Hi there, I used thermo's TMT 10plex kit to label peptides and pool them together for LCMS/MS analysis. I am analyzing my data in Proteome Discoverer 2.3 and wondering how to quantify the number of peptides that were labeled vs unlabeled to make sure my labeling strategy worked.
When I run the processing and consensus workflows recommended by thermo for this data, I get protein IDs and quantitation for most of the proteins- I have the TMT label set as a static modification for N-terminus for the peptides and on lysine residues. So, all of my identified peptides and proteins have the TMT label identified as a modification. Surely there are some peptides that the program can identify that weren't labeled, so how do I find those peptides and determine the ratio of labeled:unlabeled peptides in my sample?
Also, even though all of the identified peptides in this workflow are marked as having the TMT label, not all of them are quantified. Does anyone know why this happens?
Thank you!
Talia
You may use a spiked in peptide to estimate your labelling recovery and perform normalization...Spike a known amount of peptide or mixture of known synthetic peptides and subject them to TMT labelling including your target digested proteome. Afterwards, use targeted analysis (MRM) or DIA to compare labelled and unalabelled responses. By doing this you can get your labelling efficiency in absolute numbers and decide whether your unlabelled fraction is negligable or not...
Hi Talia,
I have looked into this particular question in the Methods paper I have published ; please see the link below. I did not use Proteome discoverer - I used MaxQuant and configured each of the TMT channel modification as a variable modification so that I could search my mass spec file to see how many peptides were not labeled.
Let me know if you can access the paper link below, if not I can also just email it to you.
All the best,
Hediye.
Chapter Sample Preparation for Relative Quantitation of Proteins Usi...
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