Monday, December 23, 2013
Use of quantitative mass spectrometric analysis to elucidate the mechanisms of phospho-priming and auto-activation of the checkpoint kinase Rad53 in vivo
Rad53 is a protein that is involved in DNA repair in yeast. An extremely similar protein in humans, Rad51, is shown above. You irradiate some cells and probe with an anti-Rad51 antibody and you get the distinct foci shown in green above.
Despite years of research, DNA break repair is something that is poorly understood. A really nice paper in MCP uses a spiked in SILAC approach and high res mass spec to try to pull back the curtains.
In this multi-lab (heck, multi-nation) study, this team uses Rad53 deficient yeast strains and MMS (methyl methanesulfonate, a chemical that induces DNA double strand damage) to try to fish out the pathway leading to Rad51 activation.
Minor comment on the paper. The MS1 search tolerance was set at 25ppm in the MaxQuant/Andromeda runs. Particularly in a SILAC study, I think that this window is a little too big and might lead to mismatched pairs and maybe a raised FDR. Otherwise, this paper is a nice solid look at using a good classic genetics (knockout) approach coupled with HR-MS to fish out pathway differences. This is also one of those rare systems approaches successfully using spiked in SILAC.
You can pull up the original paper while it's still open access here.