Top down proteoform analysis of kinase inhibitors in an approachable method!

 

Wow. This new study of kinase inhibitor treatment of cancer cells - using top down (intact protein/ no digestion) proteomics is 

1) Super legit

2) Seems really approachable

3) Kind of resets the bar in my head for what we can do right now with today's off-the-shelf technology.

And I might have a surprise for you. While Neil Kelleher's name is here because it is part of a special issue in his honor - this isn't a Kelleher lab study! 

Generally when we see a super impressive top down study I flip through it and then think - cool - maybe I'll be able to replicate it in 10 years? There is often modified instruments or things where you think - if I was able to keep my core scientific team together in a group for a decade we could pull off something this hard. 

Not to say there isn't some legit technical firepower on this study (Kevin Gao is a pro's pro mass spectrometrist), but you can read through this protocol and think - wait - could I totally do this? 

Instrumentation is an Exploris 240! (Approachable, affordable, clean it yourself hardware!) 

The HPLC is a custom Accela....okay, well...I don't have one of those, but it is running at 400nL/min with an interesting combination of buffers. I assume any U3000 RSLC, Eksigent or whatever could assume those same performance metrics. 

Custom nanoLC source. Details in references, but you can make a nanoLC source from Legos. Probably not that tough to reproduce (or necessary). There are funny little bars that are necessary for the Exploris systems when you make your own source and those can set you back several hundred $$

They used TopPic suite for the data analysis, which you can get for free here, as long as you sign stuff saying you won't be a jerk. For some of the focused proteoform specific (is the phosphorylation site at this place or this place) they (interestingly) used BioPharma Finder. I've never loaded more than 5 proteins in that at a time and it's super slow with that many. I assume they put in one sequence and a narrow time window in order to really lock down that one target they're trying to localize. 

The results are well displayed - really pretty and clear - and, again, really might just change your mind about doing top down proteomics. Bravo to this team, I legitimately loved reading this paper from beginning to end. 

Wait - found something to complain about! Whew, I was worried. They haven't unlocked the PRIDE repository so I can look at the files. It was just accepted (JPR ASAP).