Dear all,
Hello everyone, I am going to start the experiment with 6 plex TMT for my studies. Anyone, please tell me what column should I look for my label check experiment. Initially, to know my label efficiency in the peptides what should l see. Is it the reporter intensity in protein table that should come equally after summation of all identified intensities in all the six samples? or something else. Any help will be appreciated.
Thank you
With Regards
Syed Azmal Ali
Syed,
I cant answer with specific reference to MaxQuant, but in more generic terms, you will need to ensure that your labelling efficiency is 100% or very near. To this end, a search using any available search engine (Mascot, X! Tandem, Protein Prospector or MaxQuant) should enable you to allow variable modification of your peptide N-termini and lysine residues with your TMT. If you see any under-derivatised peptides (this is particularly relevant to K-terminated peptides, where there are usually two sites which are available for TMT/iTRAQ tagging), then your labelling efficiency is off, and you need to check your conditions. You would not expect to get a precise 1:1 ratio of each sample for every peptide, due to systematic error in protein loading, as well as the biological variability which is the experimental goal.
I hope this goes some way towards addressing your question, although clearly there will be other users who have MaxQuant experience and can contribute on which column is the relevant output.
Sarah
Hi Syed, I have exactly the same question as yours - did you ever find a solution? Would you share it with me?
Thanks, Martina
Yes, actually, there are multiple ways to follow up the label binding efficiency of individual peptides. One way or I can say, common procedure was already described by Sarah in the above answer. For analysis point of view it is also depends that what software and search engine you are using. If you are not satisfied with above answer and still has doubt, please feel free to email me ([email protected]) directly with elaborated question(s). I will be happy, if I am able solve your query.
Azmal
Hi. Do you know if is there any way to add iTRAQ/TMT as variable modification in MaxQuant ( there is not in the list)? I tried through the configuration list but i did not find the correct way.
Yes, instead of using it in isobaric labels in the MS ion MS2 tab; change it to standard label using configuration-modification tabs and restart the maxquant you will find them in the variable modification tab.
Dear all,
Do you add the TMT both as type in group specific parameters with the correction factors and as variable modification or only as variable modification for calculating the labeling efficiency? Your help is appreciated.
Best wishes,
Sidrah
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