Friday, July 12, 2024

More single cell histone PTMs (epiproteomics!) label free, targeted WITH derivatization!

 


I've whined about this before, but I went to an epigenetics meeting last year and had a lightning talk (and awesomely lucky poster location by the coffee) and pitched single cell histone PTMs ("come talk to me by the coffee"). 

No one did. 

For real, every single single cell proteomics dataset I've analyzed (and I've looked at just about everyone's RAW data - except the people who don't make theirs public even after I request it - and you know who you are and you suck) - has histone PTMs in it. 

If you really want to do histone PTMs right, though, you need to do something about all those basic residues. The gold standard is propiprolypropylianation (spelling?) where you derivatize every lysine that is unmodified and then you digest. If you've never done it, it sucks. You can't buy the reagents. You have to buy some reagents and then create the active (reactive) form and then you add it to your sample before it expires. Not fun and I didn't think - for even a single second - that the reaction could be controlled well enough to do it for single cells.

I was wrong. 


This group did some super high precision work on the CellenOne and not only isolated single cells well and lysed them in tiny volumes, but they also dumped that live reactant into the single cell lysates. Derivatizing their lysines from single cells. When I said that I see histone PTMs in every single cell data set, it's not hundreds of them. It's the easy ones. K27/K28 (depending on whether you count the M, which UniProt does) and K80 are friendly and tryptic. Unfortunately, there is also a lot of homology in all the histones so finding a modification site is generally easy at a peptide level....tryptically digested it is tough to figure out what histone it came from. If you propionylate (spelling?) then you get a longer sequence that can help break those populations up and assign them to the original correct proteoform.

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