Import MS2 spectra from Waters Xevo TQ-XS (Survey Scan)

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Import MS2 spectra from Waters Xevo TQ-XS (Survey Scan) gioele visconti  2022-03-07
 

Hello,

I am using a Waters Xevo triple quadrupole (TQ-XS) instrument that contains MRM and MS2 scans (Survey Scan, which is the equivalent of the full scan).
I would like to use Skyline to search for specific m/z, so created an import list with the name of the precursor, its m/z and the polarity. When importing the RAW file, I can see the preview of the imported spectra (so the import step was fine) but when i select the precursor I don't have any spectral information. By looking the details with SeeMS, the Survey Scan spectra are settled as MS level "2". How can I extract this information in Skyline? Should I modify something into the transition list?

I also tried to convert the full scan spectra usign ProteoWizard but the issue was still present (and spectra are settled as MS level "2").

Thanks in advance and best regards,

Gioele

 
 
Nick Shulman responded:  2022-03-07
I do not know the answer to your question but if you send us your Skyline document and one or more of your .raw folders, we might be able to come up with a solution.

This sounds like it might be a bug in the way that we are interpreting these spectra from your machine.

If all you wanted to do was extract chromatograms for your precursors from these survey spectra, you could go to:
Settings > Transition Settings > Full Scan
and in the MS1 filtering section set "Isotope Peaks Included" to "None"
and in the MS/MS filtering section, set "Acquisition Method" to "DIA" and the Isolation Scheme to "All Ions".

Then, Skyline will extract chromatograms for your precursor from all of the spectra in your document.

However, since your document contains both MS1 and MS2 spectra, and the MS1 spectra are incorrectly labeled as MS2, I cannot think of any way to do what you need to do.
Maybe we will be able to come up with a better solution after we see your data.
-- Nick
 
gioele visconti responded:  2022-03-07
Hi Nick,

Thank you for your answer.

I set the transition setting as you suggested but no chromatogrm was shown (e.g. for m/z 121.1).

As requested, I uploaded the RAW file into the file sharing section (https://skyline.ms/project/home/support/file%20sharing/begin.view?). The file is a compressed archive named "Gioele_Waters_TQ-XS_MRM_and_Survey_Scan.RAW.zip", and the password to encrypt it is "Un1GE2123".

Thanks again for your help! Just let me know if you need any further information.

Gioele
 
gioele visconti responded:  2022-03-17
Hello Nick,

Did you have time to look at the file?

Best regards,

Gioele
 
Nick Shulman responded:  2022-03-17
Sorry about not getting back to you.
Your data file has SRM chromatograms in it. Whenever a raw file has SRM chromatograms in it (specifically, whenever a raw file contains more than 2 chromatograms), Skyline will ignore all of the spectra.
In order to get Skyline to pay attention to the spectra, you would need to create a file which did not have any chromatograms in it.
One way to do that would be to use MSConvert to convert the raw file to .mzXML. Since .mzXML cannot contain chromatograms, the chromatograms would be removed.
There might be a better way to do this by maybe checking the "SIM as Spectra" checkbox in MSConvert.

If you send me your Skyline document I might be able to try some of these ideas out and give better suggestions.
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files.

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

Sorry I do not have any good ideas at the moment. Maybe someone else on the Skyline support board can give you better suggestions.
-- Nick
 
gioele visconti responded:  2022-03-18
Hello Nick,

Thank you for your reply. I converted the Waters RAW file using the setting you suggested (see screnshot attached) but the import in Skyline still does not provide have any spectral information.

If you want to give a look at the Skyline document you can find in on https://skyline.ms/files.url under the name "Gioele_Waters_TQ-XS_MRM_and_Survey_Scan.sky.zip" - no password is required to unzip. The RAW spectra used was the same as the one the I uploaded in the previous answer ("Gioele_Waters_TQ-XS_MRM_and_Survey_Scan.RAW.zip"), so you can use it and covert in case it is required.

Thanks a lot for your support! Best regards,

Gioele
 
gioele visconti responded:  2022-04-06
Hi Nick,

Did you have time to look at the Skyline project?

Best regards,

Gioele
 
matt.chambers42 responded:  2022-04-20
Hi Gioele and Nick,

Gioele's data file's MS2s have an odd precursor "Set Mass": a constant 50 m/z. Would that make Skyline ignore them, Nick? Msconvert isn't making a mistake here, that's really what the data file says the precursor m/z is. And likewise, the data file really says they are MS2 scans, not MS1 (survey) scans.