TMT balance

support
TMT balance tehmina bharucha  2021-11-04
 
Thank you for all your work! I have been using Skyline to perform PRM of TMTpro (16plex) labelled peptides. Is it possible to set up Skyline to analyse transitions with TMT balance regions attached? i.e. look at the MS/MS spectra to identify transitions (product ions) that are modified by TMTpro balance regions?
 
 
Nick Shulman responded:  2021-11-04
I am not sure that I understand your question.

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.

If that .zip file and your raw file are less than 50MB you can attach them to this support request.
You can upload larger files here:
https://skyline.ms/files.url

-- Nick
 
tehmina bharucha responded:  2021-11-04
Dear Nick,

Might it be possible to speak to you to explain?

I have shared my files as you suggested.

My best wishes and thanks, Tehmina
 
Nick Shulman responded:  2021-11-04
Thanks for sending your Skyline document.
I see that you have all of your precursors have the TMT pro special ions.
If you want to see the peak areas for your different TMT ions, you can use the Document Grid (view > Document Grid) and then choose the report called "Transition Results".

I will send you an email in case you have other questions I can answer.
-- Nick
 
Nick Shulman responded:  2021-11-04
Thanks for clarifying your question in email.

If you want to monitor the part of the peptide that is left behind after the reporter ion is cleaved off, the feature that you are looking for is "Neutral Losses".
You can tell Skyline that a modification has potential neutral losses by editing the modification definition ("Settings > Peptide Settings > Modifications > Edit List > Edit") and then push the "Loss>>" button in the "Edit Structural Modification" dialog.

You can add all 16 of your reporter ions your TMT modification definitions.
Then, you can add those neutral loss transitions to your document by right-clicking on precursors in the Targets tree and choosing "Pick Children". The neutral losses that you want will look like "precursor -##.#" where "##.#" is the mass of the neutral loss.

After you add these new transitions you should tell Skyline to extract the chromatograms again by choosing "Edit > Manage Results > Reimport".
-- Nick