Improved Mass Spectrometric Analysis of Membrane Proteins Based on Rapid and Versatile Sample Preparation on Nanodiamond Particles

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.