Gel-absorption-based sample preparation method for shotgun analysis of membrane proteome

Abstract

Membrane proteins solubilized in a starting buffer containing high concentration of sodium dodecyl sulfate (SDS) are directly entrapped and immobilized into gel matrix when the membrane protein solution is absorbed by the vacuum-dried polyacrylamide gel. After the detergent and other salts are removed by washing, the proteins are subjected to in-gel digestion and the tryptic peptides are extracted and analyzed by CapLC-MS/MS. The newly developed method not only avoids protein loss and the adverse protein modifications during gel-embedment but also improves the subsequent in-gel digestion and the recovery of tryptic peptides, particularly the hydrophobic peptides. Thus, this method facilitates the identification of membrane proteins especially the integral membrane proteins. © 2012 Springer Science+Business Media, LLC.