how to analyze prmPASEF data with skyline

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how to analyze prmPASEF data with skyline guo xue  2024-12-26 23:23
 

Hello Skyline team, I have generated a prmPASEF data with timsTOF flex. However when I loaded into the the skyline, the precursor for M, M+1 and M+2 are 3 straight lines instead of forming peaks. I wonder why it is like this? and is there a general protocol for how to use skyline to analyze the prmPASEF data?

 
 
Nick Shulman responded:  2024-12-27 07:35
When a chromatogram has long straight lines it often means that there were very few spectra from which to extract chromatogram points.
This happens a lot with DDA data where there might be a long time between points where the mass spectrometer happens to have chosen to fragment a matching precursor.
This is not something that we see very often with MS1 chromatograms, because, usually, MS1 spectra are collected at regular intervals. However, I could imagine this happening because of something to do with ion mobility filtering.

Another thing that might be going on is that Skyline might be getting confused about whether an MS1 spectrum is a real MS1 spectrum or is actually a SIM scan. This had been a problem with Agilent centroided data, but it's conceivable that it might happen any time that Skyline is unable to figure out the m/z range over which data in MS1 spectra was collected. It's a long shot, but here is a support request which talks about what happens if Skyline is confused about the m/z range of MS1 spectra:
https://skyline.ms/announcements/home/support/thread.view?rowId=52933

If you send us your Skyline document and your Bruker .d folder we would probably be able to figure out what is going on.
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.

That Share Document dialog gives you the option to include the .d folders in the .zip, or you can send those to us separately.

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
 
guo xue responded:  2024-12-29 18:41
Hi Nick,
thank you very much for your responds. I tried to upload my files but it kept showing undefined error. Would you mind to download from the google drive?
https://drive.google.com/drive/folders/1NRmTNOcRyyxFuVHK4bx7RJHqJgLiLgKi?usp=sharing
there is only one raw file and skyline file. thank you very much.
 
Nick Shulman responded:  2024-12-30 05:45
Thank you for sending those files.

I think the chromatograms might make more sense if you right-click on the chromatogram graph in Skyline and choose "Transform > None".
The default is "Transform > Interpolated" which shows a chromatogram that has been resampled in the time dimension so that all of the points have the same time interval between them. The MS1 chromatogram in this "interpolated" view have long straight horizontal lines.

If you choose "Transform > Raw" you can see that the actual MS1 chromatograms that Skyline managed to extract have only two points in them: one at 5.86 minute and one at 5.88 minutes.
The MS2 chromatograms have lots of points from 5.36 minutes to 5.84 minutes.

I used ProteoWizard SeeMS.exe to look at the spectra in the .d folder that you sent.
I see that there really are no MS1 spectra from 5.24 minutes until 5.85 minutes.

I do not know very much about how mass spectrometers acquire data, but I wonder whether the mass spectrometer was so busy collecting MS2 spectra for your PRM method over that time range that it was unable to acquire any MS1 spectra. Or maybe the instrument method really did specify to not collect MS1 spectra over that time range.

When Skyline extracted chromatograms for this dataset, Skyline did originally extract an MS1 chromatogram which went all the way from zero minutes to ten minutes. When Skyline extracts MS1 and MS2 chromatograms in DIA or PRM data, and Skyline notices that the MS2 chromatograms cover a narrower time range than the MS1 chromatogram, Skyline truncates the MS1 chromatograms so that they match the MS2 chromatograms. After doing this, the MS1 chromatograms were left with only the two points at the end of the MS2 chromatogram time range.
-- Nick
 
guo xue responded:  2024-12-30 18:27
Hi Nick, thank you very much for your reply. In this case I will work on the PRM method I am using. I didn't specifically not collect the MS1 spectra but maybe the method setting is not correct. about the transform, I dont have Transform > Raw, is it a new feature?
 
Nick Shulman responded:  2024-12-30 18:46
Oops.
I meant to say "Transform > None".
I wrote that correctly in one spot in my message, but then I incorrectly said "Raw" a few lines later.
There is no "Raw" menu item.

By the way, I do see that you are using Skyline version 22.2.
The latest released version of Skyline is 24.1.
We always recommend using the latest version of Skyline unless you are trying to reproduce results from earlier experiments.

Yes, Skyline 22.2 also has the "Transform > None" menu item.
-- Nick