Skyline for small molecules

Skyline for small molecules zhong li  2023-07-13 12:05

Have a question for the small molecule quantitation (MRM data from Triple Q instrument) : I need to quantify Compound A (with Internal Standard B), but there is no standard Compound A. I want to use the standard/calibration curve from Compound C’s signals (with Internal Standard B) to quantify Compound A. Is it possible in Skyline? Thanks.



Nick Shulman responded:  2023-07-13 12:46
It sounds like the feature that you want is "Surrogate Standards".

There is a small description of how to do that here:
-- Nick
zhong li responded:  2023-07-13 13:01
Thanks for your response, but surrogate standard does not work here. My situation may be similar to the recent post "Surrogate External Calibration". But mine is for Internal Calibration since I will try to use the same internal standard (Compound B) for both Compounds A and C. Want to quantify Compound A (signal ratio A/B) with signals from Compound C (signal ratio C/B). But since Compounds A and C are different (MRM), Skyline does not allow to do so.
Nick Shulman responded:  2023-07-13 13:14
I do not understand what you are trying to do but if I could see your Skyline document it might make more sense.

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:

-- Nick
zhong li responded:  2023-07-14 06:09
Thanks, Nick. The file is attached (We need to quantify Compound C23 which has no standard. Want to use Compound C22 signal to quantify. Compound d7-C24 is the internal standard in all samples).
Will Thompson responded:  2023-07-14 07:57
I agree this would be a pretty useful feature to have in Skyline, especially in the case where only a limited set of authentic standards are available of a particular class of compounds. This is very common in the lipid space; you want to perform a calibration curve in order to get an idea of the 'response function' of a compound class, but you don't have a standard for that exact molecule (in other words, the runs defined as "standards" for that particular compound are pretty much useless in the document). So what you want to do is use the response curve from the "standard" runs for one compound, and apply that calibration curve to the "unknowns" for a different compound.

This currently does not exist in Skyline, so I'll share one idea about how it might work:

I think would need to happen is an extra column in the Molecule Quantification document grid, which allowed the user to define a "surrogate calibrator". The list would simply be a list of all of the molecules in the document. In this example, the user would be able to choose "C22-0" in the "Surrogate calibrator" location, for "C23-0" line in the grid. If this was selected, the calculation that Skyline would do is to take the "normalized peak area" for any "unknown" sample for C23-0, and would use the calibration regression from the C22-0 line in the document to calculate the concentration.
Will Thompson responded:  2023-07-14 08:18
I just read Enders post on Surrogate External Calibration, and seems like the combination of what he requested (workflow recommended above is a way to maybe do it) and using the current "surrogate Internal standard" approach would address Lucas' request. d7-C24 assigned as the surrogate internal standard to both C22 and C23 molecules, and then C22 assigned as a Surrogate External Standard (or surrogate calibrator) to C23.
zhong li responded:  2023-07-14 10:56
Thanks, Will. Currently Skyline does not allow to add "surrogate calibrator" in Molecule Quantification document grid. Hopefully it may in the future. What you proposed is exactly what I need.
Brian Pratt responded:  2023-07-17 09:39
I'll have a look at that. Perhaps if you switch to the "Mixed" user interface mode, as a workaround?

- Brian Pratt
Nick Shulman responded:  2023-07-17 10:17


What do you mean when you say:

Currently Skyline does not allow to add "surrogate calibrator" in Molecule Quantification document grid.


In the "" file that you attached, I see that the "Standard Type" of "d7-C24-0" has been set to "Surrogate Standard" and the "Normalization Method" of C22-0 has been set to "Ratio to surrogate d7-C24-0".
It sounds like all you need to do is set the "Normalization Method" of "C23-0" to "Ratio to surrogate d7-C24-0" as well.

Are you seeing something different in your Skyline?
-- Nick

zhong li responded:  2023-07-17 11:32

Thanks, Nick. C22-0 is fine (has C22-0 standard. Yes, use surrogate internal standard d7-C24-0 here). My question on the post is how to quantify C23-0:
C23-0 has no standard. Thus explore to see if I can use C22-0 signal as surrogate standard for C23-0. Also use d7-C24-0 as surrogate internal standard. (C22-0 and C23-0 are different compounds.) Skyline seems not be able to do such things for now.