No product ion chromatograms found error - crosslinked data - 24.1version David26  2024-08-07 07:50
 

Hello,

I have the following error message : "No product ion chromatograms found" and indeed I don't see any fragments for any ion in any of the proteins monitored. Sometimes the error message don't appear but the signal associated to the fragments is extremely low (a line on the 0)

What I tested :
• crosslinked peptides / non-crosslinked peptides
• changing transition settings / peptide settings (and re-importing the results each time that I was doing significant changes) for the transition/peptide settings modified I tried to stick with what you said in this similar request : https://skyline.ms/announcements/home/support/thread.view?rowId=43123
• using different raw files with different protein concentrations to verify if it was a sensitivity problem (I can send the data if needed)

Thank you in advance for your help,

Best regards,

David

 
 
Nick Shulman responded:  2024-08-07 09:21
Can you send us a .sky.zip file and one or more of your .raw files?
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.
The Share Document dialog also gives you the option to include .raw files in the .zip or you could send those .raw files to us separately.

Files which are less than 50MB can be attached to this support request. You can always upload larger files here:
https://skyline.ms/files.url

When you see a chromatogram with no signal, you can often figure out what is going wrong by clicking on a point along the chromatogram.
When you click on a point along the chromatogram, Skyline will show the spectrum which contributed to that point in the extracted ion chromatogram. Skyline will highlight the region around the transition m/z values which show the m/z channel that Skyline summed across to obtain the chromatogram intensity.
If you want to need to make those m/z channels wider, you can usually do that by changing the resolution or mass accuracy value at "Settings > Transition Settings > Full Scan".
After you change that setting you would need to tell Skyline to extract chromatograms again by using the "Reimport" button at "Edit > Manage Results" in order for the setting change to actually have an effect.

After I see your .sky.zip file and your .raw files I will probably be able to tell you what is going wrong.
-- Nick
 
David26 responded:  2024-08-08 00:48
Hello, thank you for your fast response,

I am sending you the .zip document as you described with one raw file (David_crosslinking_07_08_24.sky.zip)

There is signal for the chromatogram but only for the precursors, fragments don't show anything as you can see on the following image and in the attached document. This is not only happening on the sended raw file but also on other raw files coming from other MS instruments. The problem persists after chaging the resolution and mass accuracy- on the Transition settings menu and re-importing the results.

Thanks again for your help,

David
 
Nick Shulman responded:  2024-08-08 01:22
When Skyline extracts chromatograms from MS2 spectra in a DDA dataset, the chromatograms do tend to look like that with long straight lines connecting the places where the mass spectrometer happens to have isolated something which matches the m/z of the precursor in the Skyline document.
For this reason, DDA MS2 chromatograms are not very useful in Skyline.
DDA MS2 chromatograms can be useful for showing you the time points where the mass spectrometer isolated a matching precursor, and you can click on points along the chromatogram to bring up Skyline's spectrum viewer.
DDA MS2 chromatograms cannot really be used for quantification because you have no control over where in the elution profile spectra were collected.
If you want useful MS2 chromatograms you should either acquire PRM data where you tell the mass spectrometer to repeatedly collect MS2 spectra for the analytes you are interested in, or DIA where you tell the mass spectrometer to collect MS2 spectras for the same precursor m/z ranges.

The screenshot that you posted is too low resolution for me to be able to see which peptide you are looking at.

What were you hoping the MS2 chromatograms in this dataset would look like?
-- Nick
 
David26 responded:  2024-08-08 04:27
Ok I will run a PRM or DIA method in that case, thanks for your help.

David