Tuesday, April 19, 2016

Integrate top-down with the other kind of proteomics!

Top-down proteomics is something I think the whole scientific community finds really exciting. Its that goal off in the distance of what proteomics will one day become that we're solidly and steadily progressing to. All of our current techniques are, well, compromises. By necessity we have to make our experiments way more complex and throw away lots of information --by digesting stuff.

It might be easy sometimes to dismiss the Kelleher group a little as futurists. Yes, they're absolutely pushing the technology futher...but we'll be able to solve biological problems with their awesome tools and techniques in a decade or two after a few big mass spec advances.  Come on, don't tell me you haven't sat back and thought that (or said it).

This recent paper from Ioanna Ntai et al., shows that there is stuff that we can gain from integrating top down proteomics into our work, right now.  Does current technology limit our ability to really do comprehensive top down proteomics? Sure, but this shows that even around our current limitations there is so much to be gained from top-down that it can still add some clarity to biological problems right now!

What'd they do? They took data from some xenografts (human tumors grown in mice) and studied those tumors with RNASeq, bottom up proteomics and fractionated intact proteins for top-down. There aren't many details on the sequencing in the paper, but the bottom-up definitely wasn't the emphasis here as it was performed on an old instrument (having personally visited all these authors labs I know they have nice stuff, but I guess it was all busy) ;)  they do get points from this noisy blogger by employing PEAKS Studio so they could easily look for mutations and PTMs. (Which I LOVE when you've got secondary confirmation methods like they have here!)

The top-down was done on an Orbitrap Elite using two different methods depending on the protein size....which makes a lot of sense....and makes me feel like I should read more papers from this group. Ugh. Okay.

I might be behind the curve, but I just learned about this thing.  If you have a Google account you can set up Google Scholar alerts. So every time you go into Google Scholar a little bell will be in the side of the screen that will show you that someone published something on your interest list. You can customize Chrome to show that alert in different ways. So from now on I'll get a little red alert in my browser when the Kelleher lab publishes a new paper. That's pretty cool, right?!?


Just kidding. Something neat in the methods is something called "The Advanced Protein Assay" from a company based in Colorado. Hunted it down and it looks pretty cool.  Load concentration on mass specs is something I think we've all kind of...guessed..??...on? Pierce knocked out a cool thing recently that might be similar to this that can quantify digested peptides and I've heard nothing but positive things about it. 

BACK ON TOPIC (fo' real):

What did we get out of this integrated approach? Bottom-up gave 4k proteins or so. Top-down produced about 1,000 proteoforms, mostly 30kDa and down -- and this is interesting, because these littler proteins were, in general, missed by the bottom-up approach! and, overall, the data was very complementary!!! They also seem to use the top-down data to resolve some things that don't correlate between the RNASeq and the bottom-up data, but I didn't really read that part closely. Its a dense paper and I've got to go to work!

TL/DR: Current instrumentation and techniques can boost your proteomics workflows RIGHT NOW. Maybe stop thinking of top-down as "I can't get all one Billion proteoforms today so its no good". Maybe think of it as, holy cow, I might be able to add 1,000 small protein IDs to understanding this biological problem and maybe that what I need to get to the bottom of this project.

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