Extending the capacity of reporter ion quan experiments is a big topic these days! Case in point? Check out this great study in the current issue of MCP!
In this work mouse diet is the topic, but if you aren't interested in that (if it was Pug diet you'd have my attention...we've got a problem here with one that rattles windows when he walks...) it is still worth checking out!
'Cause they really put in the legwork to make sure the methology they employed was the most rigorous and statistically valid methodology.
They start off with SPS MS3 for the individual TMT experiments for the most robust quan values to start off with. They employ the edgeR quan stats package to evaluate linearity across their samples. They use 2 pooled controls in each TMT10 set (I'm assuming this is for the proof-of-principle work, but inter-set matching is a powerful, though somewhat expensive, way to verify that your values are absolutely on target!) and they evaluate multiple normalization techniques and methods to determine the one that is the most reliable.
If you want to compare between multiple TMT experiments and need the ultimate case study of how to get the very best results across all your samples -- this is a great study to start with!
Best yet? This guy signed off on it!
Actually...I did check with someone in the lab. Not the same Larry David... I'm just going to assume that everyone in the lab has the "Curb Your Enthusiasm" ringtone and that the joke has never gotten old. Just in case me mentioning it didn't get it stuck in your head -- here is the full length song!
Thanks for the shout out on the research! Two pooled channels is not just for validation. Everything depends on how well the IRS factors can be estimated. Averaging two channels is much more robust than just a single channel. The cost is reducing biological samples from 9 to 8 per TMT. That is only an 11% hit for greatly improved accuracy.