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MRM visualisation issue Tobi H  2023-11-14 21:16
 

Hello,

I'm fairly new to Skyline and have a problem concerning the visualization (and maybe also integration?) of MRM data. I'm doing LC-MS/MS and metabolomics.
I attached an image of a compound's XIC peak (precursor; m/z 361.2010) below. As you can see the peak is interrupted. The high points are MS/MS scans of the actual compound. The low points are MS/MS scans of a precursor ion with the mass 363.2166 Da.
Now there is an overlap of monitored transitions at that retention time with the compound eluting directly before that having a mass of 363.2166 Da. I figured that there could be some "interference" on the software level, but that would be weird, because there are two more transitions being monitored at that time (precursors m/z 651.7973, 777.6940) and they aren't seen in the XIC peak of m/z 361.2010.
Furthermore, every other XIC peak looks normal, no matter how many transitions might be overlapped at their respective retention times or the masses of these. Except for a similar case of a precursor ion of m/z 315.2319 showcasing the same interrupted XIC peak pattern and a compound eluting before that with a mass of 317.2475. The XIC peaks of m/z 363.2166 and 317.2475 don't have these interruptions.

The question would be: What is happening? Why is the XIC peak of one compound interrupted by another compound and why so selectively? Is there maybe an issue with some isotope recognition option ...? Is there a way to adjust the XIC's mass width?

Thank you!
 
 
Nick Shulman responded:  2023-11-14 21:22
When a chromatogram looks jagged like that, it is usually because there are spectra with two different sets of attributes which are contributing to the extracted chromatogram.
Sometimes that is because the mass spectrometer was told to collect two different sorts of spectra, such as some with a very short fill time and some with a very long fill time, so the observed intensities are oscillating wildly.
Sometimes it is because the mass spectrometer has two different isolation windows, and Skyline believes that both of those isolation windows match the precursor in your Skyline document.

I cannot tell exactly what is going on in your screenshots, but if you send us your Skyline document and your raw file we can probably figure it out.

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/or 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
-- Nick
 
Tobi H responded:  2023-11-14 22:19
Hello Nick,
thank you for your answer!

I suspect it could be the second possible reason you named: "Sometimes it is because the mass spectrometer has two different isolation windows, and Skyline believes that both of those isolation windows match the precursor in your Skyline document."
I'm acquiring both m/z 363.2166 and 361.2010 as precursors and the dwell times overlap. The Q1 operates on a nominal isolation window, meaning the precursor mass is selected +/- 0.5 Da.

If the problem goes back to the isolation window and Skyline thinking that both belong to the same precursor, is the integrated area reliable?

See the attached zip file.

Thank you for your help!
 
Nick Shulman responded:  2023-11-14 22:58
Thank you for sending your Skyline document.
I see that the "MS/MS Acquisition Method" at "Settings > Transition Settings > Full Scan" is "DDA".
You should probably change that to "PRM" and then tell Skyline to extract chromatograms again by going to "Edit > Manage Results" and pushing the "Reimport" button.

A couple of things are happening because you have chosen "DDA":
1. The MS2 chromatograms are all considered "non-quantitative" which is why the chromatograms are being drawn as dotted lines.
2. Skyline considers that an MS2 spectrum should be used for an extracted chromatogram if the isolation m/z overlaps with the isotope envelope of the precursor. So, for your 315.2319 precursor, the chromatogram will have points that came from spectra that isolated 315.23, but also 316.23 and 317.23 because those masses could have come that precursor with a couple of heavy atoms.

If you change the acquisition method to PRM then only spectra which are close to the monoisotopic mass of the precursor will contribute to MS2 chromatograms.
-- Nick
 
Tobi H responded:  2023-11-15 15:21
Hello Nick,
 
thank you so much for noticing! After correcting that it works perfectly.
I worked with DDA data before and completely forgot / overlooked that setting.

Thank you again and have a nice day!