Sunday, April 16, 2017

Histone post translational modifications play a central role in P.falciparum development!!!

WOOOHOOOO!!!!!  I am having the best weekend. Great weather, awesome people visiting -- then my Google Scholar Alert directs me to a beautiful study revealing a major missing piece of the P. falciparum regulation puzzle.

Warning -- this is probably gonna get all rambly. I am too excited for it not to be!

P. falciparum is responsible for the most deadly form of malaria we currently have for humans (did you know we know of at least 200 vectors that cause malaria (cool podcast about it), but only 5 normally get us -- and throughout our evolution we've had to beat a few -- mostly cause a vector drove us almost to complete extinction a couple times? [Another plug for the Fever])

Laurence Florens et al., first gave the world a deep comprehensive proteomic view of 4 stages of the parasite 15 years ago and loads of awesome work has been done since -- but something always seems to be missing. No way can this dumb little protozoa be doing all this stuff -- look at that picture above -- 7 stages of life cycle -- hijacks the cells of two different species (it has different development stages in the mosquito AND in us -- occupying our livers and red blood cells) and does all this regulation with 5,369 genes. Something critical is missing in our understanding here.

500 HISTONE MODIFICATIONS would sure explain a lot of it!!!

And that is what these authors have found in this stellar study. They enriched histones out of the 7 parasite stages, digested them (I've never directly worked with histones -- the enrichment and derivatization for LC-MS is kinda fascinating -- detailed really well in the paper) and single shot LC-MS into an Orbitrap Velos Pro running a well-optimized "high/low" mode.  I seriously try not to look at the author names before I read a paper, but I sometimes get into the methods section and think "wow, they know what they're doing" [my rule is to never tell you about the other ones] --and sneak a peak -- yeah...this is one of those.

Did I warn you this was going to get rambly? (I LOVE THIS PAPER!) It might get worse.

The authors get their peptide spectral matches in Proteome Discoverer 1.4 via Mascot using a large series of preset post-translational modifications that I have to imagine are well-characterized for histones. Worth noting -- this is not the first P.falciparum histone study, they have info to guide them a little here, but there is obviously a deep knowledge of histone modifications in play to search these RAW files without apparent deltaM searching.

The data next goes into EpiProfile.

I was impressed when this paper first came out but since it didn't really affect me I never even contacted the Garcia lab to get access to it (which you can here).

From a purely "what are the current limitations of proteomics" standpoint -- EpiProfile is important. Oh -- and it is going to be a big topic this summer (did I mention I love this paper?). This is why:

Imagine you are doing XIC based label free quan on sample 1 and sample 2. In sample 1 you find peptide HAPPYK with an intensity of 1e6 that elutes at 23.4 minutes. In sample B, you only find HAPPYK at that retention time at almost baseline, but at 10.8 minutes you find HAPPphospho-YK with an intesity of 8e5. Unless you've got some awesome tools that I don't have for global analysis of this type, get ready to buckle down for a fun filled afternoon of manually linking your modified and unmodified variants in that Excel sheet!  (PD 2.0/2.1 can make the job a little easier for you -- see video 22 here, but you still have to do a lot of manual work)

EpiProfile does this automatically for histones -- and the way it does it sounds so elegantly simple that I'd argue the logic behind it stands as an important development for our field all on it's own. (Worth noting? BioPharma finder does a great job of this as well -- but last time I tried it, I couldn't realistically feed it more than 5 protein sequences. The author designed it with exactly one protein in mind).

Back to the new paper! What did they find? Nearly 500 histone PTMs in P.falciparum -- over 100 that pass stringent manual validation. 15 new histone PTMs that (so far) appear exclusive to the parasite. (Not to sound hopelessly optimistic, but anything the parasite has that we don't sounds like a potential druggable target to me!)

Perhaps most importantly -- specific histone modifications that correlate with life cycle changes in the parasite, answering important questions -- "how did it just do that?!? the transcriptome doesn't appear to change at all!!"

One more note and I'll go do something else -- this isn't it from this team on this project. 2 other publications are en route. I know I'll be looking out for them.

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