Tuesday, December 1, 2015

Can phospho- protein profiling be highly reproducible?

DNA damage response proceeds along a tightly controlled phosphorylation cascade. The main operators are very well studied and predictable enough that immuno assays are used in the clinic for these phosphorylation sites.

Could you quantify the entire cascade with a single LC-MS run? And could you do it with a high level of reproducibility?  Sure looks like it!

In this paper from Jacob Kennedy et al., out of the Fred Hutch, they use a single step IMAC pull-down followed by MRMs and the data looks fantastic. (Max CV on phosphos of ~16%?!?!)

Do 150 Western blots? Or monitor all of this pathway in a single run? Makes the mass spec seem like a pretty cheap option, right? Now, being the resolution snob that I am, I would like to point out that the relatively small number of targets here, this is something that could easily be adapted to a Q Exactive. Again, the data looks seriously fantastic here, but if I was to improve this assay in any way I'd want to see my fragment ions in PRM +/- 1ppm.

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