QUantification

support
QUantification priscillia lagoutte  2021-08-31
 

HI,

I will contact you because I try to find the linearity range of my peptide for quantification assay. So I Injected 10 different quantities of synthetic heavy peptide and then I looked calibration curve (generated by Skyline). I also export total areas (from result grid) and analyse with excel.

I found that calibration curve is different because in case of skyline, it takes peak areas (Max height) and in my case I look total areas and linearity range is not the same between both calibration curve.

Can you help me to understand ?
Thanks in advance

Priscillia

 
 
Nick Shulman responded:  2021-08-31
Can you 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.

If that .zip file is less than 50MB you can attach it to this support request.
Otherwise, you can upload it here:
https://skyline.ms/files.url

The number which gets plotted on the Calibration Curve comes from a column called "Normalized Area". It is possible to see that column on the Results Grid if you have a Peptide selected in the Targets tree. When you customize the view in the Results Grid, you can find the Normalized Area column under "Peptide Results > Quantification > Normalized Area".

If you would like to learn more about using the Document Grid and the Results Grid, you might find the Custom Reports tutorial helpful:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_custom_reports

If your peptides have only one precursor under them, then I would expect the "Total Area" values that you exported would be equal to the Normalized Area values that get plotted in the Calibration Curve. Skyline never does anything with the value "Max Height".

After I see your Skyline document I will probably be able to give you a better answer as to why your Excel graph looks different than what you are seeing in Skyline.
-- Nick