PRECISE readout of MEK phosphorylation cascades by top down proteomics!

 

.....wow.... I won't lie. I'm stunned. I didn't think we were here yet.... and, to be fair, maybe we arent,but this group is! 

Bad background by Ben: A whole lot of the central regulatory pathways controlling tons of things in cells are based on some key central phosphorylation cascades. MAP kinase and MTOR are famous ones. They modify proteins by phosphorylating them because the modification can be fast and it reversible. If you go into any oncology centric place there is probably some really really really skilled pathway scientist (or 4, if they can afford them) who can dissect these cascades, probably through western blots and FACs. These people can tell you that if MEK1 S2998 is phosphorylated but T2992 is not - that means something critically important. 

When we do shotgun proteomics, we cut this region into a small piece and peptides phosphorylated once on the same region of that fragment coelute. It's often very hard, if not impossible, to tell which site is phosphorylated - or both. 

Obviously we should do this without digesting them, right? The problem with that is that top down proteomics only really works on small proteins. If MEK1 was 16kDa, it would still be tough, but you could do it. MEK1 is almost 50kDa, though! 

Enter "Individual Ion Mass Spectrometry" IIIMS? (older post explaining what that is here) and - what?? - ETD IIMS? 

I was going to cut in parts of the materials and methods section here, but I don't want to intimidate anyone into thinking this is clearly just a proof of concept. However, I will say that the process to get to these data makes label free single cell proteomics look like a nice fun day (which it is NOT). 

However, we have to start somewhere and being able to confidently localize 4 separate phosphorylation sites on a critically important protein this big - with anything - is a step in the right direction!