Friday, August 5, 2016

Flu phosphoproteomics reveals almost 300 new phosphorylation sites!

It is seriously too early to be thinking about the flu, right? In a few months I'll be thinking about it all the time, wondering when some gross/adorable neice, nephew or friend's kid is gonna share a well-cultured virus with me.

Sandra Söderholm et al., have clearly been thinking about it for a while, cause they just knocked out a stellar phosphoproteomic analysis of flu-infected human macrophages. You can find it open access here at MCP.

They did some nice biology including mouse work on top of the human macrophages, but I mostly care about the other stuff.

The macrophages +/- virus were lysed, proteins digested, and the phosphopeptides IMAC enriched

Wait. Segway here. There are far far too many ways to enrich phosphopeptides at this point. I think we need some common nomenclature or something. For example-- extremely extremely minor criticism of this paper...but if you tell me that you IMAC enriched as you did in this paper and I go to this paper, get the PDF and find in the method section and you give me another reference to go to -- and its the exactly zero modifications of the Villen and Gygi IMAC method --

Oh no! The segway became longer than the serious stuff again!

The IMAC enriched phosphopeptides were ran out EasyNanoLC and Q Exactive (classic, I think). Method looks pretty standard, but I'd have to pull the files from PRIDE (PXD001620) to find out the gradient length and stuff (sorry. I know I'm a nerd, but this is seriously what I'm gonna look at first.)

They used Proteome Discoverer 1.3 with Mascot (big human database w/custom reverse) and PhosphoRS to get their lists. So they have macrophages +/-phosphorylation lists.

That stuff goes to PhosFox!  What's that? The website is here. They wrote it themselves (paper here) and it looks really cool. Its for +/- phosphorylation stuff. Unfortunately, it may only be available in Perl, but I can't investigate right now, I gotta get to work.

Wrap up. Honestly, I like this paper cause...
1) It is super biology centric. Here is our model and we're just doing a discovery to see what we can find
2) The mass spec just looks like another tool in the lab. Here is our standard old IMAC setup, EasyNano, QE, PD 1.3, and our cute little custom program to pull out the neat stuff --oh, and they use IPA to pull it all together.
3) And guess what?!? They found some new stuff. Phosphorylation sites with high confidence that no one has reported before that appear to be involved in our host response to viruses. Not the newest instrument, not some cutting edge new sample prep method -- solid, proven technology and methodology and some killer biology and BOOM!  A bunch of stuff we didn't know before!

(The Pug:Blog word ratio has now been returned to normal.)

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