peptide RT prediction does not work. wnstjr95  2019-07-20


I try to use iRT to predict the retention time of some peptides.
Without MRM data from QQQ, I tried to get the RT of some peptides.
But, I couldn't get any predicted retention time.

after importing MRM data, I could see the predicted and measured retention time.

I thought that I can simply get the predicted retention time since skyline uses the SSCalC.

Could you explain why it is not working? and If possilbe, let me know how to get the predicted RT without imporing MRM raw data.


Nick Shulman responded:  2019-07-22
I do not 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:

It might also help if you could post a screenshot of what you are looking at.

You might want to take a look at the iRT tutorial:
-- Nick
wnstjr95 responded:  2019-07-22
The question is simple. I just want to know the predicted RT of a peptide without LC-MS measurement.

Using SSCalC (, it can be predicted simply. But, I just want to use Skyline.

To predict RT of unknown peptides, the recent version of Skyline needs to spike-in the known RT standard into the sample. And then, we can predict the RT of interest in sample. My Question is "Is it possible to predict the RT without the RT standard?"

As I remember, previous version had this kind of RT prediction. After I made RT calculator using some peptides, I could simply predict the RT of any peptides without LC-MS measurements. But, in the new version, it seems there are some changes.


Brendan MacLean responded:  2019-07-22
Hi Jun,
Have a look at the Targeted Method Refinement tutorial on page 8:

It explains how to set up a retention time predictor using SSRCalc without any standards. You will need to measure some peptides to train the predictor to your system, but then you can just use SSRCalc hydrophobicity scores to predict other peptides.

Hope this helps. This has not changed. If you were successful with Skyline and SSRCalc before, you should be able to be again. We have added support for iRT, which requires prior measurement and designating standard peptides. This effort should give you much more precise predictions than SSRCalc, as detailed in the paper:

But, if you want to rely on SSRCalc that functionality remains much as it was 10 years ago.

Thanks for posting to the Skyline support board. Good luck setting up your targeted methods.