TMT neutral losses

TMT neutral losses aj10  2024-04-11 14:47


I am trying to quantify neutral losses from TMT-tagged peptides (the reporter tag + CO). I'd like to include the neutral losses regardless of their presence in the spectra used to generate the spectral library. When adding each tag as a neutral loss to the N-terminus modification, I set the losses to be included by default. However, these transitions are not added by default to my spectral library. I can add them manually using the Pick Children option, but I've got more than 10k peptides.

Second question. If we can resolve that, I only want the neutral losses from the precursor (i.e. not from every possible b ion). Is there a way to specify that and apply it to all peptides?

I'm running the Skyline daily build downloaded last Friday.

Thank you for your help!

Nick Shulman responded:  2024-04-11 14:56
The neutral losses in the "Edit Structural Modification" dialog would only be for if you wanted to monitor the complementary ion that remains after the TMT tag has fallen off of the peptide.
If you want to monitor the reporter ion itself you should go to:
Settings > Transition Settings > Filter
and check the checkboxes next to the TMT ions in the "Special Ions" listbox.
Those checkbox have three different states that they could be in (checked, unchecked and a gray state that is called "indeterminate"). If you put them in the checked state then the TMT reporter ions will be automatically added to all the precursors in your document. If they do not get added you might need to go to "Refine > Advanced" and check the checkbox next to "Auto select all...Transitions".

By the way, we are working on new features which will hopefully make TMT analysis in Skyline better in the near future.
Those features are:
1. The ability to specify how TMT tags are being used to multiplex replicates such that Group Comparison's can be done between TMT channels as if they were separate replicates.
2. Isotope deconvolution to deal with the fact that a particular TMT tag contributes signal into its neighbors owing to isotope distributions
3. Something yet to be determined which will make Skyline work better with DDA results.
-- Nick
aj10 responded:  2024-04-11 15:04
Hi Nick,

Yes, I am interested in the complementary ions.

Nick Shulman responded:  2024-04-11 15:15
If you really are interested in the complementary ions then I would recommend setting things up so that the neutral loss gets added to absolutely everything in your document and then use the Document Grid to delete all of the transitions which are not precursors and which have a neutral loss.

If you are not familiar with the Document Grid then I would recommend the Custom Reports tutorial:

Once the Document Grid is showing all of the transitions that you want to delete you can select all of the rows and use one of the menu items on the "Actions" dropdown at the top of the Document Grid to remove the unwanted transitions from the document.
-- Nick
aj10 responded:  2024-04-12 11:57
Hi Nick,

Thanks for the advice. Seems that it's working now!