Retention time shift and integration

Retention time shift and integration nbekhti  2023-08-15 09:31

Hello Skyline team,

I work on small molecules on Skyline and I have a retention time shift for some molecules. How can I "automatically" reintegrate these peaks with the observed RT instead of doing it manually, chromatogram by chromatogram as I'm having 200+ samples.

Can anyone help me with this please? thanks


Nick Shulman responded:  2023-08-15 09:35
If you send us your Skyline document and a few of your raw files we might be able to figure something out.

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 and your raw files are less than 50MB you can attach them to this support request. You can upload larger files here:

-- Nick
nbekhti responded:  2023-08-16 06:48
Thank you Nick for your quick reply.

I'm attaching below an example of RT shift of some metabolites in QC sample replicates along the sequence.
In this case (example of 4 samples), it's ok to modify manually the integration, but is more time consuming when doing this for several metabolites for the 200 samples.

If any further info is needed, please let me know

Nick Shulman responded:  2023-08-16 07:57
Thanks for sending that file.

For which molecules and replicates is Skyline picking the incorrect peak?

I see that you have set the "Explicit Retention Time" for some of your molecules. When the Explicit Retention Time has been set, if Skyline detects a peak which overlaps with the Explicit Retention Time, Skyline will always choose that peak, even if there is a much better looking peak right next to it. If that behavior is causing Skyline to pick the incorrect peak, you might be able to improve things by changing the Explicit Retention Time to be blank.

You can use the Document Grid to change the Explicit Retention Time of a molecule.
If you would like to learn more about the Document Grid you can look at the Custom Reports tutorial:
-- Nick
nbekhti responded:  2023-08-17 06:06
Thank you Nick for your reply.

If we consider the example of Glutamic acid, the integration is correctly done in replicate 08 and 09, but the RT shift happens later in the sequence and we notice a wrong integration of this same peak in replicates 10, 13 and 14 (where RTs are a little bit shifted). We notice the same thing in the other metabolites given in examples: N-acetyl-L-aspartic acid, Phenylacetylglutamine and 1-Methylguanosine.

In this case if I remove the explicit retention time, which peak skyline will choose to integrate?

Thank you for the tutorial.

Nick Shulman responded:  2023-08-17 08:10
If you want to see what the effect of blanking out the Explicit Retention Time is, you can use the "Molecules" report in the Document Grid to set all of the Explicit Retention Time values to blank.
Then, you can go to "Edit > Manage Results" and press the "Rescore" button to tell Skyline to do the peak detection and scoring again.

It looks like Skyline will choose the correct peaks for Glutamic acid in all of your replicates after you blank out the Explicit Retention Time values.

If you are ever wondering why Skyline chose a particular peak, you can bring up the Candidate Peaks window using the menu item:
View > Other Grids > Candidate Peaks

When the Explicit Retention Time has been set, I see that one of the peaks ends up getting a very high score in the "Identified Count" column which ends up causing Skyline to choose that particular peak.
-- Nick
nbekhti responded:  2023-08-17 08:55
OK thank you so much.
I'll test this and let you know.

Thank you again
nbekhti responded:  2023-08-22 05:42
Hi Nick,

This is working great, thank you again for your help.
Have a very great week.