Wednesday, June 26, 2019

TMT Labeling for the Masses! A thought experiment for ultra-efficient, low cost labeling!

Disclaimer: You should definitely 100% completely follow every step of your vendor provided protocols. You probably spent close to a million $$ on that instrument, what's our total reagent costs? The "next gen" people drop close to $1,000 per sample no matter what on their global assays....

A while back I noted some other (also unrecommended) thought experiments from some people at Harvard where they were using less than the ratios that you should be using (I can't find the link...) but I think they were using 1:2 ratios but typically getting 95%-ish labeling efficiency.

In this (don't do it, for real) thought experiment, these theorists find they can obtain over 99% efficiency with a 1:1 ratio of reagent after some tweaking. 

You certainly shouldn't do this. 
And you certainly shouldn't take these authors' word that this works. You can check their work, it's at ProteomeXchange here.  Definitely download the .csv file, the file naming isn't quite intuitive (and keep in mind about half the optimization was done with the TMT-0 reagent, which if you haven't used before may not be default in your software (this is where you find/add it in Proteome Discoverer. Depending on your version, you may need to Apply the modification and then close your open workflows so you can see it)

(TMT-0 is +224, TMT-duplex is +225, TMT6-11plex is +229, for reference in any other software program)

I'm definitely not going to do this myself, obviously, I'm going to follow my vendor recommended protocols, just as I will urge you to do, but -- 

-- a quick filter says that out of 5,234 PSMs labeled in this method, I'm getting 3 unlabeled PSMs from the author's data....that's >99.942% labeling this was me just processing one of the short gradient optimization TMT-0 experiments, so grain of salt here on the larger complex sets, but wow....not a bad start. You still shouldn't do it, but it's an interesting thing to think about. 

No comments:

Post a Comment