SureQuant MS/MS peak detection fails markus stoeckli  2023-07-12 01:59

We use the Exploris systems to acquire tPx data using the SureQuant method. When testing different Skyline features, we were super happy to see the implementation of support for Triggered Acquisition. But, at least with our data, we couldn't get this setting to work (ver. As you can see in the attached images, when we switch from PRM to SureQuant MS/MS peak integration, none of the peaks are detected any longer. Is this a known issue and is there a solution for this?
Many thanks!

Nick Shulman responded:  2023-07-12 02:25
Can you send us your Skyline document 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.

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:

When your MS2 transitions are missing their chromatograms like they are in your screenshot, it is because Skyline did not find any MS2 spectra which matched the precursor in your Skyline document.
There are a couple of reasons that changing the Acquisition Method to "SureQuant" might have caused this to happen:
1. When you change the Acquisition Method to "SureQuant", Skyline also changes the "Method match tolerance" at "Settings > Transition Settings > Instrument" to "0.007". It is possible that this value is too small of a number and, for this reason, none of the MS2 spectra in your .raw file match the m/z of the precursor in your document.
2. When "Triggered chromatogram extraction" is checked at "Settings > Transition Settings > Instrument", Skyline pays attention to the "Scan Description" values on the spectra. If the scan description starts with "SQ_" then the scan description has to match a very specific format in order for Skyline to use it in a particular precursor's chromatogram. You can get more information about that here:

After I see your Skyline document and raw file I will probably be able to tell you what is going wrong.
-- Nick
markus stoeckli responded:  2023-07-12 05:18
@Nick, many thanks for your reply, very informative. I didn't realize that the scan description is being read by Skyline. We had the issue, that the Thermo SureQuant template had the sub-experiments in the order R2+, K2+, R3+, K3+ and Skyline would blindly fill the tables in the order K2+, R2+, K3+, R3+. We then changed the mass shift in the tables, but did not pay attention to the scan description (see attached image). So based on your suggestion, we changed this and are currently running a test. Will report shortly on outcome...
markus stoeckli responded:  2023-07-12 07:03
One step forward, one back. When we had "SQ_ENDO_K+8_2" in the scan description, we did not get any heavy ions imported, but the parent and MS/MS of the un-labled peptides are integrated. Is there some documentation on what we should put into this field? Should it read "SQ_IS_K+8_2"?
Nick Shulman responded:  2023-07-12 08:54
Yes, I believe SQ_IS_K+8_2 would be the correct scan description for the internal standard (i.e. heavy) charge 2 precursor with K labeled with +8.

The most reliable way for you to fix this might be to make it so that all of your scan descriptions were blank so that Skyline would ignore them.

You only need to set the scan descriptions if you actually have compounds in your document whose precursor m/z differ from each other by less than 0.007 so that Skyline ends up extracting chromatograms using spectra that were intended for different compounds, which would result in spiky chromatograms as they alternate between spectra with different resolution and fill time settings.

-- Nick