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Skyline for small molecules : MS/MS visualisation

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Skyline for small molecules : MS/MS visualisation oceane delos  2025-03-25 08:28
 
Dear Skyline support,

We have a global lipid analysis method on a Thermo Exploris 240. We analyse in DDA.
We have molecules with identical monoisotopic masses that we can differentiate thanks to their fragmentation.
That's why we always look at our fragmentation spectra to confirm the molecules.
We are currently reprocessing with Tracefinder software.

On Skyline, I've tested it with ‘molecule interface’, with a list of compounds of interest. The areas are integrated in full scan, but I don't have MS/MS visualisation.
I tested Lipid creator. Theoretical fragmentations are created, with a ‘Library Match’ display with the expected fragments and red and green colour codes in the list of molecules to confirm the fragments detected.
But I think the integration is only done on the MS/MS triggered. And if not enough MS/MS are triggered, the peaks are not Gaussian.
That's why I'd prefer integration on the FS, while viewing the MS/MS under each peak.

My question is: Is it possible on Skyline to integrate the FS while displaying the MS/MS for small molecules?

Thank you for your consideration.

Best regards
Océane DELOS
 
 
Nick Shulman responded:  2025-03-25 11:27
If you have chosen "DDA" as the Acquisition Method on the "Full Scan" tab at "Settings > Transition Settings", Skyline will calculate peak areas and library dot products for the MS2 data.
You can see some screenshots of what we were looking at when we implemented this feature here:
https://github.com/ProteoWizard/pwiz/pull/2034

The important thing for getting this to work was to turn off Skyline's background subtraction for DDA data.
Once we did that, the peak areas reported by Skyline for the MS2 transitions end up being similar to the average of the intensities on the MS2 spectra that were sampled between the integration boundaries combined with the intensities from the MS2 spectra immediately outside of the boundaries if those spectra exist.

Have you tried settings the "Acquisition Method" in Skyline to "DDA"? What do your MS2 peak areas end up looking like?
Can you send us your Skyline document?
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.
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
 
oceane delos responded:  2025-03-26 03:25
Thank's for your reply.
In the attachment, I give you two data processes that I have done. It's an example with the ceramides family.
One with just full scan and one with library spectra.
In full scan process, i have the area integration as i want.
In DDA process, i think i have the area integration on MS/MS datas. But it's better with the integration on full scan.
ideally, I'd like a mix of the two processes : integration in FS and confirmation of molecules with MS/MS.

Is that possible ?

Thank's you

Océane
 
Nick Shulman responded:  2025-03-26 08:02
Usually in a DDA run your MS1 chromatograms will have a lot more points in them than the MS2 chromatograms because there will be a lot more MS1 spectra than there will be MS2 spectra with an isolation window that matches the m/z of the precursor.
In the attached screenshot, it looks like the MS1 chromatogram has exactly the same number of points in it as the MS2 chromatogram, which is weird.

The precursor chromatogram will look like that if you have told Skyline not to extract MS1 chromatograms. That is, if the "MS1 filtering Isotope peaks included" setting at "Settings > Transition Settings > Full Scan" is "None" then the intensities in the precursor chromatogram will be obtained by measuring the amount of intact precursor in the MS2 spectra.
When you make changes to the Transition Settings you usually need to tell Skyline to extract chromatograms again using, for instance, the "Reimport" button at "Edit > Manage Results".
When I look at the Audit Log ("View > Other Grids > Audit Log") it does not look like the precursor chromatogram would have ever been extracted from MS2 spectra, so I really do not know why the precursor chromatogram looks like that.
If you send me your raw file ("250205ODE_INGC12_pos.raw") it might be a little easier for me to understand how you managed to get a chromatogram that looks like that.

However, I am not sure I understand the actual question that you are asking.
-- Nick