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timsTOF PASEF PRM Import Issues

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timsTOF PASEF PRM Import Issues robwsprung  2025-04-15 08:18
 

Dear Skyline Team,

Thank you for your continued development of this excellent software package. The longevity of the project is a testament to its utility.

I am currently developing a scheduled PASEF-PRM method on a timsTOF Pro instrument and I am running into some issues when importing results. I've tried importing a reference DDA run of isotope-labeled peptides and a scheduled PASEF-PRM run of the same sample while adjusting the Transition Settings - Full-Scan - MS/MS Filtering parameters as follows, with varying outcomes:

Import as PRM – no chromatograms
Import as DDA – Few product ion traces (even in DDA run), despite spectra in library
Import as DIA – Product ions observed in DDA run, few products in PRM run

I've attached some screenshots of my observations and a zipped file of the Skyline document. Any suggestions you could provide would be appreciated. Let me know if you need the timsTOF acquisitions uploaded also. Thanks.

Robert
 
 
Nick Shulman responded:  2025-04-15 09:10
I see that you uploaded "OVOL Targets timsTOF PRM 66 Peptides - PRM 032825.sky.zip" to the Support file sharing folder.

In that document, I see that the Isolation Scheme at "Settings > Transition Settings > Full Scan" is "All Ions".
That is probably not what you want, since "All Ions" is what you would use if the mass spectrometer was doing no filtering at the MS1 level and was just fragmenting everything.
The most common isolation scheme to use is "Results Only" which tells Skyline to use the actual isolation windows for the MS2 spectra in the raw data.

It sounds like you might be planning on uploading more files and screenshots.
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
 
robwsprung responded:  2025-04-15 10:08
Hi Nick,

I changed All Ions to Results Only as you suggested and re-imported the data, but the PRM run still has the same missing chromatograms.

I've uploaded the timsTOF files as well:

Weil_100_4782_2181_70_Slot2-28_1_10945.d - Reference DDA run
Weil_100_4782_3182_70_Slot2-28_1_10949.d - Subsequent PRM run

Robert
 
Nick Shulman responded:  2025-04-15 11:31
You can use a program such as ProteoWizard SeeMS.exe to see which m/z's were isolated in your PRM run.
The mass spectrometer used a 3 m/z wide window around these targets.
If your Isolation Scheme is "Results Only" then the m/z that was isolated must be within 1.5 units of the precursor m/z in the Skyline document.
If you choose "PRM" as the Acquisition Method, then the "Method match tolerance m/z" setting at "Settings > Transition Settings > Instrument" controls how close the isolated m/z need to be to the precursor m/z.

Is there a particular thing in the Skyline document that you were expecting to have chromatograms that does not have it?
It looks like the transitions with missing chromatograms are the ones where there really were no spectra with an isolation window that matched the precursor m/z.
-- Nick
 
robwsprung responded:  2025-04-15 12:15
Hi Nick,

Thanks for the tip about SeeMS displaying scan information. It appears that an incorrect isolation list was applied to this sequence.

Robert