Missing light product ions in SureQuant experiment carmen.gonzaleztejedo  2020-09-28
 
Dear Team, We performed a SureQuant experiment in an Orbitrap Exploris 480. When we load the data into the Skyline, we can see the XIC for the light and heavy precursors and the XICs for the heavy fragments. However, we cannot see the fragments of the light precursors (see attached). I have checked all the peptide and transition settings but I cannot figure out what the issue is. I would really appreciate if you can help me with that. Thanks a lot in advance. Regards, Carmen.
 
 
Nick Shulman responded:  2020-09-28
Can you send us your Skyline document and one 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 your .raw file are less than 50MB, you can attach them to this support request. Otherwise, you can upload those files here:
https://skyline.ms/files.url

The most common reason that MS2 chromatograms are missing like that is that Skyline did not find any MS2 scans where the isolation window matched the precursor in your Skyline document. When the acquisition method is "Targeted", any particular MS2 scan will only be matched to one precursor in your Skyline document, whichever precursor had the closest matching m/z. Sometimes, there is another precursor in your document whose m/z was closer to what the mass spectrometer said the isolation window was, and so that is the precursor whose chromatogram gets the extracted point. If that other peptide were to have been removed from the document, then your intended peptide would have gotten those points.

We have seen that with SureQuant methods, the isolation window reported in the .raw file is based on what was observed in the triggering MS1 scan, rather than being a theoretical m/z based on the known chemical formula of the targeted peptide. For this reason, there is never a perfect match between the reported isolation window and the precursor m/z. For these sorts of methods, you sometimes have to change the Acquisition Method to "DIA" (and you can use the Isolation Scheme called "Results Only"). Usually, if this sort of thing is happening, what you see is that your MS2 chromatograms have some points in them, but seem to be missing a lot of points from scan which had been intended for that precursor.

There is another feature which Skyline has for SureQuant methods that involves having your method set the "Scan Description" to special values which indicate to Skyline which peptide a particular spectrum was intended for. I can give you more details about this feature if it turns out to be necessary.

For now, it would be easiest if I could take a look at your Skyline and .raw file.
-- Nick
 
carmen.gonzaleztejedo responded:  2020-09-28
Dear Nick,

Thanks for your quick reply and the explanation. I did try to change the Acquisition method to DIA but nothing changed.
I have uploaded the skyline file and .raw data in the File Sharing Folder (SureQuant_Skyline_Support_CG.zip).

Please do let me know if you need anything else.

Regards,
Carmen
 
Nick Shulman responded:  2020-09-28
Carmen,

I see that you are using Skyline version 20.1.

You should use Skyline-Daily (which will be released as "Skyline 20.2" in a week or so). We fixed a bug a few months ago where Skyline's interpretation of the isolation window of SureQuant data is sometimes off by 5 Daltons because of something to do with ProteoWizard not handling the "MS2 Isolation Offset" correctly. This is fixed in Skyline-Daily.

By the way, we added a new feature in Skyline-Daily called "Triggered Acquisition", to fix some problems that Skyline had with handling SureQuant data: background subtraction and truncated peaks. You can read more about this feature here:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=TriggeredAcquisition

We would recommend that you set the "Triggered Acquisition" checkbox in Skyline-Daily at "Settings > Transition Settings > Instrument".

Let me know if you run into any problems with Skyline-Daily's interpretation of your SureQuant data. SureQuant support is an important feature in Skyline 20.2 and we would like to make sure it is perfect.

Thanks,
-- Nick