Wednesday, November 28, 2018

Proteogenomics fills in not-so-subtle blanks in the B.subtilis proteome!

I love these studies where someone tackles a model organism! Bacillus subtilis? Really? We have to know everything about this one, right!?!? Just what I can remember from long long ago grad school...

It has a genome about the same size as E.coli -- but
It can form nearly indestructible spores, hang out for 20,000 years, and start growing again.
It can make super weird fruiting bodies.
You can knock out every one of it's penicillin binding proteins (required for it to make cell walls) and it will still grow!

Despite hundreds? Thousands? Of studies on this little organism, loads of mysteries still exist? Such as...only 50% of the proteome is accounted for...???

Ravikumar et al., from the Macek lab is on the scene, solving these mysteries in this new Nature Scientific Report!

How do you improve on the understanding of something expected to be this well understood?

You grow normal and knockout bacteria in normal media(s) and in SILAC (&Super-SILAC) ones.
You fire up the Orbi-XL and you

Like --
1) In gel digestion
2) In solution digestion
3) You phospho enrich (4 WAYS)
4) You acetyl-enrich
5) You employ the appropriate multi-enzyme approaches

You process 1,688 (!!!!!!) files through MaxQuant.
You use the canonical databases.
You supplement these with 6 frame translated genomes.
You use Motif-X and other tools to assemble everything
..and finally(!) you do this:
...tried to spell that like 12 times. Screenshot!

What do you get out of this monumental amount of work?

1) By far, the most complete proteome of this important organism ever done.
2) A really intimidating folder on ProteomeXchange/PRIDE (here)  For real, someone deserves an award for just uploading this many files.
3) An amazingly thorough understanding of how this organism tightly regulates itself through a complex combination of acetylation and phosphorylation events.
4) And some useful phylostrafibrastabhic data?
5) B.subtilis finally

(...come on...I had to use that somewhere....)

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