Wednesday, October 30, 2013

TMT 10plex on the Q Exactive Plus!

Yes, I'm re-using this image.
Why, you ask?  Because I am looking at data that you can generate from using the TMT10plex on the Q Exactive Plus.
Imagine a world where your isolation mass width is ~1 Da with no loss in sensitivity.  Imagine that the reporter ions that you have on 10 channels for quan are resolved at 50,000 resolution at the reporter ion level (35,000 resolution setting).  Wait.  Don't imagine that part, here's a picture of a randomly selected peptide reporter pair at 1:2 ratio (honest to Gus, it is random, they all look this good!)
Then imagine that you set up a series of controls in a 1:2:4:8 ratio, and they all turned out that way, with a <15% variance from expected at the protein level.  As a side note, it might be interested to know that this data came from quan on ~4,000 protein IDs.  Yup!  I'm not making this up.

That has been what my last 2 days have been like.  Lots of time thinking about how many iTRAQ 8 plex samples I have ran over the course of my career, and how little interesting data has come from them.  The future is looking pretty bright.

By the way, the method that my friends and I generated on this amazing instrument is now uploaded to the Orbitrap methods database.  Yes, the QE Plus is more sensitive and can handle a more narrow isolation width, but widen the window a little and this is how you should run TMT10plex on your Q Exactive.

Now, my disclaimer.  Did the Ting et. al., MS3 paper show that narrowing the isolation window didn't help all that much with the reporter ion suppression?  Yes it did.  Ultimately the MS3 method is better.  But narrowing the isolation most certainly does not hurt!  All it can do is eliminate co-eluting species and improve your quan.  And if you fall into my camp of people who need to run faster with more sensitive identification at all costs, even that of reporter accuracy, then we take the improvements we can get without compromising speed.


  1. Its really impressive !!

    on the other note, I got the Orbi Fusion raw file from ChorusProject (One hour proteome article). I must say the scan speed in Fusion is really really fast and thats how they got entire yeast proteome mined in less than 2 hours time...


    1. Santosh,
      Yeah, the Orbi Fusion is exceptionally fast and more sensitive than even the QE Plus. Honestly, the Fusion is going to beat everything in every category. But that doesn't take away from the fact that the QE Plus is a great series of improvements on an already great platform.
      I'm glad to hear that Tranche is working well enough that RAW files can be obtained from it again!

  2. Ben,
    Tranche is still not working. I got it from ChorusProject !

  3. Hi Ben,
    Thanks for a great post, I was trying to find the TMT method for the Q Ex Plus in the method database but could not find it.. The only TMT method in the database is for the Velos.


  4. Vojtech,
    Thanks for bringing this to my attention. I must have uploaded it to the wrong folder. I'll get that fixed.

  5. Hello,

    I know this is an old post, but we just got a QE and are trying to do TMT analyses. We are struggling with acquiring good sequence coverage and TMT label. I tried to find your method, but I don't see it in the methods database. Is it still available?

  6. Oh geez. That is totally my fault. I put them into the wrong folder (which is currently invisible to you guys)! I'm sorry about that.
    I put up a new folder "QE_TMTmethods_finally". There are 3 methods. 1 for the QE and TMT 2 or 6 plex, one for QE TMT 10 and one for QE Plus TMT10 (smaller isolation window). Sorry for the delay!