Chromatogram information unavailable

Chromatogram information unavailable liuh1  2024-02-26 13:35

I use skyline-daily to process Agilent 6495C QQQ data. With several metabolites I always have problem to integrate the peak area. It shows the message: "Chromatogram information unavailable" I have followed the answer about the same problem, but it can't solve my problem.
Last week I worked for several times to get one file with less "Chromatogram information unavailable", but more metabolites show "Chromatogram information unavailable" after I added a few data file to the project.

I have checked the data file on Masshunter Qual, the metabolite shows great peak and right retention time.

What can I do next?


Nick Shulman responded:  2024-02-26 13:45
With triple quad data, that usually means that was no chromatogram with a matching precursor and product m/z in the mass spec data.
The setting which controls how close the m/z in the mass spec file must be to the m/z in the Skyline document is "Method match tolerance m/z" at "Settings > Transition Settings > Instrument".

Also, if you have set the "Explicit Retention Time" on your molecule, you should make sure that the explicit retention time is within the time range that the chromatogram was acquired. If the Explicit Retention Time is outside of that range then the chromatogram will be ignored even if its Q1 and Q3 m/z's match what is in the Skyline document.

If you would like, you can send us your Skyline document and your raw data (i.e. the Agilent .d folders).

In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including whatever chromatograms were taken from the raw files.
The "Share Skyline Document" also allows you to include the results files, or you could zip up your Agilent .d folders separately.

Files which are less than 50MB can be attached to this support request. You can upload larger files here:
-- Nick
liuh1 responded:  2024-02-28 10:37
Hi Nick,
Thanks for your prompt reply. I just loaded my skyline file with the following name: PoolesQC_skyline_022823
I have several compounds with the "Chromatogram information unavailable". Can you please check the problem with glutamate?
Glutamate: RT 10.75


Nick Shulman responded:  2024-02-28 11:19
Can you send me one or more of your raw files, for instance "PooledQC_TM01.d"?
You can package that folder up in a .zip file.
Files which are less than 50MB can be attached to this support request. You can upload larger files here:
-- Nick
liuh1 responded:  2024-02-28 11:24
Just upload PooledQC_TM01.d

Nick Shulman responded:  2024-02-28 12:10
Your "-146.0000" precursor for "Glutamate" has two transitions: the precursor "-146.0000" and a fragment ion "-102.1000".
In your raw file, there is a Q1=-146 Q3=-102.1 chromatogram, but there is nothing in there that looks anything like Q1=-146 Q3=-146.

When you have a Precursor with more than one Transition, and Skyline is only able to find a chromatogram that matches one of those transitions, Skyline does not consider that to be a match.
If a Precursor has only one Transition, then only finding one chromatogram is fine.
But, if Skyline was trying to match more than one Transition, then only finding one transition makes Skyline think it's not a match at all.

If you were to delete the precursor transition from your Skyline document and tell Skyline to extract chromatograms again then Skyline would correctly match the Q1=-146 Q3=-102.1 to your fragment transition.
-- Nick
liuh1 responded:  2024-02-28 13:27
Hi Nick,
Glutamate only has one negative transition: 146 to 102. There is no 146 to 146 in my transition list.
 Another example is phosphoenolpyruvate (PEP), it only has one transition: 167 to 79. It also shows the same problem.


liuh1 responded:  2024-02-28 13:37
HI Nick,
Just for your information, I have only one good skyline results with this project. When I add more samples to the skyline project, I lost glutamate results with several other metabolites results.
Nick Shulman responded:  2024-02-28 13:46
Those two molecules do have a "precursor" in the targets tree. See the attached screenshot. If you delete those precursor transitions then you will be able to see the chromatograms for the remaining transition.

It might be that the original transition list did not include these precursor transitions but Skyline might have added them later on because the "Ion Types" at "Settings > Transition Settings > Filter" includes the letter "p".
-- Nick
liuh1 responded:  2024-02-28 14:02
Yes, there are two transitions for glutamate. one is positive mode with 148 to 87, another is negative mode with 146 to 102.
Nick Shulman responded:  2024-02-28 14:13
The things that are causing you trouble are the things that are highlighted in my screenshot.
If you delete those precursor transitions and tell Skyline to extract chromatograms again then the remaining fragment transitions will have chromatograms.

Those are precursor transitions where the m/z of the fragment ion is the same as the m/z of the precursor ion.
On a triple quad, that might be something where no fragmentation happens in the second quadrupole, or it might be something that is designed to measure the amount of intact precursor after fragmentation in the second quadrupole.
-- Nick