Saturday, March 17, 2018
The Beadome! (The crap that sticks in every pull-down!)
I'm new to IP pull-downs, affinity purification, affinity enrichment, all this stuff. My background in the lab is global plasma/serum stuff. I never have enough dynamic range, I never have enough peptide coverage and the instrument is never fast enough.
At my new facility, IP is the way of life. Everything is enriched with big proteins taken from mouse or camel blood or whatever and stuck to beads or something. It's a mystery to me how it works and I'm too busy fixing EasyNanos and looking for the coolest PTMs you've ever heard of to ask.
As I'm running these things my first thoughts have been -- wait -- if we're using an antibody shouldn't we pull down like, I don't know, the one thing that the antibody is specific for? Aren't antibodies for matching to one single protein? WHY ARE THERE 2000 THINGS!?!?!
Some of this isn't my ignorance. Turns out a lot of it is just crap that will ALWAYS pull down. There have been multiple awesome studies over the last 10+ years on the "BEADOME"
This is a great first starting point (2008)!
This review is more recent and has some interesting new information as well (can't take screenshots I have it in hardback only)....
Okay -- so here's the question I'm going to get to really quickly. When I do plasma proteomics the first thing I do is build the biggest static exclusion list that I can. I have exactly ZERO time to waste fragmenting albumin, transferrin, and about 45 other proteins. If you're really interested in albumin, you'd better tell me about it up front because no mass spec I'm in front of for very long will ever detect an albumin peptide.
How consistent is this BeadOme thing? And should the Q Exactive (which has a maximum static exclusion list of 5,000 ions) and the Fusion (which -- I've yet to run plasma or serum on yet, so I don't know the upper limit -- gotta be more than 5,000, right? It's got 2 PlayStation 4 CPUs inside it!) be continuously ignoring a lot of stuff to boost my dynamic range?!?!?