using one peptide calibration curve to quantify another peptide

using one peptide calibration curve to quantify another peptide ana  2019-06-06

Hello Skyline team,

following up on some of the items of this previous discussion:

I am still having problems figuring out how to quantify a peptide for which I don't have standards using the calibration curve generated for another peptide. I'll try to describe my workflow as well as possible (attached step by step snapshots):

  1. I generate a calibration curve normalizing to an internal standard and using the Ratio to Global Standards normalization method
  2. I choose one of my peptides (FGLSLVR) to be used to quantify another peptide and mark is as Surrogate Standard
  3. I change the peptides for which I don't have standards for to be normalized by Ratio to surrogate FGLSLVR instead of Ratio to Global Standards\
  4. The result is a new calibration instead of using the one for peptide FGLSLVR. I tried excluding the calibration points for the samples identified as standards (since I do not have them for these peptides) but it doesn't make a difference.

I know that this is not best practice, but instead of reporting just areas, I would like to report a concentration (even if it's far off from the real value, it can be used for comparing different samples and give an idea of how much it's present). I can do this type of analyses with the software provided by our LCMS and would love to find out if I can also do it using Skyline (since I am quantifying all other peptides for which I have standards with it).

Thanks in advance!

Mike MacCoss responded:  2019-06-06

Hi Ana,
This is our lab's strategy for performing calibration in most proteomics cases.

It has also been used by the Hoofnagle lab in a clinical assay

It is compatible with or without a isotope labeled or surrogate internal standard peptide.


ana responded:  2019-06-06

Thanks! Very interesting papers. I forgot Lindsey presented on this work at the Proteomics course in Novato back in March.

In general lines though, I guess Skyline by itself does not allow to use one calibration to a different compound? I haven't used the small molecule mode yet, but in the small molecule world, this is needed often and would be nice to have the option embedded in the software.

Thanks for the help!

Mike MacCoss responded:  2019-06-06

The issue isn't what Skyline will or won't let you do. The problem is that each analyte has a different response in the mass spectrometer. Skyline does support the use of surrogate internal standards but it is only possible to "calibrate" an analyte to itself. Skyline supports both internal and external calibration. The solution that works for everything is to use a single point external calibration to a reference sample. That way the level of that analyte can be compared between batches, between instruments, and if the reference is shared the measurement can be compared between labs.


ana responded:  2019-06-07

Yes, I agree with the fact that different analytes have different responses and it is not a good practice to calibrate one using another. Thanks again for sharing the single point external calibration solution, I really like that approach.