When you choose "Ratio to heavy" as the normalization method, Skyline expects that the heavy standard will be present at approximately the same concentration in all the samples and the light concentration will be different for different concentration levels.
For this reason, "Ratio to heavy" is not the correct thing to choose if your calibration samples have different heavy concentrations.
Your calibration curve has a negative slope because the heavy concentrations are changing in the completely wrong way in your external standards for what would be expected if the heavy peptide were being used as an internal standard.

Skyline does not really support "reverse calibration curves" in the sense that you are thinking of them.
People do use Skyline to create response curves using spiked in heavy standards, but the only purpose of those response curves is to figure out what peptide concentration is within the linear range of the mass spectrometer.

After you have verified that the peptide that you are looking at is in the linear range, then you would probably spike your heavy standard into your unknown sample at a known concentration, and then just look at the "Ratio to heavy" value to figure out what the analyte concentration is.
You can also use the Document Grid to fill in the values for a column called "Internal standard concentration" and then the "Calculated Concentration" value will be the ratio to heavy multiplied by the Ratio to heavy value.

Here is a support request where someone asks about reverse calibration curves and the answer was that the only thing you should use them for is verifying the linear range:
https://skyline.ms/announcements/home/support/thread.view?rowId=36322

If you would like to learn more about the Document Grid this is a good tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_custom_reports
-- Nick

Thank you for your response. I am not interested in reverse curve.

I just want to know the calculated concentration?? Can you please help me how should I do, please let me know the steps.
My internal standard (heavy) concentration is 10nm. Especially, which normalization method I should use (attached)??




Many thanks
Arpita

I think what you need to do is to tell Skyline not to try to create a calibration curve.
To do that, you would go to the "Quantification" tab at "Settings > Peptide Settings" and set "Regression fit" to "None".
Also, on the Quantification tab you can set "Normalization method" to "Ratio to heavy".
That is the easiest way to change the normalization method for all of the peptides in your document.
The "Normalization Method" column in the Document Grid is usually only used if a particular peptide needs to have a different normalization method than everything else in the document.

Once you have done that, I believe that the "Calculated Concentration" column will show you the Internal Standard Concentration times the measured ratio to heavy.

If you still need help you should 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

Dear Nick,

Thank you so much for your response, it worked.



Arpita