Skyline does not get used for TMT very much, but I am actively working on features to make Skyline work better with TMT.

A big reason that people do not use Skyline for TMT is that Skyline really only knows how to quantify things from extracted chromatograms, whereas, with TMT you are typically quantifying things from a single spectrum where the observed ratios between the different channels will be very precise because they were all acquired in a single scan event.

I believe that typically with TMT one or more of the TMT channels would be for your internal standard, and your absolute quantification would come from the ratio of the other TMT channels to the normalization channel. As such, a calibration curve involving a heavy peptide would not be useful in a TMT experiment.

The way to add TMT fragment ions to your document is to go to:
Settings > Transition Settings > Filter
and check the checkboxes next to the TMT fragments in the "Special Ions" listbox.
After you have done that, the TMT ions will either be automatically added to every precursor in the document, or you will be able to add them by right-clicking on precursors in the Targets tree and choosing "Pick Children".

However, after you have added those TMT fragments to your document, it might impossible to get useful information out of Skyline since you probably are not interested in extracting chromatograms for these TMT fragments.

If you would like you could send us your Skyline document and your raw files and we could make sure that the new features that we are planning on adding to Skyline in the future will be helpful for your scenario.

In Skyline you can always use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

Files which are less than 50MB can be attached to these support requests. You can always upload larger files here:
https://skyline.ms/files.url
-- Nick

Hi Nick,

Just following up on this discussion - I would be curious to know whether you have any unreleased tools for TMT-based quantitation as discussed above. We are currently developing PRM-PASEF assays for TMT labelled peptides - the idea is to have some of the reporter ion channels as an internal calibration curve using known concentrations of NAT peptides. Presumably the only way to perform quantitation would then be to use the integrated EICs for each reporter to do regression analysis in excel or is there a better way to do this?

Thanks very much for you help!
Vincent

We are still hoping to eventually support using TMT reporter ions in Group Comparisons and Calibration Curves in Skyline.

I am attaching a .pdf which I made back in March which has some description of how it would work.
There is a special build of Skyline that you can install here:
https://proteome.gs.washington.edu/~nicksh/SpecialSkylines/TMT/

There is usually a lot of spillover from one TMT channel to the other based on the isotope distribution of the reagents.
Therefore, converting from the observed peak areas for each of the m/z's into quantities of reporter ions requires some matrix math to deconvolute the overlapping isotope distributions.
It will probably be 6 months to a year before Skyline is able to do all of this for you.
-- Nick