Monday, April 17, 2017
Analysis of variability in label free quan measurements
I'm going to put this here, even though I'm not 100% sure I get it all.
Maybe I'll figure it out while I'm typing this!
The authors set out to understand where quantitative variability occurs in different global label free quantification methods. To get going they start out with something really hard to do proteomics on -- plant matter, specifically my old enemy Arabidopsis thaliana. They follow a standardized protocol to extract the proteins/peptides from the plant (which involves lots of liquid N2 and manual crushing...) and do proteomics on it.
Edit: (cause I want there to be something that shows up in Google Images if you type "scumbag arabidopsis" from now on.)
They use a big EasySpray column and use an Orbitrap Velos Pro running "high/low". The data processing is done with Proteome Discoverer 1.4/Scaffold, Progenesis IQ and MaxQuant LFQ. To get a feel for variability they focus on about a dozen specific proteins.
What is interesting:
They find the 3 software packages get different results -- which makes sense to some degree, they are all doing their calculations very differently. And you are in a super complex matrix and focusing on a small number of targets.
A lot of the variability they see is between the point they extract the proteins and the point the data report comes out. I honestly started reading this because I thought this would be an analysis of the differences between harvesting/digestion techniques, but one technique is used here. One really complex technique.
But...a measurable amount of variability appears to simply come from the individual variability of these clonal plants...which should have the same genetic makeup.... Makes sense to me -- I always like these images @PastelBio uses for illustration...
What I don't get (and this isn't a criticism at all, this is probably me just not being smart enough to get it!): I don't fully understand how they separated upstream technical variation out of the equation to get to the individual biological contributions from the clonal plants. Still an interesting paper, and something that might be consuming some of the idle process time in my brain today.