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Dilution factor and accuracy

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Dilution factor and accuracy jeroen kooistra  2023-10-06 00:22
 

Hi Skyline team,

We are currently setting up an Quantitative bottom-up method for a therapeutic protein. We apply different forms of the protein to investigate the parallelism. Because one form has lower molar weight than the other, more surrogate peptide will be formed per µg of protein. We apply a dilution factor to correct for that. Skyline calculates a corrected concentration as required, but the accuracy is not corrected. Could you please advise how to generate accuracy's corrected by the dilution factor?
We apply a calibration curve and a global standard as internal standard.

Many thanks and with kind regards,

Jeroen
 
 
Nick Shulman responded:  2023-10-06 00:37
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. You can upload larger files here:
https://skyline.ms/files.url

This sounds like a problem that was reported two years ago:
https://skyline.ms/announcements/home/support/thread.view?rowId=50690

It definitely looks like we did make a code change to fix that problem back then, so I am not sure what might be going wrong now.
-- Nick
 
Nick Shulman responded:  2023-10-10 11:23
Jeroen,

I just noticed that you were using the "Explicit Analyte Concentration" column on each of the peptide results.
Normally, you would have set the "Analyte Concentration" value on each of the replicates.

The "Explicit Analyte Concentration" column is something that you would use if your individual peptides did were all diluted by completely different amounts in the different replicates and you need to specify the "Full Matrix" of all of the concentrations.

If you have a standard dilution series, you would normally set the "Analyte Concentration" on each of the replicates, and, if your peptides had different starting concentrations, you would set the "Concentration Multiplier" on each peptide.

It looks like Skyline doing something wrong if you have specified both the "Dilution Factor" and the "Explicit Analyte Concentration". But, if you just stick with "Analyte Concentration" and "Dilution Factor", you will get the results you expect.
-- Nick
 
jeroen kooistra responded:  2023-10-10 23:33
Thanks a lot Nick!

I will try it out.
But on the other hand, "Explicit Analyte Concentration" should be corrected by the dilution factor also, am I right?
 
Thanks for the cooperation.