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Unable to generate calibration curves r yagoubi19  2022-01-22 03:42
 

I am trying to generate a calibration curve but after following all the steps I get an error message: "Error: All of the external standards are missing one or more peaks. The selected replicate has missing or truncated transitions." I don't understand this error message as all of my standards have clear well-defined peaks.

I have designated all of my external standards as "Standard" and filled in their concentrations in the document grid.

I also paid attention to light and heavy parameters.

Can anyone help me? Did I forget something?

Thanks
 
 
Nick Shulman responded:  2022-01-22 07:06
If you send me your Skyline document I will be able to tell you what is going wrong.

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.

If that .zip file is less than 50MB you can attach it to this supported peaks.
You can upload larger files here:
https://skyline.ms/files.url

Sometimes you get that that error message if some of your transition peaks are "truncated". That means that the peak boundary is at the edge of the extracted chromatogram. When a peak goes all the way up to the end of the chromatogram like that, Skyline considers that the peak area is an unreliable number, and so it does not end up getting plotted on the calibration curve. If you adjust the peak boundary of that peak so that it is no longer at the end of chromatogram, then Skyline will all its area to be used on the calibration curve.

After I see your Skyline document I will be able to tell you what is going wrong.
-- Nick
 
r yagoubi19 responded:  2022-01-22 08:34
Hi Nick,

Thank you for your quick answer. I think you are right, it might be the case here, I do think that the peak are at the edge of the chromatogram, but I don't know how to change it, but you're right it might also be something else!

 I have attached my file. Thank you so much again!
 
Nick Shulman responded:  2022-01-22 09:36
Thank you for sending your Skyline document.
The problem is that you have marked "Light" as the internal standard, and the only thing that you have in your document are light precursors.
You should go to:
Settings > Molecule Settings > Labels
and uncheck the checkbox next to "Light".

-- Nick
 
r yagoubi19 responded:  2022-01-23 02:32
It worked! Thanks so much Nick!