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

Abstract

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.

Inactivation of the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath) by acetylene

“…The growth of Methylococcus capsulatus (Bath) (M. capsulatus) strain ATCC 33009 and purification of the pMMO from the bacteria were adapted from our previous publication [7,19]. Protein samples were…”

Sample preparation strategies for improving the identification of membrane proteins by mass spectrometry

“…Among all available nanoparticles, surface-oxidized nanodiamond particles (NDs) [67][68][69] have demonstrated high efficiency for membrane protein purification, thereby avoiding time-consuming steps during sample preparation. The entire process is known as SPEED (solid-phase extraction and elution on diamond).…”

“…Moreover, NDs tend to attract proteases from the solution so the protein digestion can be carried out directly on the surface of the nanoparticles in a more efficient manner. This technology has provided stable protein-ND complexes, thus facilitating SDS-PAGE separation of membrane proteins, improving MALDI and LC-based MS analysis, and allowing large-scale shotgun proteomic studies [69].…”

Investigation of bovine serum albumin-surfactant aggregation and its physicochemical characteristics

“…BSA is mostly preferred because of its stability, low cost, and inactivity in many biochemical reactions. Although BSA is highly stable, the balance between folded and unfolded structures can be changed delicately by various factors such as temperature, pH, inorganic salts, organic solvents, detergents, and pressure [24]. Therefore, we assume that a systematic investigation of surfactant-BSA mixtures would be highly prospective and imperative.…”