Multifunctional nanoreactor for comprehensive characterization of membrane proteins based on surface functionalized...

An integrated protocol is proposed here for efficient analysis of membrane proteins based on surface functionalized mesoporous graphene foams (MGF). The inherent hydrophobic nature of MGF and surface modification with hydrophilic chitosan (CS) make it highly suitable for the enrichment of hydrophobic membrane proteins from organic solvent, while remaining well-dispersed in aqueous solution for subsequent proteolysis. Therefore, such a multifunctional reactor ensures a facile solvent adjustment route. Furthermore, as a chitosan modified nanoporous reactor, it also provides a biocompatible nanoenvironment that can maintain the stability and activity of enzymes to realize efficient in situ digestion of the enriched membrane proteins. The concept was first proved with a standard hydrophobic membrane protein, bacteriorhodopsin, where a high number of identified peptides and amino acid sequence coverage were achieved even at extremely low protein concentration. The mesoporous reaction system was further applied to the analysis of complex real-case proteome samples, where 931 membrane proteins were identified in triplicate analyses by 2D LC-MS/MS. In contrast, with in-solution proteolysis, only 73 membrane proteins were identified from the same sample by the same 2D LC-MS/MS. The identified membrane proteins by the MGF-CS protocol include many biomarkers of the cell line. These results suggest that the multifunctional MGF-CS protocol is of great value to facilitate the comprehensive characterization of membrane proteins in the proteome research.