Analysis of Sciex MRM3 data from 7500 QTRAP

Analysis of Sciex MRM3 data from 7500 QTRAP h l elfrink  2024-02-08 08:01

Dear Skyline team,

For our publication we are considering uploading the targeted MRM3 data (Sciex 7500 QTRAP) that we generated to the Panorama repository. If I understand correctly, then compatibility of the data with Skyline is a prerequisite, but this is not true for our data. When trying to open a data file Skyline cannot find any chromatogram. This issue was raised previously by a colleague of mine a couple of years ago, the proposed solution, using the the MS3 fragment instead of the MS2 fragment did not work for us.

Could you provide another round of feed back on this issue, please? Should we look for an alternative repository to share our data for now?


Nick Shulman responded:  2024-02-08 08:12
Skyline usually does a good job with MS3 data.
When Skyline is extracting chromatograms, by default, Skyline does not care whether a spectrum is MS2 or MS3 and will extract a chromatogram point whenever the precursor that was isolated in the MS1 scan has an m/z which matches the precursor in your Skyline document. This change to enable extracting chromatograms from MS3 data happened in 2019.

A new feature "Spectrum Filters" was added in Skyline 23.1 which gives you more control over exactly which spectra will contribute to a chromatogram. You can read about that feature here:

I do not know whether these features will enable you to succeed at analyzing your data using Skyline. If you run into any trouble you could try sending us your data and we can try to figure out if there is a workaround. If you want to send us files you can either attach them to this support request if they are less than 50MB or you can upload them here if they are big:

-- Nick
h l elfrink responded:  2024-02-16 07:50
Dear Nick,

Indeed your tip seem to have worked, though I am struggling a bit to apply the right modifications for some of the other target peptides (some neutral losses). But at least we are supposed to be able to upload our MRM3 data to the Panorama repository, so we will consider dropping the data there.

Have a nice weekend.

With kind regards,

Nick Shulman responded:  2024-02-16 09:45
The place to tell Skyline about the TMT tags that you are interested in is on the "Filter" tab at:
Settings > Transition Settings

"Neutral losses" are only intended to be used to monitor things that are still attached to some of the amino acids in the peptide. For things that are completely detached you should list them in the "Special Ions" list in the Transition Filter Settings.

Skyline knows about most TMT reporter ions so you probably just need to check the checkbox next to them.