surrogate peptide as global peptide for several light peptide from a previous thread but it is not working for me. It mentioned to set the Standard Type of each of these heavy peptides to "Surrogate Standard" and then set the "Normalization Method" for all the other peptides to the appropriate "Ratio to Xxxx". However, I can't see the options to Ratio to Light or Ratio to Heavy after setting the whole peptide as Surrogate Peptide, do I have to change something under the "peptide setting" "modifications" tab? There I have the internal standard type set as "heavy". Do I set the peptides with no heavy counterpart as Standard Type "none"?

Thank you very much,

HHB!
 
 
Nick Shulman responded:  2025-04-08 13:16
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.

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

I am not sure why Skyline is not offering you the normalization option you want, but after I see your document it will probably be easy to figure out.
Can you also send me a screenshot of what you are looking at?
-- Nick
 
yurubi responded:  2025-04-08 14:28
Here it is, thank you
 
Nick Shulman responded:  2025-04-08 15:03
Thank you for attaching that Skyline document.

Where are you looking that you cannot find the "Ratio to Heavy" option.
The usual place to set the Normalization Method of molecules is in the Document Grid.
If you choose the report "Peptide Quantification" there will be a "Normalization Method" column where you can specify the normalization method for each molecule.
The place to set the default normalization method is on the Quantification tab in the Peptide Settings dialog, and the place to set overrides of that default normalization method is in the Normalization Method column in the Document Grid.

Note that you cannot choose "Ratio to Surrogate Xxx" as the default normalization method.
If you had a surrogate standard that you wanted to use as the default normalization method then you should make it a Global Standard instead of a Surrogate Standard.

You can learn more about the Document Grid here:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_custom_reports

Here is the help page for Surrogate Standards:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=Surrogate%20Standards

I hope this answers your question. If not, can you post a picture of what you are looking at?
-- Nick
 
yurubi responded:  2025-04-08 15:14
Nick, I can see "Ratio to Heavy" but in your explanation in the other thread I understood that if I set the whole peptide as Surrogate Standard then I should see "Ratio to Light" and "Ratio to Heavy" options. Let me try what you suggested first. Thank you!