Thank you for the diagram.
I see that you have these concentrations:
|Replicate||Compound A Conc||Compound B Conc
|STD0||5 ng/mL||8 ng/mL
|STD20||25 ng/mL||48 ng/mL
|STD40||45 ng/mL||88 ng/mL
|STD60||65 ng/mL||128 ng/mL
|STD80||85 ng/mL||168 ng/mL
|STD100||105 ng/mL||208 ng/mL
In this case, there is no simple ratio between the concentration of compound A to compound B across the replicates.
Skyline does not currently support this, so you would have to deal with compound A and compound B in separate Skyline documents, or you would have to lie to Skyline about what the actual concentrations were and then add or subtract an appropriate number from the final result.
We have anticipated for a long time that Skyline might need to allow users to specify the "full matrix" of concentrations of compounds and replicates instead of just relying on the Replicate's "Analyte Concentration" and the Molecule's "Concentration Multiplier".
Are you planning on using the calibration curve in Skyline to calculate the concentration of unknown samples? If so, you should definitely send us your Skyline document. The main reason that Skyline does not have this "full matrix" feature yet is that we have not seen a dataset that required it, and so I was not confident that I could implement this and get the correct numbers without a datset to test it on.
Usually when scientists are requesting this "full matrix" feature, they just want to determine the linearity of the response curve. When it actually comes to using a calibration curve to quantify unknown samples, they always have been intending to use a single point calibrator.
Either way, it would be great to see your data.
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files.
If that .zip file is less than 50MB you can attach it to this support request. Otherwise, you can upload it here: