Wednesday, November 27, 2013

What does a good TMT or iTRAQ MS/MS spectra look like?

Holy cow!  I haven't posted anything in almost a week.  Normally there are very good reasons for this, like 1) I changed my password and forgot it or 2) It was nouveau week, or 3) The super cool projects I'm working on in my spare time are either a) something I can't yet tell you about cause its secret or b) something that didn't actually work.  Possibly a combination of all of these, but I'd appreciate it y'all would assume it isn't primarily 3b!

But now I'm back, full of espresso and I'm excited to throw this one out here.   I may have written about this before, and I plan to actually do another entry later on "this is a good spectra, this is not" but this one is pertinent to a lot of people due to the explosive popularity of the (fantastic!) TMT10 reagents.

Here is the question:  When I'm looking at an MS/MS spectra of a reporter ion tagged peptide, what am I looking for to tell that I have a good one?  I.e., how can I tell that my HCD collision energy is too much or too little?

Disclaimer:  I totally made the following up.  I ran iTRAQ for years for my own research and help  at least one person a week optimize their reporter ions.  This is the way I do it.  People have probably published other better ways of doing it, but this one is faster.

I base my opinion of whether I'm looking at a good MS/MS spectra on only 2 things
1) Are there reporter ions
2) Can I find my parent ion at <5% base peak intensity

Randomly chosen example from a friend's TMT10 run:

This is on an Orbi Velos.  The chromatogram isn't ugly because of spray stability issues (Patricia doesn't mess around when it comes to technique, the spray stability is great).  It is ugly because of the relatively high amount of time it takes the Orbi Velos to do a Top15 method with MS/MS at 30k resolution.

Lets look at criteria #1

Reporter ions!  Check

What about criteria #2?  The base peak is 1.2E6

How about my parent ion?

Hard to read, but the parent is 4E4.  Less than 5%, but still there.  HCD can be a little tricky to optimize.  It can be easy to over-blast your peptides and not have enough left to sequence.  If there is still a small percentage of the parent around, then I can feel pretty confident that I didn't hit the peptide too hard.  If there is a lot of parent around then I didn't hit it hard enough.  The 5% rule is a crude estimation.  Is there parent?  Is there just a tiny bit?  Perfect.

So this brings into play the big advantage that I perceive between the iTRAQ 8 plex and TMT10, and why every person I've seen do the comparison has switched to TMT10.  This is much easier to optimize.  The iTRAQ 8 chemistry is tricky.  It takes several passes to get your collision energy where you have reporter ions AND you have enough peptide left to sequence.  It is significantly easier to get this right with the TMT10, because the reporters come off with at least the same efficiency as the breaking of the peptide backbone.  When in doubt process the data!  I bet you'll find that spectra optimized like this will end up sequenced with good quan data at a pretty high efficiency.

TL/DR:  Its a good reporter ion MS/MS spectra if you have reporter ions and you can still find some of your parent ion at a low level in the spectra.


  1. Great post.
    It would be interesting to check how the new TMT affects the charge states of the peptides. For instance, iTRAQ 4-plex and 8-plex are know to increase the proportion of 3+ peptides which makes Mascot scores to drop and consequently less IDs

    1. What year is it?!?! Okay...well...