Reverse calibration curves for determining LOD and LOQ in PRM assays resulted in some kind of paradox

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Reverse calibration curves for determining LOD and LOQ in PRM assays resulted in some kind of paradox sofia farkona  2019-11-08
 
Hello all. I have a question which is not limited to Skyline software. However I would appreciate it if you could have a look. I have developed PRM assays targeting of some proteins of interest. The biological sample into which I apply the PRM assays is bronchoalveolar lavage. I have constructed REVERSE calibration curves to determine LOD and LOQ because the majority of the endogenous peptides I look at are present in most of the samples. Basically the heavy peptides are spiked in, in different dilution in the same BAL sample (or in a pool of BAL samples). In that way our calibrator sample have a constant concentration of the light or endogenous peptide while the heavy counterparts are in different concentrations. What we plot is the H/L ratio over the theoretical concentration of heavy peptide spiked in the sample. The paradox is this: While in this way I manage to "see" the heavy up to a concentration of for example 0.05 fmol/uL (or up to an amount of for example 22 fmol) when I apply my PRM to monitor the light peptides in other BAL samples, following the calculations, it seems that the light peptides exist in lower concentrations than this 0.05 fmol/uL (or in a lower amount than this 22 fmol).

This has confused me very much and I don't even know how to look for this in the literature.

 
 
Nick Shulman responded:  2019-11-08
I am not sure I understand your question.

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

It might also help if you could post a screenshot of what you are seeing.
-- Nick
 
sofia farkona responded:  2019-11-08
I am trying to explain in the attached document what happens.
I am not sure if the mistake is in calculations....
I am already aware we have spiked in too much heavy, but there was a reason we did this, not because we don't read the literature....
 
Nick Shulman responded:  2019-11-08
Thank you for attaching that PowerPoint.

It looks like something weird happens on the third slide of your PowerPoint.
For most of your samples the peak height of the light peptide is 300,000, but on slide 3, it jumps up to 3,000,000.

I think I would need to see your Skyline document in order to make sense of this.

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

-- Nick