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Thermo Stellar Adaptive RT with Skyline Issues

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Thermo Stellar Adaptive RT with Skyline Issues asilverberg  2026-02-25 07:01
 

Hi Skyline team,

I'm using a Thermo Stellar mass spec for lipidomics (PRM method), and I'm having some trouble when I use the Adaptive Retention Time feature. I notice that certain product ion chromatograms aren't found by Skyline even though the raw data shows clear peaks. I'm guessing it's an issue with Adaptive RT adjusting the collection window/explicit retention time that Skyline uses to match transitions. I've tried adjusting the m/z tolerance and RT prediction settings in transition settings, but it doesn't seem to be changing anything. Is there another setting/adjustment that I'm missing? In the attachment, the left data set is without Adaptive RT and the right is with Adaptive RT. No other adjustments to the transition list or targeted list.

Thanks,
Annika
 
 
Nick Shulman responded:  2026-02-25 07:21
Can you send us your Skyline document and your .raw files?

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.

The Share Document dialog gives you the option to include the .raw files in the .zip file. Alternatively, you could send those .raw files to us separately.

Files which are less than 50MB can be attached to these support requests. You can always upload larger files here:
https://skyline.ms/files.url

Note that if you have specified an Explicit Retention Time for a molecule, Skyline will discard the extracted chromatogram if its retention time does not overlap with the Explicit Retention Time. If this happened, you would see a message about that in the Immediate Window during chromatogram extraction.

After we see your Skyline document and .raw files we will probably be able to tell you what is going wrong.
-- Nick
 
Brian Pratt responded:  2026-02-25 07:29
Hi Annika,

I'd just add that if I understand the idea of Adaptive RT, it suggests that you don't actually have highly repeatable chromatography. That's a problem for Explicit RT, which assumes that you do. But I should say that this is the first I've heard of Adaptive RT, so I may be completely wrong here.

You might try increasing the explicit RT window in Skyline, or just not using Explicit RT at all for the troublesome peaks and let Skyline find the peaks without that hint.

But mostly, yes, please do send an example. I think this might be the first time we've dealt with that kind of data.

Brian Pratt
 
asilverberg responded:  2026-02-25 08:41
Hi Nick and Brian,

Attached is the zipped Skyline result file.

Yeah, I was also thinking it may be a matter of removing explicit RTs. I realize it's not a widely used method so if that's the easiest way around it then I can definitely just do that. I can also use some other ways pre-Skyline to adjust my RTs rather than the Adaptive RT for peaks that have shifted. Just covering my bases to make sure I'm not missing something trivial.

Thank you!
Annika
 
asilverberg responded:  2026-02-25 08:42
Oops, I had to upload the .zip to the file sharing folder.

Thanks,
Annika
 
Nick Shulman responded:  2026-02-25 09:58
Yes, the reason that the MS2 chromatograms are being discarded for that molecule in that replicate is that the matching MS2 spectra go from 14.5 minutes to 15.2 minutes and the Explicit Retention Time is 15.3 minutes.
You should change the Explicit Retention Time to be blank on that molecule.
You can do this by right-clicking on the Molecule in the Targets tree and choosing "Modify" and then setting "Retention time" and "Retention time window" in the "Explicit values (optional)" section to blank.
-- Nick