The idea is this: When you are looking at reporter ions, such as TMT and iTRAQ, the resulting ions that you are using for your quantification are in the low mass range where there is lots of interfering junk. Also, if you have co-isolated more than one ion species, then you may be looking at reporter ions from both your ion of interest as well from these co-isolated ones.
I'm going to take a step back. The following figure is an ideal iTRAQ experiment.
In the ideal set up, you only get your ion of interest fragmented and you can trust the above statement. More reporter ion 1 means that this protein is up-regulated in patient #1.
The problem is this: Although we have selected a specific ion, sometimes within 1 part-per million (0.01 Da or less, the instrument can only isolated as well as its weakest component can isolate. This often means that when you think you are fragmenting ion 515.01 +/- 0.01, you are actually fragmenting 515.01 +/- 2.0. This allows other contaminating ions in to screw up your ratios.
This method goes one step forward and selects the ion again, and does MS3 on it, with the correct assumption that the majority of contaminating ions that slipped through for MS2 will not be able to repeat the trick and be selected for MS3.
The benefit is vastly improved quan values. If this is what is important to you, I strongly recommend you read this paper!
Unfortunately, there is a drawback. The extra step in getting and isolating the MS3 takes additional time. This is why I doubt I'll ever use this method. In my work, I need to dig deep. The proteins I am looking for are the lowest possible copy numbers. These are proteins expressed by a percentage of parasites that are infecting a percentage of human cells.
Would I like better quan? Of course! But when I do an iTRAQ/TMT experiment, I am averaging (automatically, in Proteome Discoverer) the reporter ions from a number of peptides from the same protein and then I am looking for pathways, not individual quantifications.
I believe that this pathway is implicated in my disease of interest because multiple proteins in this pathway seem to be affected during infection. Using a method that is more precise, but gives me fewer of these proteins is going to adversely affect my results, in the way that I use reporter ions -- as a filtering method for implicating pathways.
Don't get me wrong. I absolutely love the paper from Ting et al., it is just that the drawbacks outweigh the positives for my application of reporter ions.
Also,MS3 method does not allow one to use HCD for MS2. Its always CID followed by HCD. Do you know how to tweak it to do both MS2 and MS3 by HCD method?ReplyDelete
That is a great point. Due to patent infringement issues, HCD spectra can only be read in the Orbitrap. The instruments are capable, but the software must disable this ability. Therefore there is no way to do HCD for both MS2 and MS3. CID must be first until this can be worked out legally.
Thanks Ben for this info. I hope this can be worked out as it ASAP so that it will allow researchers like me to take full advantage of MS3 feature.ReplyDelete
Santosh, I will let you know if I hear anything. It is always sad when business gets in the way of science.ReplyDelete