Thursday, October 22, 2015
Quantitative proteomics and lysine acetylomics of astrocytes
Apparently, antioxidants are super important in brain astrocytes. Maybe cause they use a lot of energy and that results in the formation of dangerous oxygen free radicals?
To investigate this, Mariana Pehar et al., did just an awesome job of profiling astrocytes that had a major antioxidant pathway knocked out (or down) sorry, I skimmed the biology here, I was really interested in the method for my own selfish reasons.
The cells were SILAC labeled. So they end up with really nice up/down regulation of their whole proteins. For the whole proteomics they did in-gel digestion with 40ug of protein and cut out 12 sections and double digested (LysC and Trypsin).
For the quantitative lysine acetylomics, the proteins were mixed, SCX fractionated and the peptides were incubated with a bead with an antibody that recognizes acetylated lysines and pulled down. Everything was LC-MS/MS'ed on an Orbitrap Elite.
On the data processing side, the files were ran through once with a recalibration algorithm (similar to the Recalibration node in PD 1.4). Once recalibrated, the files were reprocessed. The data was processed with various tools including Andromeda in MaxQuant, and MS-Viewer in the Protein Prospector and Perseus. The combination of these analyses is a solid output that gives the changes at the whole protein level as well as the changes at the lysine acetylome level.
Oh, and they worked out a cool variation of anti-oxidant response that appears to be mediated by the super cool Nrf protein(s). Solid paper! Having trouble getting the PRIDE depository reference number listed in the paper to lead me to anything, unfortunately, cause I'd love to see this RAW data, but super cool paper.