Wednesday, January 7, 2015

ATM and ATR in Arabidopsis

In grad school we had a series of guest lectures every Friday. In order to get the 1 credit for attending you could miss no more than 2 lectures per semester.  I planned mine out so that  I didn't have to hear anything about Arabidopsis.  Yes, I know it is an important model organism.  And yes, I know GMO crops are necessary to the continued habitation and population growth of this planet...I just always found plant research sooooo boooorrrrrriiiiinnngggg.

Now, this is weird, cause I've got like 10 papers open on my desktop here and I'm going to tell you about the Arabidopsis one.  Why?  Cause this study is so good it makes this ugly little plant interesting!  The paper is from Elisabeth Roitinger and Manuel Hofer, et. al., and is a study on quantitative phosphoproteomics of ATM and ATR knockouts in this plant.  (In press and open access, at least for now,  at MCP here.)

If you aren't familiar with ATM/ATR, you've definitely never done cancer research.  ATM and ATR are massive central regulators of DNA repair. If something happens to your DNA where it needs repaired, one or both of these guys is going to start a phospho- chain reaction that will make the damaged cells stop dividing and initiate proteins to do DNA repair.  If you knock out either of these genes (some people actually live...though not for long...with mutations in these genes) it almost always means your mouse gets cancer and dies real young.

See why this is interesting?  What the heck are these proteins doing in plants?!?!?  Turns out, the same exact stuff!

This group made ATM and ATR knock out (or knock-down...I'm not going to study the genetics part) Arabidopsis plants and then FASP digested out the protein. There were surprisingly few extra steps involved, getting protein out of plant tissue is tough, but the protocol provided here is one of the most straight-forward ones I've seen and the one I'll follow in the future. Next, they iTRAQ labeled their WT and mutant strains and phosphopeptide enriched with titanium dioxide and IMAC.  The enriched samples were SCX fractionated and the resulting fractions were analyzed on an Orbitrap Velos.

The Orbitrap method is interesting. Every precursor was fragmented once with CID and once with HCD.  Which is better for phosphopeptides? CID or HCD?  Who cares!  On a hybrid instrument you can do both, so why not!  Is ETD better for phosphopeptides? Sure. But CID+HCD will, under most conditions, still be faster, and 2 scans may be almost as good, and faster -- which should result in more total PSMs.

How'd they do with this method?  15,000+ total quantifiable phosphorylation sites.  Boom.

By the way, I mentioned above that if you induce DNA breaks that's when ATM and ATR are kicked into play. They tested this model system by irradiating these knockout plants.  This study showed differential regulation of the knockouts with radiation over the wild-type plants with radiation that closely mirrored those that we know occur in humans and mice.

In the end?  A really nice phosphoproteomic study AND a great new model system for cancer researchers who don't mind working with plants...

...which may not be so bad, after all.

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