Integration of single masses in a DIA experiment on Thermo Scientifi Orbitrap ID-X Tribrid Mass Spectrometer

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Integration of single masses in a DIA experiment on Thermo Scientifi Orbitrap ID-X Tribrid Mass Spectrometer harald schoeny  2022-05-12 06:56
 

Dear Skyline Team,
I am trying to integrate a single mass (184.07332 Da) on MS2 level of a DIA experiment, in which I selected a specific precursor mass range (670-900) and measured a specific product mass range (180-190). I want to obtain a sum value for a lipid class and I have already applied this method on a Thermo QExective HF (see attached publication- AIF method).
Now I have measured the same on an ID-X instrument and the MS2 was recorded in an ion trap instead of the orbitrap.
Unfortunately, I am not able to get anything in skyline although these settings worked for the HF experiment. I have tried to set the product mass analyzer in the transition settings to QIT (but also every other option) but nothing worked.
Do you have any idea, what I can try?

Thnak you very much! All the best,
Harald

 
 
Nick Shulman responded:  2022-05-12 07:12
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 see that on the Full Scan tab of the Transition Settings dialog, you have "Isolation Scheme" set to "All Ions". You should probably change that to "Results Only".
"All Ions" is intended to be used with Waters MSe data, where there was no isolation window and everything got fragmented at the same time.
"Results Only" tells Skyline to trust whatever the mass spectrometer said the isolation window was.

After I see your Skyline document and .raw file I will probably be able to figure out exactly what is going wrong.
-- Nick
 
harald schoeny responded:  2022-05-17 04:27
Dear Nick,
 thank you for your fast reply.
I have attached the zip file.

I have tried both settings "Results only" and "all ions" but nothing worked. In theory "Results only" should be the right one because the data I have generated follow this MSe principle. I don't know what is written in the mzML files but it seems that already in the raw files from Thermo it is unclear what the precursor mass is. In the header it shows the mass exactly in the middle of the selected precursor mass range (785 Da in attached screen shot for 670-900 Da range). In the QExactive HF data I could have a work around for that but now the MS2 is generated in the ion trap instead of the orbitrap in the ID-X instrument and I guess this is a problem for skyline as well.

Thank you very much for your help!
Best,
Harald
 
Nick Shulman responded:  2022-05-17 05:17
Harald,

It sounds like you intended to attach some files to this support request, but we did not receive any files.
This often happens if the files that you are trying to attach are too large. The maximum file size that you can attach to these support requests is 50 MB.
You can upload larger files here:
https://skyline.ms/files.url
-- Nick
 
harald schoeny responded:  2022-05-17 05:24
I am sorry. I have uploaded the zip file "Skyline Upload" as you have suggested.
Best,
Harald
 
Nick Shulman responded:  2022-05-17 10:10
Harald,

Thank you for uploading your Skyline document.
Can you also upload your .raw file and your .mzML file?
It sounds like you have used msconvert to deconvolute the msx data in your .raw file. It would be good for us to see your .raw file and your .mzML file so that we can see whether something went wrong during the msconvert step.
-- Nick
 
harald schoeny responded:  2022-05-19 00:28
Dear Nick,
I have tried to upload both file types but nothing worked.
Best,
Harald
 
Nick Shulman responded:  2022-05-19 04:16
Thank you for uploading that .raw file and the .mzML files.

The original Skyline document that you uploaded did not have any precursors or transitions in it.
Skyline documents have four levels of things displayed in the Targets tree: Molecule Lists, Molecules, Precursors and Transitions.
Your document has molecules, but nothing below that.
There must have used to have been some transitions in your document, because Skyline will not allow you to do "File > Import > Results" unless you have at least one transition.
Do you have another version of the Skyline document which has some transitions in it?

I think what you need to do is go to:
Settings > Transition Settings > Instrument
and change the "Min m/z" and "Max m/z" to a wide range of numbers (e.g. 100-2000).
Currently you have that range set to 241-341.

I imagine that what happened was that your document used to have precursors and transitions in it, but when the Instrument m/z range was changed, Skyline removed all of your precursors and transitions from the document.
After you have changed the instrument m/z range you can add precursors and transitions to the document.
You can either do this by right-clicking on molecules in the Targets tree and choosing "Add Precursor" and then right-clicking on the precursor and choosing "Add transition".
Alternatively, you can paste in a list of transitions by using the menu item:
Edit > Insert > Transition List

I see that your raw file was collected in negative ion mode. If you use the "Add Precursor" right-click menu to add a precursor to your document, make sure that you specify an adduct which has a negative charge.

I see that your raw file has isolation windows 450-600, 500-790, and 670-900. With the isolation scheme "Results Only", if Skyline were extracting a chromatogram for a precursor with m/z 525, Skyline would extract points from both the 450-600 and 500-790 windows, since both of those windows contain 525. If you don't want Skyline to do that, then you should change the isolation scheme at "Settings > Transition Settings > Full Scan" to something else. If you changed that to an isolation scheme which had a margin which was greater than 25, then Skyline would no longer consider that 525 was included in the 500-790 isolation window.

In summary, you should go to:
Settings > Transition Settings > Instrument
and change the "Min m/z" and "Max m/z" to a wider range.
Then you should add transitions to your document again.

-- Nick