Improved mass spectrometric analysis of membrane proteins based on rapid and versatile sample preparation on...

We have developed a novel streamlined sample preparation procedure for mass spectrometric (MS) analysis of membrane proteins using surface-oxidized nanodiamond particles. The platform consists of solid-phase extraction and elution of the membrane proteins on nanodiamonds, concentrating the membrane proteins on the nanodiamonds and separating out detergents, chaotropic agents, and salts, and other impurities that are often present at high concentrations in solubilized membrane preparations. In this manner, membrane-protein extracts are transformed into MS-ready samples in minutes. The protocol is not only fast, but also widely adaptable and highly effective for preparing generic membrane protein samples for both MALDI-MS studies of membrane-protein complexes and shotgun membrane proteomics studies. As proof of concept, we have demonstrated substantial improvements in the MALDI-MS analysis of the particulate methane monooxygenase (pMMO) complex, a three-subunit transmembrane protein solubilized in various detergent buffers. Enzymatic digestions of membrane proteins are also greatly facilitated since the proteins extracted on to the nanodiamonds are exposed on the surface of the nanoparticles rather than in SDS gels or in detergent solutions. We illustrate the effectiveness of nanodiamonds for SDS removal in the preparation of membrane proteins for MS analysis on the proteome level by examining the quality of the tryptic peptides prepared by on-surface nanodiamond digestion of an E. coli membrane fraction for shotgun proteomics.