Tuesday, August 8, 2017

Global SILAC-SPROX shows protein stability patterns in cancer cell lines!

I looked at this new paper at JPR for an embarrassingly long time this morning before I got it.

This strategy totally helped!

Honestly, I had to go to the original SILAC-SPROX paper at MCP here as well.

But I've got it now, and I'm impressed and surprised that this works.

SILAC-SPROX is a strategy for determining the protein stability over all the proteins in 2 (in this instance -- should be compatible with 3-plex) cell lines. I'd assume that global protein stability would be pretty much stable across one human cell line to the next.

They way they assay this is taking total protein aliquots and exposing them to different concentrations of urea. Urea exposed SILAC labeled protein from cell line A and cell line B exposed to X concentration of urea are combined, digested and quantified using normalize SILAC type proteomics.

If cell line A is less stable (unfold more readily in that concentration of urea) then there will be more linear protein available for tryptic digestion and it will look like protein up-regulation. Of course some proteins will be more resistant to denaturation by urea than others, so you have to look at a global picture.

This figure from the MCP paper linked above explains the idea pretty well. Remove the gel at the bottom and replace that with nanoLC-quadrupole Orbitrap analysis instead.

Naively...I would think, sure this would work when comparing something "simple" like bacterial strains...but human cells are so complicated that the protective machinery would never allow a read out from something like this.

And I would be wrong. This group shows 3 separate experiments with different cell lines that demonstrate this as a measurable readout for different cancer cell lines. They make some biological interpretations of the data based on the protein denaturation patterns, but I used up all my free time this morning working out the method.

Seriously interesting technique!  It does remind me of some other global studies based on temperature such as this recent one in Science and an older one I can't think of this morning and I do wonder how these techniques would correspond in readout. 

No comments:

Post a Comment