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Fragmentation efficiency of TMT-modified peptides
Posted: Sun Dec 09, 2012 12:39 pm
I'm trying to get a TMT-protocol up-and-running and I've been doing a bunch of experiments with CID versus HCD on TMT-labeled and unlabled peptides. So far, all the experiments I've done indicate that the TMT-labeled peptides don't produce as many peptide-spectral-matches (PSMs) as the corresponding un-modified peptides. I've done side-by-side experiments with TMT0 versus unlabled peptides using both CID and HCD at a couple different fragmentation energies. I observe ~75% as many peptide IDs and ~60% as many spectral counts for TMT0-labeled peptides versus the control, unlabeled peptides using CID or HCD.
I'm wondering if this is normal, or if it indicates that I'm doing something wrong? Is there anything that can be done to optimize the efficiency of peptide-IDs for TMT/iTRAQ-labeled peptides?
Also, I have not been cleaning up the peptides after the TMT protocol, so there's still unincorporated tag in the sample... could that be a problem?
PS - This is on a Velos Orbitrap Pro.
Posted: Mon Dec 10, 2012 8:54 am
My experience has been mostly with iTRAQ reagents. And we normally observed decreased numbers of identifications as well (~10% less). I think there are a few things going on:
1) The tag changes the chemistry of the sample. The changes can have both positive and negative impacts on the identification of the peptide depending on the sequence. The tag changes the mass distribution of the sample (all the the peptides get larger) and the charge distribution (slight increase in average charge state). In addition, you are adding a very labile bond (or multiple) to the peptide. If you are using HCD you probably need to increase the collision energy compared to unmodified peptides. How much depends on the pressures and other nuances of your instruments. I would recommend shooting a sample several times and testing different collision energies and searching the data. My guess is if you used 35 for normal peptides you would want 40 or 45 for iTRAQ/TMT labeled peptides (based off of experience with the Velos Orbi, not the Pro).
2) The sample complexity increases. Yes, that unincorporated tag is hard to get rid of. We have tried different RP and SCX methods but it generally is still there. Maybe someone else on the forum has done further work in figuring out how to get rid of it. During SCX it generally gets fractionated into just one or two fractions. I do expect that it has some negative effect on IDs but I dont know how to quantify it. You also increase the sample complexity bc of partially modified peptides (100% is difficult to achieve) partial modification of Tyrosines, and other side chains reactions.
3) Sample loss during labeling. I don't know for sure but this step is the one that concerns me the most. It is done in high concentrations of organic solvent and it seems like it could result in precipitation or other sample loss.
Posted: Mon Dec 10, 2012 9:30 am
Thanks for the detailed response, Doug. It doesn't really fix the problem, but it makes me feel better to know that it's not just me. I also found this paper:http://pubs.acs.org/doi/abs/10.1021/ac100890k
which doesn't directly compare tagged-versus-unmodified peptides, but it does compare proteomic sensitivity of several different isobaric tagging reagents (iTRAQ4, iTRAQ8, and TMT6) to eachother. The difference in sensitivity with the different reagents is dramatic! If you can see a ~3-fold loss in spectral counts just switching from one tagging reagent to another, then I'm not surprised to see a drop in sensitivity with TMT0 tag versus no-tag. I was just surprised initially because I haven't seen this described in the literature anywhere... in fact, some of the promotional material for iTRAQ reagents suggests that sequence coverage and ionization are improved!
Posted: Thu Dec 27, 2012 12:54 pm
I agree with most of what Doug has said above. You need to turn the collision energy up a bit to still get good fragmentation of the TMT modified peptides. I usually use an NCE of around 30 for labeled peptides on the QExactive. I also see a bit of a decrease in the identifications, but find it is quite minimal. We have even played around with hyperplexing using MS3 scanning, and the decrease in IDs is still only minimal.
If you look back in the literature you will find a lot of papers about the shortcomings of isobaric labeling methods, but they offer some unique advantages.
I find that SCX gets rid of most of the unused label. You can also reduce the amount of unused label by decreasing the amount you use in the labeling procedure. The kits are sold as single use tubes for each label, and we have found that you can really reduce the amount you use and not see any decrease in labeling efficiency. You can also fiddle with the organic concentrations when you do this to avoid problems such as precipitation.
Posted: Tue Feb 05, 2013 2:30 pm
As pointed out before, one issue is a shift to higher charge states leading to a reduction of IDs, while 2+ peptides also yield higher reporter signals than +3 etc. Have a look at this paper from the Larsen group. They use ammonia solution to compensate for iTRAQ-based charge increases: http://pubs.acs.org/doi/abs/10.1021/pr100230q
In our hands this works fine.
Posted: Sun Mar 17, 2013 3:27 am
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Posted: Sun Mar 17, 2013 7:22 am
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