Saturday, May 28, 2016

Structural characterization of missense mutations(!!) by Native intact protein analysis!

[Edited on 5/30/16 to take out some of the stronger language.]

Characterization of mutations is one of the next big things coming in proteomics. I know 4 or 7 groups working on it right now that have all sorts of cool things to show soon.

But this study in the new JASMS in my bathroom is something I hadn't even considered!  Its from Gili Ben-Nissan et al., and you can find the abstract here.

In this study they show that they can find missense mutations (change in one amino acid) in NATIVE INTACT PROTEINS!!! (Sorry for shouting, this is rad!)

What they are interested in is a protein involved in Parkinson's disease. And there is a mutation associated with it that changes the native protein structure. To approach this analysis they break out 3 instruments -- two TOFs and the Exactive Plus EMR.

Very early into the study discover that the EMR is doing such an amazing job that they continue with it and drop both the TOFs (even the fancy pants IMS-TOF) because the EMR provides the data that they need. I seriously love that little box. Someone in Maryland buy one so I can pop in just to see it!

What they do say is that they end up using the EMR exclusively because even the ion mobility TOF can't do this kind of analysis

[End Rant.exe]

Since they're down to the EMR, they have to do some sample prep before they go into the instrument.
 I know there are some fancy IMS things that you can do and some people have found that they can take the effect of the ion mobility field as reflective of some sort of a function of protein stability, but
you can definitely argue that the effect of an electric field on an intact protein in the gas phase isn't exactly the most direct way to understand something meant to happily exist in an extremely complex aqueous buffer system.
 Here they do some really nice degradation assays and thermal stability stuff (in liquid) before the EMR and --- they show they can absolutely track the native protein changes from a single amino acid substitution.

What?!?!  I know!

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