Friday, July 28, 2017
Tube gel digestion?
In-gel digestion appears to be making a comeback in the literature (it works so well, it's hard to imagine it ever going away, despite the technical challenges). There is even a spiffy new term for it that eludes me right now that I should add to the translator when I remember.
(BTW, I consider the biggest challenges of SDS-PAGE in gel digestion to be time, loading control, reproducibility, enhance sample handling leading to contamination, etc.,)
This recent paper suggest a tube-gel digestion protocol that looks like it minimizes a lot of these!
They spike UPS1 protein standards in controlled amounts into a yeast lysate background and compare in-solution, standard in-gel digestion, and their tube based protocol. Label free quan is performed on a quadrupole-Orbitrap and that data is processed in MaxQuant.
Not only do they minimize the sample handling, but they appear to retrieve more yeast protein identifications than the other two methods while consistently identifying more of the UPS1 proteins at the lower dilution spike-ins.
I really like this method because it doesn't take too much to imagine automating several of their steps.
All the RAW data is available at ProteomeXchange here (PXD003841)