This is such a great paper! AND its Open Access. Several people who occasionally read my ramblings here who need to see this right now are about to get this link emailed directly to them! You're welcome!
The paper is from Lichao Zhang et al., and some guy named Don Hunt was apparently involved which might explain some things about it.
When I visit people who profile antibodies, they are doing 2 things. First they are getting intact masses on the antibody. In big facilities, maybe its a whole group of people figuring out what intact protein masses are there. The second thing is digesting with trypsin and peptide mapping. Between the two groups they pretty much figure out what they're looking at. Groups that use multiple enzymes get better coverage, but you're looking at a ton of runs.
This approach? Kind of a lower-middle down approach with just enough awesome tweaks to maybe get the whole antibody figured out in one shot!
They start with the whole antibody and then they reduce and alkylate it (more on that in a minute). Then they run it through or over an immobilized enzyme I've never ever heard of, aspergillopepsin I, which instantly cuts the antibody to pieces around 3-9 kDa long. See? Lower-middle-down! What else would you call it?
What else would you call peptides that are 3-9kDa long? Perfect for ETD! In this case they used an LTQ Orbitrap Velos with ETD. And these perfectly-sized fragments give off amazing levels of coverage. They process everything with ProsightPC BioMarker search functions.
Okay. Neat, right? But it gets better.
The digestion occurs with a bioreactor. The antibody goes in and comes out digested...and the reaction quenched. Want bigger fragments? Increase the flowrate. Smaller? Decrease it.
One last thing. They alkylate the cysteines with a new reagent. Its called NAEM
Not only does it alkylate in 10 minutes, but it also puts a positive charge on the cysteines which aids in fragmentation.
How's it work out? Absolutely ridiculous levels of coverage of these huge and hugely important proteins and their PTMs in record instrument time!
There is another paper coming out soon, using chemical cleavage of cysteine for middle-down antibodies analysis. the nice thing about it is that you know you're getting even sized chucks, and a known quantity of fragments, regardless of the sequence of the antibody.
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