Thursday, October 31, 2013
Do we still need to fractionate our samples for deep proteome coverage?
Here is a good question: Are we still in a place, technologically, where we gain more from pre-fractionation than we lose?
At first, we had to, right? If we didn't cut gel slices or fractionate a sample by SCX or IEF or some other manner all we'd ever see was albumin and keratin from Pan troglodytes. Maybe that was just me. My first several proteomics experiments and that's all I got. Chimpanzee keratin, because mine is more similar, apparently, to chimp skin than human. I have proof, btw, I'm not just being funny.
Fractionation seemed to be the key. 20 fractions yielded more than 10 fractions and we dealt with the fact that we went from a 24 hour run time to a 48 hour one, because we were getting some sample depth.
Here is the question, though, is it still necessary? Or have we attained the speed and sensitivity in MS and the quality of nanospray chromatography separations gotten to the point that the inevitable losses incurred by pre-fractionation and the staggering increase in run time are far worse than the gains?
The literature this month would go in two completely different directions on this one. Two weeks ago I summarized a paper in Proteomics that used a pretty unique 3D fractionation method and a lot of you guys really liked that paper. Going in the complete opposite direction, the paper from Josh Coon's lab with the one hour proteome showed that you can get into the high numbers with just 1D, if you optimize the LC really well (oh, and have a Fusion).
Here is a new one for camp number 2: "Rapid and Deep Human Proteome Analysis by Single-dimension shotgun proteomics" by Pimoradian et. al., out of Sweden, the home of the world's greatest band (In Flames), and also the home of Roman Zubarev's, who is corresponding author on this work. From the title, you might be able to guess a little bit about the paper...but I'll still tell you more. By using a 50cm column and optimizing the LC conditions to the ideal dynamic exclusion settings (see, I told you this was super important! see rant #1, rant #2) , they were able to knock out 4,800 unique proteins out of an A375 cell pellet.
I'm going to reserve my opinion for now. Nope, I lied. I think that if you're currently doing lots of prefractionation or 3D separations, I think that you owe it to yourself to step back away from your system and give one of these optimized 1D separations a shot. The evidence is building.
You can link to the abstract for Pimoradian et. al., here, but the pre-print release link has expired.