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Can't get a Calibration Curve - Error: All of the external standards missing one or more peak”

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Can't get a Calibration Curve - Error: All of the external standards missing one or more peak” markus kellmann  2026-02-27 05:18
 

I have a data set with a dilution series of heavily labeled peptides and would like to create a calibration curve. The individual transitions look good; I have reduced them to the point where all transitions are visible and integrated. However, I am getting the following error message: “Error: All of the external standards missing one or more peak”.
Unfortunately, other reports here along the same lines have not helped me. Could you please take a look to see if I am doing something wrong?
Zip is attached.
Thank you in advance for your help.
Markus
 
 
Nick Shulman responded:  2026-02-27 07:33
Sorry that the error message is misleading.

What you actually need to do is go to the "Modifications" tab in "Settings > Peptide Settings" and change "Internal Standard Type" to "None".
Skyline expects that the internal standard will be spiked into your replicates in equal amounts, so it assumes that you can't possibly want to use your heavy peptides for quantification. Skyline can't find anything that is not an internal standard to plot on the calibration curve and unfortunately gives you an error message about missing peaks. But, what you really need to do is tell Skyline that your heavy label is not being used as an internal standard.

-- Nick
 
markus kellmann responded:  2026-03-01 22:39
Hi Nick,
Thanks for the quick reply. Yes, I must have overlooked that. Now it's working fine for me.
Sometimes you just don't look closely enough.
Thanks again,
Markus