Tuesday, July 4, 2017

MS-Westerns - Like W.B., but specific, sensitive & reproducible, with dynamic range!

Disclaimer: I hate Western blots.

Although somewhat less common, the idea still exists out there that mass spectrometry quantification data needs to be "validated" with extract from platypus blood followed by putting wasabi peroxidase sauce on it and letting a camera stare at it in the dark for an hour...or something....

The idea of directly confronting the Western blot with alternative mass spec techniques is in no way new, and I've clung to every one of them. This new paper at bioRxiV has a bit of a different take than I've seen before, though!

Let's talk about the results the authors present first!
1) Better protein specificity (shocker!)
2) Better dynamic range. Doesn't take much to beat a Western in dynamic range, but I'll take it! I've got a figure somewhere from years ago -- 6 SILAC peptides for one protein that showed clear differential regulation and Westerns that looked exactly the same. Western dynamic range...

3) Better sensitivity than Westerns!!!  This myth is still out there that Westerns are more sensitive than mass spectrometry. Someone said it to me just a few weeks ago. It'll take more papers like this contesting this fact to change the perception.

How'd they do it?

They start with SDS-PAGE gels. They use heavy protein standard in these gels (QconCAT proteins) that are controlled in abundance. Standard data dependent acquisition is tested on both a quadrupole Orbitrap and linear ion trap Orbitrap device and both show the results mentioned above.

The ratios of the heavy/light peptides are used for the quantification. The math they employ allows absolute quan of the proteins of interest. To validate this, they use a Zebra fish and human cell line model.

All the RAW data is available at ProteomeXchange here, PXD005654, following publication. Information is available within the paper for accessing the data prior to publication.

Honestly, I'm still just a little fuzzy on some of the details, but I wanted to get this out there before I ran out of time! My favorite part of this is that you don't need anything new. Just order a heavy protein for your target and you're good to go!

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