Thursday, August 31, 2017

Great study on premature aging and SILAC technique for protein turnover!

Hutchinson-Gilford progeria syndrome (HGPS, often just called progeria) is a premature aging disease that is driven by a mutation in a scaffolding protein. DJ Solarize was one of the longest surviving people with the disease in history and he only made it to 36 years old (want to see some talent? This is a video he posted of him messing around with a single channel mixer and two obviously damaged random LPs from the 80s).

In this great new study --

--this team takes a look at the downstream protein effects of the lamin A/C scaffold proteins using SILAC based approach. I'm gonna start typing this and hope that this helps me pull my thoughts together on how this all works.

They used silencing RNA technology to interfere with the proteins
They induce the fibroblast cells into quiescent state (by what looks like starving them with 1% serum?)
Then they pulse label the cells with SILAC media by putting it in every other day. Control cells appear to be just grown normally. 
Then the cells were made quiescent again. 

The cells were ruptured and nuclear proteins enriched and normalish SILAC experimental technique was used (mixing heavy and light and using an Orbitrap Tribrid (high-low mode) or a Q Exactive for the mass spec work. 

All the analysis appears to have been done with this Integrated Proteomics Applications software.

Interestingly -- they search the data with a large mass tolerance (50ppm!) and post search they rule out their options by using a much smaller/tighter window (10ppm, I think). All the data is deposited at PRIDE/ProteomeXchange, via too many identifiers for me to list here. (I'm very curious how PD handles this data -- in the queue for later, I guess...)

Besides this super cool technique -- they find systematic effects of the lamin mutations that lead to this premature aging effect including accumulation of proteins in the nucleolar area that mucks everything up. They use ICC or IHC (I get them mixed up still) imaging techniques that 1) look cool and 2) back up their findings. 

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