Missing transitions in timsTOF data

support
Missing transitions in timsTOF data matt  2020-05-26
 

Hi

I have some timsTOF Pro PRM data which I am having problems with. For some of my targets Skyline does not find the MS2 fragments but I can clearly see them using Bruker DataAnalysis software. It only seems to be selected targets, some work fine.
Any suggestions?
Thanks!
Matt

 
 
Nick Shulman responded:  2020-05-26
Are you saying that the MS2 chromatograms that Skyline extracts for your are flat and don't have any signal?
If that's the case, then the problem might be that Skyline is not summing across a wide enough m/z channel when extracting chromatograms. The width of the m/z channel that Skyline sums across is controlled at:
Settings > Transition Settings > Full Scan > MS/MS Filtering
and then the setting that controls that is either called "Mass Accuracy" or "Resolution", depending on whether you've told Skyline that your data is centroided.
You can click on a chromatogram in Skyline, and it brings up the "Full Scan Spectrum Viewer" which allows you to see how wide of a m/z channel Skyline is summing across.

Or, are you saying that the MS2 chromatograms that Skyline extracts do not have any points in them. It might be that Skyline is being too strict about assigning MS2 scans to the molecules in your document. One setting that controls this is the "m/z match tolerance" at Settings > Transition Settings > Instrument. It is also greatly controlled by the Isolation Scheme settings at Settings > Transition Settings > Full Scan
If you have chosen "Targeted" as the acquisition method, then Skyline will associate any particular MS2 scan with the molecule whose m/z most closely matches what was isolated in that scan. Some PRM methods are designed so that MS2 scans are intended to be used by more than one analyte. If that is the case with your method, then you would need to change the acquisition method to "DIA", and probably choose "Results Only" as the isolation scheme.

We would probably need to see your Skyline document and raw files to really identify the problem.

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.

If that .zip file is less than 50MB you can attach it to this support request.
Otherwise you can upload it here:
https://skyline.ms/files.url

You should probably zip up at least one of your raw file directories and upload that there too.

-- Nick