Fail to integrate small molecule peaks ho-tak lau  2023-02-22 11:45
 

Hi Skyline Team,

I am trying to analyze some small molecule data acquired on TimTOF pro2 in MRM mode (Skyline-daily 22.2.1.391 (1e3a4e917)). However, Skyline is not able to integrate the peaks. DataAnalysis from Bruker showed that the peaks are there. What change can I make to have Skyline to integrate the peaks?

Thank you in advance.

Ho-Tak
 
 
Brian Pratt responded:  2023-02-22 12:57

Thanks for providing all the files.

That's unusual data - for MRM we'd expect to see chromatogram data rather than full scan. And for full scan data we'd expect to see MS1 data along with the MS2 data.

Is there a different way to export the data?

Thanks for using the Skyline support board,

Brian Pratt
 
ho-tak lau responded:  2023-03-08 13:09

Hi Brian,

Sorry for the late reply. I have been struggling with changing the method. I also tried to build a prmPASEF method by adjusting the default Bruker prm method just to fit the molecules I am interested in, but the data look the same as the MRM data.

What do you mean by exporting the data in a different way?

Ho-Tak
 
Brian Pratt responded:  2023-03-08 13:41

I think I just didn't look at your settings closely enough. Have a look at the attached image and see if that works better for you.

Best regards,

Brian
 
ho-tak lau responded:  2023-03-08 14:21

Thank you! It works now.

I also want to report a parsing bug without opening a new thread.

When I tried to build library from Fragpipe 19.1 interact-xxx.pep.xml with the xxx_uncalibrated.mgf using Skyline 22.2.0.351. Skyline reported an error, I have to rename the .mgf to xxx_uncalibrated_uncalibrated.mgf.

Ho-Tak
 
Brian Pratt responded:  2023-03-08 14:28

It's really better to open a new thread, if you'd be so kind.

Thanks!

Brian
 
ho-tak lau responded:  2023-03-09 14:53

Hello Brian,

What is the difference between PRM and DIA in Transition Settings -> MS/MS filtering. I have another set of data with two molecules. Using PRM, Skyline will only pick up 1 molecule, but when I switch to DIA, All ions. It will pick up both molecules.
 
Nick Shulman responded:  2023-03-09 15:32
Ho-Tak,

I see that in your Skyline document that you have a precursor with the m/z "873.2409".
I also see spectra in your Bruker .d folder where the isolated precursor was "873.3297".

When you choose "PRM" as the acquisition method, Skyline will extract a chromatogram point from a spectrum if the m/z of the isolated precursor in the spectrum is within the "method match tolerance" of the m/z of the precursor in your Skyline document.
You can change the method match tolerance at "Settings > Transition Settings > Instrument".

The default value of the method match tolerance is 0.055, but if you changed it to a larger number such as 0.2, then you will find that PRM works as an acquisition method.

You would not want to use "DIA/All Ions" for this data. That setting would only be appropriate for things like GC MS where the mass spectrometer fragmented everything without first filtering by precursor m/z.

If you hover the mouse over the Acquisition Method dropdown at "Settings > Transition Settings > Full Scan", Skyline will display a tooltip which tries to explain the difference between PRM, DIA, etc.
-- Nick