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Peptide quantification with single point internal calibration

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Peptide quantification with single point internal calibration ahat erpan  2025-03-21 08:59
 

Hi Skyline Team,

In general I use calibration curve for quantification but needed to use single point internal calibration using a heavy standard spiked into each sample at a constant amount. In the document grid, I have a ratio to standard tab with value and my internal standard concentration tab with the given value. However, the value in the quantification tab for the endogenous analyte value is different from a simple multiple of the ratio to standard with internal standard concentration.

I thought they should match? Is there a regression fit enabled in internal calibration, is that why they don't match? Or did I make a mistake somewhere.
 
 
Nick Shulman responded:  2025-03-21 09:10
If you want Skyline to calculate the concentration by multiplying the Internal Standard Concentration by the ratio to heavy make sure that "Regression Fit" on the Quantification tab at "Settings > Peptide Settings" is "None" and "Normalization Method" is "Ratio to Heavy".

Other than that I cannot think of why you would be seeing the numbers you are seeing.
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
-- Nick
 
ahat erpan responded:  2025-03-21 09:51
Thank you. I think my settings are correct based on your description. I attached the zip file in the attachment. Please let me know if you find anything.
 
Nick Shulman responded:  2025-03-21 10:11
The problem is that you have two label types "heavy" and "heavy_p" and even though Skyline knows that the normalization method is "Ratio to Heavy-P" there is no explicit way to tell Skyline what is supposed to go in the numerator of that ratio.
When calculating the "Normalized Area" value, Skyline adds the peak areas for all of the label types which are not specified as internal standards on the "Modifications" tab at "Settings > Peptide Settings".
So, the values that you are seeing in the Quantification column in your screenshot are (light + heavy) / (heavy-P).
One thing that you could do to get those to be the correct number would be to mark all of the Transitions for that heavy label type as non-quantitative.
Alternatively, you could check the checkbox next to "heavy" in the "Internal standard types" list box on the Modifications tab at "Settings > Peptide Settings".

-- Nick