Wednesday, June 24, 2015

Reproducibility in peptide prep, FTW!

So, I've been somewhat unabashed (context correct? WV grammar?) regarding my love of reproducible digestion techniques.  Look.  I understand. I know the one time that you did that experiment and you accidentally used 37mM AmBiC that you got 3 extra peptides and one of them was the peptide you critically needed.  Seriously, I'm not joking, I understand.  I know I've got a thing where I don't like to alkylate my peptides when someone is using the printer. It never seems like things work out when its running. I don't know.

But the truth of the matter is that we HAVE to get onto the same protocols. The survival of our field is at stake here.  According to the numbers I'm looking at in the next tab the NIH/DOD/NSF gave $12 to the next gen sequencing people for every $1 they gave us and the biggest reason was (wanna guess?) seems to be the fact that we can't reproduce what we are doing.  We can't.  And its cause we're probably the most stubborn field in all of science right now (random insert, lol).

We know without a doubt that proteomics can be reproducible (check out Sue Abbatitiello's paper here!). But the fact that we all stick to dumb things like George uses 16 hour digestions at RT and I do 30 minute at 50C and the fact that George and I have a 10% overlap in our data makes us seem just plain silly and not a great bet for giving funding to.

But there are alternatives.  Ones that are easy and awesome.  The first one (one that I'm kind of in love with) is Perfinity.  Digestion kits that rapidly provide reproducible proteomics data.  A new alternative are the SMART digestion kits from Thermo.  Lets be honest here.  If you knew the sample inside and out, could you probably alter some parameter and get a few more peptides out of your sample? Probably. But could a researcher on another continent get exactly the same results you did?  No. Kits like these are the future of the field and the sooner we hop on these bandwagons the sooner we're gonna show the next gen sequencing people that DNA and RNA are only a fraction of the answers that they need.

Sorry if this is all preachy!  I know as well as you do that proteomics is the future, but we need to work out some of our demons before we can convince the old guys who dole out the money that it is!

1 comment:

  1. Dear Ben,

    I always enjoy reading your posts. Regarding the SMART digestion kit - In my opinion, based on the info provided by Thermo, the methodology could be useful for the batch analysis of protein mixtures of low complexity (e.g. immunoprecipitates) or single proteins. It is not going to be a match, however, to the detergent-based processing methods for complete/unbiased proteome profiling from cells and tissues, let aside membrane proteomes.