Wednesday, November 16, 2016
Proteomics of microdissected neurons!
I totally read this whole paper and found out it was from last year! What?!? Geez... Fortunately, it is still awesome!
You can direct link to it here.
While the paper is definitely geared toward a specific nefarious disease, it is a beautiful exercise in optimization of how to extract the most proteins/peptides out of a tiny amount of fixed tissue. Cause...if you can take a block of wax that has something as tiny as neurons in it and detect the proteins that ought to be there, you're doing it right!
The procedure isn't trivial, and the authors are quick to warn you -- the laser microdissection (sp?) needs to be done really really well. Sample preparation methods and instrument methods need to be employed that focus on minimizing peptide loss above all else.
And...they pull it off (of course!) and get almost 2,000 protein IDs from some areas they cut out with the laser. Others aren't nearly as high...but honestly, maybe that isn't a limitation of their methodology, it may really be biology. When you are looking at areas of anatomy this specialized will we need expression of the whole proteome?
Some details -- the LC-MS was single shot (no fractionation, but technical replicates when possible) analysis on 50cm EasySpray columns onto a Q Exactive. The QE was geared up for sensitivity -- allowing up to 120ms for MS1 fill time and up to 250ms at the MS/MS level. They were willing to take a massive hit in cycle time, if necessary, to get good fragmentation spectra.
While it seems like I'm focusing on this as if its just a methods paper, they did serious runs on tissues from Alzheimer's diseased and control brains and have all the differential data in the open access supplemental info!
Wait -- this deserves an extra sentence or two -- the supplemental tables are so freaking logical. I might be baised since I've been looking at PD output tables for...a long time...but they are so smart. For example table S4 has the protein IDs charted against the surface area of the tissue that was dissected out! S2 is the actual comparison between the normal and diseased tissues and whether the proteins were detected or not.
This is a killer little study showing how much we can get from those little waxy blocks of tissue those pathologists have been stockpiling!