QQQ with transition list - Chromaogram information unavailable

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QQQ with transition list - Chromaogram information unavailable jihyunk  2025-03-31 13:19
 

Hi,

I'm doing lipidomics with truple quad and have transition list for the compounds.
I can't see the chromatogram in some compounds out of ~600 compound list. I tried like wider retention time window and selecting nclude all matching scans, but still It show chromatogram information unavailable (also did re-import).
I can see the peak in masshunter if I extract the transition that does not show in skyline.
Could you please help me with this?
Thank you so much

 
 
Nick Shulman responded:  2025-03-31 13:32
When Skyline is reading SRM chromatograms from the raw file, Skyline does not typically make use of the settings on the "Full Scan" tab at "Settings > Transition Settings".
You should make sure that the MS1 filtering Isotope Peaks Included is "None" and MS/MS filtering Acquisition method is "None".

The usual reason that you will see "Chromatogram information unavailable" with SRM data like you have is that there really was no chromatogram in the .raw file whose Q1 and Q3 values matched the precursor and product m/z values of the transitions in your document. The closeness that these numbers are required to be for a match is controlled by the "Method match tolerance m/z" setting on the "Instrument" tab at "Settings > Transition Settings".
Another thing to keep in mind is that when Skyline is trying to figure out which group of SRM chromatograms belong to a particular group of transitions in the Skyline document with the same precursor, the Q1 values of the chromatograms in the group must exactly match each other.
Also, if you have set the "Explicit Retention Time" on any of your molecules, Skyline will ignore any SRM chromatogram whose time range does not overlap with the explicit retention time. If you are using Skyline 24.1 then you will usually see a warning about this output to the Immediate Window but earlier versions of Skyline will do this silently.

If you send us your Skyline document and your raw data I could probably figure out what is going on.
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including imported chromatograms.
The Share Document dialog also gives you the option to include the raw data in the .zip file, or you could zip those up separately and send them to us.

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
-- Nick
 
jihyunk responded:  2025-03-31 14:30
Thank you for the detailed explanation,

I attached skyline zip which includes raw data.

Thank you so much!
 
Nick Shulman responded:  2025-03-31 14:41
Thank you for attaching that .sky.zip file.
I see that you are still using Skyline 23.1.
Is there a reason that you are not using Skyline 24.1?

If you have specified an Explicit Retention Time for a molecule, Skyline will discard chromatograms which do not overlap with that explicit retention time.
In Skyline 24.1 it will output a message when it does that, but Skyline 23.1 will do that without telling you.

For all of the molecules that are missing chromatograms you should either set the Explicit Retention Time to blank, or make sure that it is a number within the time range of the acquired chromatogram.
-- Nick