Tuesday, November 15, 2016

Pulsed SILAC + Ribosome profiling = New Variation on proteogenomics!

(Image on Ribosome profiling (or Ribo-Seq) borrowed from this review article)

Ribo-Seq is a powerful technique the genomics world has now. It gets them even closer to the proteome by characterizing what messenger RNAs (mRNAs) are currently protected by ribosomes. They are protected by the ribosomes because they are, right that second, making some proteins!

I'm a little foggy still, but I think they pull out all the RNA like they would for RNA-Seq but then degrade everything that is free floating. Then they just have to destroy the ribosomes to release the protected mRNA.

In bacteria, the central dogma applies pretty clearly. The regulations systems are pretty simple...cause you've (normally) got one tiny chromosome. And its been shown that RNA-Seq and proteomics line up pretty great in bacteria, but we're a little more complicated.

Check these brand new results out!

This team uses pulsed SILAC (pSILAC!) and this technology together to assess stress response in human cells treated with bortezomid. Its a proteosome inhibitor that is used for some cancers. Actually, on its own this drug is super fascinating. Some cancer cells protect themselves from immune response by making tons of proteosomes and just eating up the immune response. This drug drops a boron right in the catalytic site of one of the major proteosome proteins and shuts 'em down.  Now...proteosomes are pretty important to our cells functioning so this creates a lot of stress!  Hence this paper!

Here is the setup from the main experiment in the paper. On the proteomics side, they do something interesting with the pulsed SILAC. They use HCD and high/high mode on an Orbitrap Velos to develop their SILAC SRM transitions for their heavy and light peptides. Then they do the rest of the study with targeted SRMs. I've never tried this approach, but it does seem kind of smart. They pick 4 transitions for each peptide to monitor -- and since the heavy label should be on the y ions, they should be reasonably easy to get to. With 4 separate SRMs, its hard to argue about interference effects, even on a device with quads that can only isolate 0.7/1.0Da at Q1/Q3, respectively.

What did they find out?  Human biology is seriously complicated!  Even this fancy-pants new Ribo-Seq thing can't accurately tell you how much protein is there or how much is being made. However, using the two together can give you an understanding of the stress response in the cell. They propose the use of this methodology for understanding other chemotherapeutics -- get a deep mRNA-Seq, go get a deep Ribo-Seq, and then get accurate information on protein levels from the proteomics to make the rest of it make sense.

If you're wondering why you wouldn't just cut out the two expensive genomics techniques from the experiment and just do good proteomics on it -- well...so am I, but you don't just want that sequencer sitting there do ya?......ummm...got one!  The pSILAC could tell you how much protein is there at each point in your time course, but the Ribo-Seq can give you an extremely rough estimate a measurement of what is being produced so you could better infer whether the change in total protein concentration is because of a change in production (translation) or degradation!

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