In an interesting recent trend, everyone seems to be emphasizing how small of a cell that they can do single cell proteomics on.
Do we have a new winner? No, Akos did single E.coli. Even if it was only like 25 proteins, that's clearly the winner for craziest tiny cell idea.
How much protein is in a PBMC?
14 picograms! (These author's math, not mine). FOURTEEN?
My group has recently struggled with some human immune cells... and from the TICs I was guessing we were starting with less than 50 picogram. FOURTEEN? Geez.
How'd they get there?
NanoPots. Ouch. Okay, so something you have to build yourself, but something with incredibly ridiculously low sample loss.
Then TMT 32plex.
If they were able to recover all 14 pg, and lets just say that they used 30 channels for actual single cells. To the mass spec, that looks like an MS1 signal of 14 x 30 = 420 picograms. Ouch. That's still not much at all....
The instrument used was a FAIMS (2 voltage) Orbitrap Fusion III (Eclipse). Real time search (ion trap matching) was used to determine was ions to analyze in the Orbitrap for MS/MS. Dual columns and emitters were also used here, and they did have to fabricate a bracket to make that work.
For samples this low in concentration there was some painstaking optimization, in particular of the "carrier" or (here) "bridge" channel. Sometimes called "boost" or "orgeano" because mass spectrometrists are still a bunch of cantankerous assholes who like to make up new terminology so that we seem as annoying and unapproachable as possible. I'm pretty sure it's just because we all hate research money and being taken seriously and we'd get a lot more of both if we'd quit making up stupid new terminology.
The bridge channel was kept very very low. The highest tested appears to be 1ng. So...1.4ng on column....
You have to dig for the HPLC stuff in the supplemental but it's about an hour. I'm a little confused about how this version of the dual column parallelization works. It is detailed, but I didn't take time to draw it out, but it looks like each sample is about 60-75 minutes. 60 minutes for 30 cells gives you 720 per day and 75 minutes for 30 cells gives you about 575. The authors report 660 cells/day, so it's somewhere in the right range! They squeeze extra signal out with a ridiculously tiny column. 50um internal diameter and 25cm length. I think this is =>100 nL/min to keep the HPLC from leaving craters where labs used to be. Real time search with spectral libraries made from these samples do some heavy lifting here. And once the authors get it all optimized out they run the system for about 3 days to report data on over 2,000 single cells.
Really truly impressive work and another great resource that demonstrates we could do high numbers of single cells if we really put the effort in!
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