Single cell proteomics by SDS-PAGE!

 

Okay, so what if you did proteomics on a sample and you were concerned that you'd homogenized too much tissue. Maybe you had an obvious phenotype and you just can't see anything differential in the proteome? And maybe you don't have a single cell sorter in your lab and a very very clean Astral, Exploris, Excedrin, 8600 or TIMSTOF sitting around. (For real, yo, I don't think we're past the requirements for very clean or almost dedicated instruments for single cells. This is from feedback from friends with Asstrals and 3 separate Ultra2s. Run 2 days of 50ng samples and that next 50 picogram single cell is garbage. Did I just come up with a smart community study idea right now? I think I did.)

Could you, with the help of a sorting core or with some quick dilution math, and some stuff you've definitely got sitting around, find out if you have single cell heterogeneity? 

Cue the skepticism, but I think this is absolutely worth trying. Good thing I've got a lab meeting in a couple of hours! 

They straight up do SDS-PAGE with single cells! The dye is something weird, and they're clearly doing some serious magnification on their gels, but its 1) A commercial gel and 2) It's the same ol' gel dock that you've got sitting around. There was a box in my lab bay with like 10 of them and the name of a professor who retired a while back! 

Here is the workflow in my mind, though. 

1) DIA proteomics

2) ...ummm....volcano plot with like nothing in it?? 

3) Repeat with DDA proteomics

4) Process with MetaMorpheus (or Bolt if you have that $20 a file) MSFragger open search would work if you have an Orbitrap or Astral probably. FDR shuts off if you have anything else. It still seems to work but not as well. 

5) ....ummmm....even with PTMs the proteomes look the same? 

6) Dilute cells (with membranes) down to statistically 1 cell per PCR tube (or have the FACs core do some sorting for you?) 

7) SDS-PAGE a bunch! 

8) Do you have clusters like this? 

9) You've got an over-homogenization issue! See if you can do some cell sorting and do single cell TYPE proteomics (cell sorted populations) OR

 10) Bite the bullet, clean the shit out of one of your instruments. Especially if you like literally study feces. Or talk to someone who does SCP through a core or collaboration. We do single cells at Pitt for like $50 internal for 40SPD. It's more unsubsidized, but we're trying to get that $ down with more hardware.