De novo analysis of poly(!!) clonal antibodies from human blood!

 

I can't seem to get this picture in higher resolution, but the paper is open access here! 

I have a list of things in my head that I don't think you can do with a mass spectrometer. Or should do. Or, maybe, if you do it, it's definitely going to suck.

Mixtures of polyclonal antibodies? Definitely on that list. Mixtures taken from human serum? Yeah, good luck with that! 

There are a LOT of steps here from the best antibody characterization group I personally know of, but solving the absurd mixture of proteoforms that are present in human serum following a viral infection? 

It's hard to quantify KRAS when you know you've got a copy of the WT and one copy of one of the convenient mutant proteoforms on the other chromosome. That's 2 proteoforms on a little (though annoying) protein. mABs class switch and glycosylate and crosslink in weird places if you look at them funny. So you end up with these 10x larger proteins with a big conserved region from all the different variants and then a mixture of craziness from the variable regions way down in the low abundance region! 

To do it required all those enzymes above with both bottom-up and middle-down proteomics. An Exploris 240 and Orbitrap Eclipse were both used. I'm a little unclear from the methods, but I'd assume the middle down definitely went on the Eclipse for ETHcD, though it may have been used for most things. Also, this is the first time I've seen an EvoSep used for mAB mapping and I'm totally psyched that it works well for it. (You know, it's kinda tuned for global proteomics and in peptide mapping you want the little tiny peptides and the big ones as well). 

And - this is a paper from a company, right, and - GASP - all the data is up on MASSIVE if you want to check it out for yourself. No joke, the 3 papers I had in front of this one to read were a 0/3 for publicly available data, so you won't hear about them here!