Hi everyone,
I am planning to do TMT labeling on digested peptides. I read the manual from Thermo. It seem that the manual doesn't mention anything about whether the digestion reaction should be quenched using 1% TFA before adding TMT reagent.
For my understanding, it seems that the basic ph need to be maintained during the process of labeling. Adding the TFA buffer may result in acidic ph. But I think the digestion reaction also need to be quenched to prevent over-digestion.
So, does anyone know whether TFA should be added before labeling?
the labeling is completed within an hour, thus you do not run the risk to over-digest proteins....TFA should be used at the end of labeling
Hi Andreas,
Thanks for your very detalied suggestions. In my case, after the initial TMT labeling, I will do phosphopeptides enrichment. Thus, I prefer to use HEPES buffer because I am worrying that ammonium bicarbonate may interfere the TiO2 column capture. I am also wondering whether C18 clean up is indispensable before the phosphopeptides enrichment. Because the enriched phosphopeptides samples will subject to C18 clean up in the end before LC-MS analysis. Do you have any suggestions on that? Thank you very much!
Best,
Xiaomeng
Hello Xiaomeng,
In my opinion, after TMT labeling it´s important the clean up by C18 tips or sep pack columns, because you will always have a lot of reaction side products that can interfeere in your phosphopeptide enrichment. I noticed this behaviour when I analized a proteome that needed to be previously fractionated by HPLC (1D), and it was really difficult to run the sample because of the high pressure of the HPLC column.
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