Thursday, May 10, 2018
BOXCAR -- It's time to stop and redesign most proteomics experiments!!
Okay. I'm going to try not to freak out. This is so simple and brilliant that every one of us sitting in front of an Orbitrap should have come up with it on our own.
We've spent all our time focused on how to get more MS/MS signal that we've ignored the fact that our 120,000 resolution; MS1 scan (that might take 250-500ms to actually do) only had ONE millisecond of fill time.
What happens if we stop being dumb and use all that wasted time for something?
10,000 proteins in a single injection? Hmmm....that wouldn't be too bad. That's, I dunno, 5 to 10 times what I'm normally getting per injection. WHAT!?!?!?!?!?!?!?
That's what happens when you unbias your MS1 acquisition and distribute it across the mass range. The authors use the quadrupole to isolate a series of little windows across the mass range. The next MS1 scan will have a different set of windows. All the sudden that one single albumin peptide isn't consuming the full 3e6 charges that the C-trap can take. It wasn't isolated in that analysis, and in the one it did it was equally distributed between the other boxcar windows. It CANT waste all of your AGC!
What they get? An average of 20-60x higher signal to noise! Which kind of results in more peptide ID's per unit time than anything I've ever seen.
If you're thinking -- wait -- they just butchered the MS1 scan by cutting it into little sections -- you're right. However, the authors are somewhat involved in the MaxQuant project and have written some clever code and optimized the isolation windows to make the quan reproducible and reliable. This might mean, however, that if you aren't using MaxQuant you're going to need to tweak your LFQ software to make this work. I'll look at the ones I use and let y'all know if I can make it work.
In a human clinical study they quantify around 6,200 proteins with no missing values across 10 samples. That's -- impressive.... On a less limited sample (mouse brains) they crack 10,000. Single shot. 10,000 proteins.
Man -- this paper is awesome, logical and I still feel really dumb -- but I'd feel a lot dumber if I wasn't using this method for some projects next week when I'm back in the lab....
EDIT: Thanks for the comments guys -- I'm out of the office till Monday. I'll be working on standardizing BOXCAR methods so that they can be distributed through www.massspectrometrymethods.org. I hope to have something so people not on this great new study can be running next week.