defining same retention times for Promega 6 × 5 LC-MS/MS Peptide Reference Mix

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defining same retention times for Promega 6 × 5 LC-MS/MS Peptide Reference Mix Lauri Peil  2025-07-15 21:45
 

It's probably just me not being able to find and use correct settings but the issue I have is following. When using Promega peptide mixture, three to four of the more abundant heavy isotopologues are easily detected and quantified, however the lower abundant peptide signal(s) is/are often picked from a wrong retention time range. Is there any way to define that the same RT window should be used for all peptides, where correct RT range is determined from the heaviest isotopologue peptide? It looks to me that defining opposite is easy where RT for heavy standard is defined by light peptide but can I do it the other way round?
Thanks a lot for your help, Skyline is by far one of my favorite programs and tools for MS!

 
 
Nick Shulman responded:  2025-07-15 22:36
When you have multiple heavy labeled forms of the same peptide, you should go to the "Modifications" tab at "Settings > Peptide Settings" and choose "Edit List" in the "Isotope label types" dropdown.
In the "Edit Label Types" window that pops up you should tell Skyline that you have five different label types and maybe call them "heavy1", "heavy2", "heavy3", "heavy4" and "heavy5".
After you have told Skyline that you have five label types, you should right-click on one of your peptides in the Targets tree and click the "Modify" menu item.
You should use the "Edit Modifications" dialog to tell Skyline which modifications should be applied to "heavy1", "heavy2", etc.
There should be a node in the Targets tree that represents the peptide, and the Peptide node in the tree should have five Precursor children representing the heavy1, heavy2, etc.

In your screenshot, the different modified forms of the peptide are separate peptide nodes in the Targets tree. The problem with having things that way is that Skyline treats those peptides as if they had structural modifications which would cause them to have to have retention times which are unrelated to each other.
If you instead make it so that it's a single peptide node with five Precursor node children, then Skyline will know that they all have the same retention time.

Also, if this is an isotopolog response curve, where the different labeled forms of the peptide have been spiked in at different concentrations, you should tell Skyline what those concentrations are.
You should create a custom report in the Document Grid which includes the "Precursor Concentration" column and then you should fill in the values in that column with the concentrations.
You can learn more about custom reports and the Document Grid here:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_custom_reports

-- Nick