NON stable isotope internal standards for absolute quantification.

NON stable isotope internal standards for absolute quantification. MP  2018-04-29
Dear Skyline team,

I have got a collection of small molecules which I am quantifying using a targeted approach. One part of those molecules has got a 13C internal standard in the sample (so all works as usual, peak rations are automatically determined). However, the other half does not have a stable isotope labelled standard, but only a chemically very close internal standard.

The question is: Can I assign the non-labelled internal standards to certain analytes, so that peak ratios are automatically calculate, just as this is done for the 12C/13C pairs? And if yes, does this work in the same transition file?

Many thanks for helping.

Best Regards,
Brendan MacLean responded:  2018-04-29
Hi Mart,
We call those Surrogate Standards and you can find an explanation here:

If that isn't enough for you, then we would love to update this page so that does work for you and anyone else with this question.

Thanks for posting to the Skyline support board.

MP responded:  2018-04-30
Dear Brendan,

Many thanks for your fast response. I have made the changes as described in your link (above), but finally I do not see any peak ratios. For example when I blot the peak ratio graph, there is no way to normalize to surrogate, only to "Heavy", "Maximum" or "Total", or "None"). The "Targets by Accession" list does not show peak ratios (behind the molecule names) and the same is true for the reports.

Sorry, I tried hard to find the issue. Any hint to solve this would be very much appreciated. I have shared the zip skyline folders on your sharing page (called "").

Best regards,
Brian Pratt responded:  2018-04-30
Hi Mart,

This (draft version, not yet officially published) tutorial might be helpful:

It's MS-Word format, hopefully that works for you.

- Brian Pratt
MP responded:  2018-05-02
Dear Brian, Skyline-team,

Many thanks, it works now!

ana responded:  2019-01-29
Hi Skyline team,

Continuing on this discussion, I am dealing with a slight different problem. I am trying to quantify peptides ~130 and have synthesized 48 of them (light, not heavy). I want to use these as external standards and surrogate standards for all other peptides and to that point all is clear, thanks for the guidelines above! The problem comes to me when I want to normalize ALL peptides to another standard, the one I am calling my ISTD (which is a commercial RT reference standard) to compensate for possible losses during steps like sample clean-up by SPE. I haven't been able to find the way to normalize to this. The steps I want my quant processing to follow are:

1) Normalize peptide areas by ISTD areas (Area_peptide/Area_ISTD)
2) Build calibration as concentration peptide(X) against Area_peptide/Area_ISTD (Y)
3) Calculate concentrations of peptides using the curves obtained from identical synthesized peptides or using them as surrogate standards for other peptides.

Thanks for any help yo can lend!
Nick Shulman responded:  2019-01-29
If you want to normalize your peptides againts "ISTD", then "ISTD" is your Global Standard.
You should right-click on "ISTD" in your Targets tree, and choose "Set Standard Type > Global Standard".
Then, you can go to:
Settings > Peptide Settings > Quantification
and set "Normalization Method" to "Ratio to Global Standards".

This sets the default normalization method for everything to "Ratio to Global Standards".
Skyline will use this this normalization method when it draws calibration curves for the peptides in your document, unless you have overridden the normalization method. If you have some peptides for which you do not want to use Ratio to Global Standards then you can use the Document Grid to override the Normalizaton Method for individual peptides.

I'm not sure what you're talking about in step 3. Skyline has to use the same normalization method to draw the calibration curve, as Skyline uses to map from normalized area to concentration. It sounds like you want something else to happen, but I do not understand what that could be. Can you explain?
-- Nick
ana responded:  2019-01-29
Thanks Nick! That did the trick. Step 3 is easy from here. It's just using a peptide for which I have a standard to quantify another for which I don't have it. So for that I can use the surrogate standard.

Thanks a lot again!