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Quantitation Using the Sum of Two Different Peptides with Different Retention Times

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Quantitation Using the Sum of Two Different Peptides with Different Retention Times jay johnson2  2023-03-30 11:24
 

I am trying to set up a Skyline that will allow us to quantitate the sum of two forms of a peptide, a version with an additional amino acid at the c-terminus and a version lacking this terminal amino acid. I can set up the target list fine by entering an adduct for the peptide version that has one less amino acid. However, the integration for the two peptides is linked and due to the structural difference the retention time is not identical. Is there anyway to independently integrate each peptide but have the calibration curve be a sum of the two peptides?

Best,
Jay
 
 
Nick Shulman responded:  2023-03-30 12:37
There is no way to do this right now in Skyline but we do have plans to make this possible soon.
Specifically, a future version of Skyline will allow you to have protein-level calibration curves (which will also work for any list of peptides) where the things plotted on the y-axis will effectively be the sum of the transition peak areas across the two peptides.

With the current version of Skyline you can find the Protein Abundance column in the Document Grid which would be the correct number for you if your Protein in the Targets tree has the two peptides that you are interested in. However, the current version of Skyline cannot plot those Protein Abundance values on a calibration curve.

If you would like to learn more about the Document Grid you should look at the Custom Reports tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_custom_reports
-- Nick