peptide absolute quantification from PRM data sanity check

peptide absolute quantification from PRM data sanity check manguy jean  2022-09-23 07:44


I learned how to use Skyline on the job for a peptide absolute quantification project. I read a lot of the tutorials, some of the posts I found here from different google searches, and looked at the methods sections of diverse papers. I think what I did makes sense I just want to make sure I didn't make a mistake because of some blindspots, so any help or advice is welcome.

We want to quantify some peptides from a hydrolysate (not for protein quantification but just for peptide quantification so we didn't have the choice of which peptides would be best). We have heavy variants of each of them. We built an internal calibration curve for each peptide using varying concentrations of heavy peptides. The first series of runs (PRM on a QE) uses a wide scale of concentrations to find the approximate concentration of light peptide, then another series of runs to adjust the calibration curve for each peptide to quantify and then replicated that again.

I used Skyline to analyze these data. I used the retention time of these peptides in previous DDA runs using the same LC parameters and the presence of peaks for the heavy and light products to select the correct peaks. Removed the product ions interfering with the peak. I then extracted the ratio of heavy/light for each analytical replicate and for each concentration. I used these ratios to make a calibration curve (linear regression) and I looked for the ratio heavy / light == 1 to find out the concentration of light. I had to go outside Skyline for this step as all my samples are marked as Standard, no sample being just Unknown, but being both at once, and as far as I know I cannot ask Skyline to look for a ratio of 1.

I hope someone can tell me if I am completely off the tracks or not.

Thank you


Nick Shulman responded:  2022-09-23 13:14
That all sounds pretty good.
Skyline does not really have any features to help you with a "reverse calibration curve", so doing the calculations outside of Skyline seems like the right thing to do.
If you would like, you could send us your Skyline document and we could tell you whether everything looks good.
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:

One thing to be mindful of if you are changing the heavy concentration and keeping the light concentration constant is that you should probably go to:
Settings > Peptide Settings > Modifications
and make sure that "Internal Standard Type" is set to something other than "heavy" (either "light" or "none" would be acceptable).
If there is an internal standard type, Skyline assumes that the peaks for the internal standard are going to be much easier to detect than the non-internal standard, so Skyline only does peak detection on the internal standard chromatograms. If you have a replicate where the heavy is very difficult to detect because it was spiked in in a very small quantity then Skyline will pick peaks badly if you have told Skyline that heavy is your internal standard.
-- Nick
manguy jean responded:  2022-10-07 05:16
Thank you Nick,

Sorry it took me so much time to answer

> make sure that "Internal Standard Type" is set to something other than "heavy"

yes, that's what I did, I forgot to write it there, I put light as my "standard".

I had to correct slightly some of the peak picking for the lowest concentrations, baed on the highest concentrations but mostly it was ok.