cannot see MS/MS data

cannot see MS/MS data naymin saw  2023-03-08 03:52

Hi Skyline team,

I am trying to develop a standard library with DDA data but I cannot see the MS/MS data in Skyline. I can check only MS1.
Could you please check and advise if somewhere is wrong?

Thanks and regards,
Nay Min

Nick Shulman responded:  2023-03-08 06:44
Skyline is usually not a useful tool for looking at small molecule DDA data.

Skyline is good for extracting chromatograms. In order for a chromatogram to be useful, the same thing needs to have been measured across multiple spectra so that the area under the curve will be a meaningful number for the purpose of quantification.

If you want to Skyline to do something useful with MS2 spectra, you might need to do your experiment again with either a PRM or DIA method.
The Skyline menu items "File > Export > Isolation List" and "File > Export > Method" are helpful for creating instrument methods.

I think the "Hi-Res Metabolomics" tutorial might be a helpful tutorial to look at:

I see that your Targets tree contains small molecule targets with precursor transitions.
If you wanted Skyline to extract chromatograms from MS2 spectra, you could right-click on one of the precursors in the Targets tree and choose "Add Transition" and tell Skyline the chemical formula or m/z of a fragment that you expect to find.
Note that there are four levels of things in the Targets tree:
Molecule List

If you want to add a transition, you need to right-click on something at the third level, such as the thing that says "125.0469[M-H]".
The thing that says "precursor - 125.0469[M-H]" is a transition, and would not have the "Add Transition" menu item if you were to right-click on it.

After you have added new transitions to the Targets tree you can tell Skyline to extract chromatograms by using the "Reimport" button on the "Edit > Manage Results" dialog.

When Skyline extracts chromatograms from DDA MS2 spectra, the chromatograms usually look very strange with long straight lines connecting the places where the precursor happened to have been sampled.

By the way, if you want to send someone a Skyline document, it is best to use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

-- Nick
Brian Pratt responded:  2023-03-08 09:24
As Nick says, the most obvious problem is that you haven't given Skyline any information about the fragments you're hoping to see. Keep in mind that Skyline is a targeted tool so it only looks for the things you ask it to look for.

If you can provide more information by using Skyline's File>Share>Complete option to create a file, and include one or two of the mass spec files, we might be able to offer a little more insight. You might be closer than you think!

Best regards,

Brian Pratt