Saturday, November 8, 2014
Automate your phosphoproteomics!
We get a lot of flack from people outside our field when it comes to reproducibility. Most of it is our fault. The genomics people all prep their samples the exact same way using the exact same kits, QC'ed mass produced reagents and the exact same protocols. We tend to be a little more artsy...and cheap... when it comes to sample prep. Everybody has their own little variations and most of us will try saving $2 here and there where we can before we load samples onto the most sensitive analytical instruments on earth. In the very near future we're going to have to standardize all this stuff or the genomics people are gonna be eating our lunches.
Within proteomics, phosphoproteomics is even worse in this regard. This is mostly because getting phosphopeptides is just hard. Even if you are using an easy kit like the Pierce spin tips, you can expect to spend your whole day getting samples ready for the instrument. The number of steps you have to go through introduces a lot of chaos into your system. It's tough reproducing those steps exactly, even yourself, on Monday and Thursday of the same week. Impossible? No. Hard? Absoutely.
I'm having this sleepy rant because I'm looking at a cool paper with a really appealing solution. Let's just automate the entire thing! In this paper from Christopher Tape et al., in this month's Analytical Chemistry (and open access), these researchers demonstrate a fully automated phosphopeptide enrichment strategy that 1) speeds everything way up and 2) makes this process amazingly reproducible.
To validate this method, they use SILAC and spiked in standards. Post enrichment the well-to-well variability in their phosphopeptide recovery was as low as the variability in their SILAC ratios!
If you do phosphoprotoemics or are interested in a good hint in the direction proteomics needs to be heading, I definitely recommend checking out this paper.