Saturday, October 18, 2014
Want to monitor global redox response? Try cysTMTRAQ
Redox states are big regulators in biological systems. Those annoying cysteines that we are always reducing and alkylating so we can ignore them are actually big regulators (regulateees?) of global redox state. One example that pops very rapidly to my mind is the NRF regulator. The cysteines in that protein can give you an awful lot of information about what is currently happening in cells and some labs deliberately target this protein to get early information regarding drug toxicity.
In global proteomics, however, we haven't really every taken a shot at monitoring the cysteines and their capabilities. Heck, we make it almost impossible to do so. When we put our cysteine alkylation as a static mod (something I don't do, btw) we are saying that 100% of the cysteines in our sample were successfully reduced and successfully alkylated. If something else was going on with the cysteines we will never ever know it.
Some researchers at the University of Florida have taken a completely opposite approach. In this paper from Jennifer Parker and Kelly Balmant out of Sixue Chen's lab (currently in press at MCP here), these researchers show us that we can be monitoring the global redox information in a cell with full quantitation as well.
Now, this technique isn't for the faint of heart. The technique is called cysTMTRAQ because it employs two sets of isobaric tags. The first is one of my favorites, the cysTMT reagent (if you dig through the blog you'll see how we used this reagent in my postdoc to get at drug mechanism-of-actions...such an awesome and underutilized reagent!) They use this to get quantitative information on the state of the cysteines. Hint: If you don't reduce and you tag, then you get quan on what cysteines are currently in an un-linked "active" state. Then they do reduce/alkylate/digest and label with iTRAQ. In this way you can get quantitative information on the global protein distribution.
I have a couple comments about this approach. The first is that this is really seriously smart. This is the first approach I've ever heard of for monitoring redox and this is great purely for that reason. The second is that I see a ton of potential in this whole approach for some us out there who really won't be going after redox states. For example: When I review a paper and it says "drug treatment leads to a decrease in phosphorylation in this list of proteins" my first thought is how do we know protein abundance didn't shift? Sure, its tough to rapidly up-regulate proteins (transcription/translation take time) but caspases kick in crazy fast and degrade proteins both rapidly and sometimes selectively).
Again, I really enjoyed this paper, but the whole time I was thinking "can I double tag all of my PTM quantification experiments?"