MSX SIM QExactive MS1 data missing scan data

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MSX SIM QExactive MS1 data missing scan data simondbourque  2022-07-08
 

Hello,

I'm running a method which combines scheduled MSX MS1 scans with scheduled non-MSX PRM scans and the MS1 scans don't seem to import properly. In the attached image the MS1 data cuts out partway through the peak, this corresponds to where the scheduled acquisition window of one target ends and so the scan filter in the raw data changes. The data is there as extraction works fine in Xcalibur.

This is running on a QE+ with xCalibur 4.0. Skyline 21.2.0.536. Let me know if there is s private location I can upload the raw and project file to.

Thanks,
Simon

 
 
Nick Shulman responded:  2022-07-11
Can you send us your Skyline document and .raw file?

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 and your .raw file are less than 50MB you can attach them to this support request.
You can upload larger files here:
https://skyline.ms/files.url

I am thinking that the behavior your are seeing might have something to do with the way that Skyline tries to extract one for "SIM" spectra, and a separate chromatogram for regular MS1 spectra. The way that Skyline decides that a spectrum is "SIM" spectrum is Skyline looks at the width of the m/z window that was scanned. If the width of that window is less than 500 Daltons, then it goes in the "SIM" chromatogram instead of the regular MS1 chromatogram. (It used to be that the 500 Dalton cutoff was a much smaller number, but for various reasons we have increased that number over the years.) If the SIM chromatogram has any points in it, then that is what Skyline ends up using instead of the MS1 chromatogram.

In Skyline-Daily there is a checkbox "Ignore SIM scans" on the "Settings > Transition Settings > Full Scan" dialog. It might be that checking that checkbox will fix your problem, or it might be that checking that checkbox will just result in your chromatogram looking like the missing half what you have in the screenshot.

It is likely that the behavior your are seeing has nothing to do with Skyline's SIM spectrum behavior. I cannot think of a reason that the chromatogram would suddenly jump down to zero the way that it does in your screenshot.

After we see the files that you send us we will probably be able to figure out what is going wrong.
-- Nick
 
simondbourque responded:  2022-07-11

Hi Nick,

Find the shared skyline file attached and the raw file (MSX_schedulued_Bug.raw) is on your upload site.

These are narrow windows (2 Da) and the peak drops to zero right where the scheduling changes. For instance if acquire peptide 1 from 2-4 mins and peptide 2 from 3-5 mins the signal drops right at 3 minutes (and 4 minutes). In xcalibur quan browser you would see 3 scan filters for this setup: Peptide 1 m/z from 2 to 3 mins, Peptide 1 m/z and peptide 2 m/z from 3-4 mins and then peptide 2 m/z alone from 4-5 mins. Skyline doesn't seem to be interpreting the changing filter properly. If I set up a method where the windows either don't overlap or completely overlap it works fine.

I don't have daily running here but I'll try to scrounge up an install and let you know if "Ignore SIM scans" works.

Thanks,
Simon

 
Nick Shulman responded:  2022-07-11
Thanks for sending those files.
Yes, the behavior that you are seeing has to do with the fact that you have some MS1 spectra where the scan window was less than 500 Daltons, and some MS1 spectra where the scan window is larger than 500 Daltons.
If you were to use Skyline-Daily and check the "Ignore SIM scans" checkbox, then you would end up seeing the other half of the chromatogram, but it still would not be what you want.

Unfortunately, I don't think there is anything you can do in Skyline to get your chromatograms to look right. We have plans in a future version of Skyline to give you much more control over exactly which spectra should contribute to a particular extracted chromatogram.
-- Nick
 
simondbourque responded:  2022-07-12
Thanks Nick,

I grabbed skyline daily and "ignore SIM scans" fixes some transitions but breaks others.

It does seem to be SIM/MS1 confusion on the part of Skyline. However, I'm not sure it's quite as you describe, the scans are all 300 to 2000 m/z and only the quad isolation window is narrow. Digging into the data a bit, there are no precursor chromatograms whenever a single quad isolation window is being acquired, as soon as a second isolation window starts it extracts the precursor properly, when a third isolation widow starts it also behaves as expected. The windows are less than 500da apart.

A quick patch will be to manually modify the isolation list so there is always at least 2 being acquired.

Thanks again,
Simon