Single cell proteomics of the developing human brain!

 

Big thanks to Matt MacDonald for sending this last night with a "Wow" as the total email content so that I got to sleep at like 1am after I felt like I'd finally gotten through it. 

Honestly, "wow" is still the correct word for it. 

Before I get into it, this is the paper. 

Single cell proteomics still feels new, but maybe I'm just old, but we're still learning what assumptions we need to make to get to real biological discovery. 

Something I argued for years was that I'd much rather have more cells than more coverage, but I think I've fallen headlong into the coverage race along with everyone else.... this paper is a solid smack in the face because they did A LOT with a few hundred proteins per cell. 

They say they get 800 on average in most of the cells. I'm spot checking in DIA-NN and before bioinformagic, I'm getting 450 or so. Probably by the time you match between runs and stuff you probably can double that. I ain't reprocessing 1,500 files, so I have a clear sample bias. 

And - this is going to sound critical - and I don't mean it to be that way, because this is just a stunning work - but mass spec proteomics people may really just care far too much about quantitative accuracy. This isn't the first time one of our key tenets of proteomics has been really challenged. A Slavov lab study made the heretical decision of not fully resolving TMT reporter ions at baseline. Something that has been unthinkable for a decade or more. It still totally worked. We may try it ourselves sometime here. 

This study did around 2,000 single cells, about 1,500 of them from "brain cell types" by cranking their resolution and ion injection time to the moon on an Orbitrap Fusion III (Eclipse) with 40SPD "Whisper" on an EvoSep (the 100nL/min one)

I'm not looking at the paper now, but my notes say that it was DIA with 50Da windows and 250(!!) milliseconds of fill time at 120,000 resolution per MS/MS event. With 12(!!) windows.

12 x .25s x 1 MS1 (which might have been 240,000 resolution) so 3.25 SECONDS? per cycle. Someone somewhere in Seattle was shown this line I just typed - and threw up. All over the place. 

But hear me out. For real, what if your quan doesn't have to be good? 

Whew! Files finally downloaded so I could look at some of them and --- yeah -- you're going to get a lot of peptides, maybe most of them, with 3 scans/peak...

One of a pile of peptides I've pulled out, but look, I'm a blogger and I have a lecture due today for a class I'm teaching next week and 1-2 feet of snow between me and work, download 'em yourself here if you don't believe me - 

ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/12/PXD071075

Here is the point to stick output of the peptide above. It looks like a triangle, but it isn't actually as good as a triangle would be. 

Is the area under the curve of this peak a reasonable approximation of the signal of the peptide? Who knows? Not me, and not these authors. But is it probably reflective of whether one of the 800 proteins in this cell is higher than the 800 proteins in another cell? Probably! At this depth you're going to be doing a lot of presence/absence stuff. And in this model that is probably a lot of power! 

OMG, and I have laughed for real multiple times about S.Table 2. Man, did they throw some shade at just about every label free single cell study that did fewer cells than this one did! Wow. I would like to thank these authors for not citing me, therefore I did not appear on their Table of Shame. They wouldn't want my stuff there because with SCoPE-MS/SCoPE2 this is actually a very normal number of cells analyzed, but the authors made it very clear that label free quan, regardless of how poor, is the superior option. They might be right.

Okay, but the take aways here should be 

1) You can do a lot with a lot of cells 

Even if!

2) You only get a few hundred proteins per cell

3) And the proteins you detect aren't all that well quantified! 

A good experimental design and cool samples and solid informatics can push you through to an amazing study. 

Quick math, btw, at 40SPD these 2,300 runs or whatever ran - with no blanks, no QCs, and not stopping to calibrate and no failed cells (there are always failed cells) a little more than 2 months on a system that is a couple generations back. That's...not bad....

And they used FACs so the cell prep was inexpensive. I don't have our calculator in front of me, but I'm going to go with this being in the $20/cell range in total costs/cell before any labor. Possibly less. 

Kid's up, gotta run. Super super super cool paper you should check out!