For most calibration curves, the X-Axis will say either "Analyte Concentration" or, if "units" have been specified on the "Quantification" tab in the Peptide Settings, it will say "Analyte Concentration (units)".
However, if you have specified an "Internal Standard Concentration" for the peptide, then the X-axis will say "Heavy:Light Concentration Ratio" like it does in your screenshot.
I am not sure exactly why Skyline does this.
The primary purpose of the specifying the "Internal Standard Concentration" for the peptide is for the scenario where you do not have any external standards, and you just want Skyline to calculate the concentration by multiplying the observed ratio by the Internal Standard Concentration value that you have provided.
The Internal Standard Concentration value is usually left blank if you have external standards.
If you were to change the Internal Standard Concentration to blank, then the X-axis would say "Analyte Concentration".

Does the calibration curve end up looking correct if you change the Internal Standard Concentration to blank?
-- Nick

Hi Nick,

If I change it to blank then the curve is blank as there is an error "Matrix must be positive definite". I can restore this by switching normalisation method to "none" and then I get the analyte concentration on the x-axis. However, the reason I want to normalise to light is so that the peak area is accounted for by any change in the light which is set to 100fmol. Therefore any change in the heavy peak area is a true change and not because of more was loaded/issue with MS detection - which the light will also be effected by.

So ideally I would like the peak area to still be normalised to light but not the analyte concentration.

Do you think this is possible?

Thanks,

Sam

Can you send me 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

I believe you can get that error if Skyline is confused about what the internal standard is, and so Skyline thinks you have no data.
On the "Modification" tab at "Settings > Peptide Settings", make sure that "heavy" is not chosen in the "Internal standard type" list.
Skyline expects that the internal standard will be present in all of your replicates, and, therefore, those internal standard peak areas will never be used in the numerator of the values that are plotted in the calibration curve, even if you have chosen "ratio to light" as the normalization method.

After I see your Skyline document it will probably be obvious what is going wrong.
-- Nick

Hi Nick,

Thank you for your help, I have attached the zip file of the calcurve document to the file sharing document

Sam

Thank you for uploading that Skyline document.
When I changed the Internal Standard Concentration of all the peptides to blank, I was able to see calibration curves for every peptide and the label on the X-axis was "Analyte Concentration (fmol/run)"
I am not sure what you might have done differently.
I am attaching the document that I made (I removed the background proteome so that it would be small enough to attach to a support request).

If you still cannot get it to work, please send me the Skyline document where it is giving you the "Matrix must be positive definite" message.
-- Nick

Hi Nick,

Ahh yes this has fixed it, before i set "Internal Standard Concentration" to zero not blank which caused the error.

Thank you

Sam