TMT quantification tutorials

TMT quantification tutorials TK_1234  2024-04-09 07:42

Hi Skyline users, a new user here.

I was able to follow the tutorial on "Absolute Quantification.' Now, I'm interested in learning about TMT-based quantitative proteomics. My questions are the following: 1) Is there a skyline tutorial on TMT quantification? 2) If the TMT-labeling is applied only to the samples but not the heavy standard target peptide (used to generate calibration curve), what is the correct way to quantify the labeled samples?

Thank you so much!

Nick Shulman responded:  2024-04-09 08:01
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:
-- Nick