Full-MS/ddMS2 data import

Full-MS/ddMS2 data import ingus perkons  2021-11-08

I have encountered an issue that I'm unable to solve.
So far I have used SkyLine ( to process Full-MS small molecule data and I have found it to be the most user friendly and well designed tool so far. _Thank you very much, developers because SkyLine has truly helped to ease our lab's daily tasks _ :-)

However, I wanted to process some Full-MS/ddMS2 data acquired on Orbitrap using an inclusion list. The problem I encounter: I cannot extract (see) the corresponding ddMS2 product ion scans. The data files are not corrupt since I can extract ddMS2 scans in both XCalibur and MZmine and there are no import errors whatsoever in the Skyline itself.

I suspect the problem is either with (a) Transition list or (b) Transition settings.

I have attached an example data file that I want to process (" Example_Full-MS-ddMS2.mzML" ) and corresponding screenshots from the imported Transition list (Capture_a.png), Transition settings (Capture_b.png) + the screenshot of what I see once everything is imported and extracted (Capture_C.png).

_I have enabled View --> Transitions --> All. _

Interestingly, when I set the mass accuracy settings in " Transition settings --> Full-scan" to 100 ppm in both “MS1 filtering” and “MS/MS filtering” panels, I can see ddMS2 scans very well (see "Capture_D.PNG"). Yet, if I set 10 ppm in “MS1 filtering” and 100 ppm in “MS/MS filtering” or vice versa - ddMS2 data again disappears after re-import.

I'm puzzled because the raw data are of good quality and the mass errors are well below 10 ppm (see Capture_E.PNG). Tried to find the answer in tutorials and the forum, but, apparently, could not notice it.

Nick Shulman responded:  2021-11-08
The mass accuracy settings at:
Settings > Transition Settings > Full Scan
tell Skyline how wide of a m/z window to sum across when extracting chromatograms from spectra.

It sounds like you are saying that when you set the mass accuracy to a low ppm number, your chromatograms end up having no signal. This means that the intensities that are present in your spectra do not match the m/z of your transitions.

If you click on a point along a chromatogram, Skyline will bring up the full scan spectrum viewer. That spectrum window will show you the m/z's of your transitions, and will have shaded boxes around them indicating how wide of a m/z channel Skyline summed across when extracting chromatograms. It sounds like what you will see is that the signal that is present is just outside of those shaded rectangles.

This might mean that your molecules of interest are not actually present in your samples. It also might mean that your mass spectrometer is improperly calibrated and the signal that is being detected does not actually match the m/z of the molecule that is generating that signal.

Can you send us your Skyline document?
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 is less than 50MB you can attach it to this support request. Otherwise you can upload it here:

-- Nick
ingus perkons responded:  2021-11-08
Hello Nick

Thank you for the rapid response. I have attached the shared .zip file as you requested. (I attached both 10 ppm and 100 ppm versions).
In accordance to "Capture_E", where I have displayed the MS1 and MS/MS data that I expect to extract they seem alright and well below 10 ppm error.

Nick Shulman responded:  2021-11-08
When your MS/MS acquisition method is "DDA", a spectrum will be included in the product ion chromatogram if the precursor m/z of that spectrum is within the isotope envelope of the molecule. In this case, the isotope envelope is a fuzzy region around the monoisotopic, M+1, M+2 m/z's of the molecule. The width of the fuzzyness around those masses depends on the MS1 mass accuracy or resolution that you have specified at "Settings > Transition Settings > Full Scan".

The reason that you cannot see the MS2 chromatograms in your 10ppm Skyline document is that there was only one spectrum that was within the 10ppm of your precursor molecule (237.10222 at 7.4 minutes). In your 100ppm Skyline document, many more MS2 spectra fell inside of the MS1 isotope envelope (e.g. 239.14m/z at 4.37 minutes was close enough to the M+2 m/z 239.1082).

When a chromatogram only has one point in it, it appears that Skyline ends up throwing away the entire chromatogram. I was not aware that Skyline did that, but it certainly makes sense since it is difficult to draw a single point chromatogram on the graph.

I hope this answers your question. If you want to look at the MS2 spectra in your data files, you can use ProteoWizard SeeMS.exe.
-- Nick
ingus perkons responded:  2021-11-09
Thank you, Nick.

Yes, this seems the most probable explanation for the observed issue. This would mean that there is no way to process such data in SkyLine to perform, for example, library matching based on a single ddMS2 spectra.
Will try to find some workaround to force Orbitrap to measure at least two ddMS2 spectra per precursor. Will update the thread, if I'll manage to figure something out. :)

ingus perkons responded:  2021-11-09

A quick update.
I managed to force Orbitrap to measure more than one replicate transition per precursor.

Yet, it did not solve the problem. =/

For instance, I want to extract MS1 and MS2 features for a compound (Benalaxyl) with an m/z of 326.1751.
In the example file I have the following scans: #1155-#1165 (see "Capture_F.PNG"), that
-For MS1 data, the precursor error ir ~2.5 ppm;
-two ddMS2 scans have been triggered for m/z 326.1737 (-4.3 ppm, #1159) and m/z 326.1742 (-2.78 ppm, #1161).
-both of them fit in the 10 ppm range and the product ions at m/z 148.1121 (measured at m/z 148.1119, -1.3 ppm) and m/z 91.0542 (measured at m/z 91.0546, -4.4 ppm) are also well below 10 ppm threshold and have been detected in both ddMS2 scans.

However, I' m not able to see them in Skyline no matter how hard I try to play around Transtion settings (see "Capture_G.PNG")
I have attached the mzXML file ("ddMS2_two_segments.mzXML"), the shared file ("2021-11-09-ddMS_Example_10ppm.sky.zip") and the transition list I'm using to illustrate the problem ("Transtion_list_example.xlsx").

I thought that 2 scans would be sufficient to perform MS2 data extraction.


Nick Shulman responded:  2021-11-09
Oops. My earlier answer was incorrect.

The actual reason that those MS2 chromatograms are disappearing is that you have the explicit retention time set to 9.98 minutes, and the chromatogram that was extracted does not overlap with that time (because all of the matching MS2 spectra came before 9.98 minutes). You would only be able to see the MS2 chromatograms if at least one matching MS2 spectrum had a retention time that was less than the explicit retention time and at least one MS2 spectrum had a retention time which was greater than the explicit retention time.

I think it's a bug that Skyline is doing this with chromatograms that Skyline extracted from full scan data. I believe it was our intention that this sort of "rejecting chromatograms because they do not overlap with the explicit retention time" was only supposed to happen when Skyline is looking at SRM data and needs to decide which chromatogram in the raw file was intended to be used by which molecule in the document.

It might take us a while to fix this, so, for now, I would recommend that you use the Document Grid to change the Explicit Retention Time of your Molecules to blank.

-- Nick
ingus perkons responded:  2021-11-09
Thank you very much. You just made my day much better.
I followed your advice and it indeed solved the issue. Never have thought about that myself.