Membrane protein isolation and identification by covalent binding for proteome research

A novel method to achieve highly efficient identification of membrane proteins (MPs) has been developed based on a covalent binding (CB) strategy. For this purpose, magnetic nanoparticles coated with a PEG layer were synthesized. The PEG chain end was functionalized to form the PEG-tresyl group, which is an octopus-like long arm to capture the free amino groups of MPs. The long arm could be used to bind proteins in a high concentration of the SDS medium. Then, the SDS and interfering substances were completely depleted by washing. The CB proteins could form a molecular monolayer on the surface of the nanoparticles in the denatured state, which was significantly favorable for the proteolysis of MPs. Therefore, isolation with CB and highly efficient digestion resulted in a larger scale of MPs. The method has been verified by a proteome identification of mouse liver samples. A total of 2946 MPs were identified in an MP fraction. A total of 1505 proteins were characterized as integral MPs, and 735 MPs were identified beyond the largest database summarized by PeptideAtlas. This approach has great potential for membrane proteome research.