Chau-Chung Han

While mass spectrometry (MS) plays a key role in proteomics research, characterization of membrane proteins (MP) by MS has been a challenging task because of the presence of a host of interfering chemicals in the hydrophobic protein extraction process, and the low protease digestion efficiency. We report a sample preparation protocol, two-phase separation with Triton X-100, induced by NaCl, with coomassie blue added for visualizing the detergent-rich phase, which streamlines MP preparation for SDS-PAGE analysis of intact MP and shot-gun proteomic analyses. MP solubilized in the detergent-rich milieu were then sequentially extracted and fractionated by surface-oxidized nanodiamond (ND) at three pHs. The high MP affinity of ND enabled extensive washes for removal of salts, detergents, lipids, and other impurities to ensure uncompromised ensuing purposes, notably enhanced proteolytic digestion and down-stream mass spectrometric (MS) analyses. Starting with a typical membranous cellular lysate fraction harvested with centrifugation/ultracentrifugation, MP purities of 70%, based on number (not weight) of proteins identified by MS, was achieved; the weight-based purity can be expected to be much higher.

Building upon recent success, diamond nanoparticles or nanodiamonds (NDs) for short have emerged as a promising new nanomaterial for biological applications. The material holds several unique properties, such as low chemical reactivity, small particle size, large specific surface area, wide optical transparency range, and facile surface functionalizability, making it a useful substrate for affinity purification mass spectrometry. Experiments have shown that the surface of NDs can be readily modified with diverse functional groups for either covalent or noncovalent conjugation with biomolecules. Additionally, NDs conjugated with bioactive ligands or polymers can be used as high affinity probes for selective extraction and enrichment of molecules of interest for characterization by mass spectrometry. This article summarizes the results of our work in employing surface‐functionalized NDs as a unique solid‐phase extraction and elution platform for pre‐concentration, selective extraction, and purification of targeted biomolecules prior to gel electrophoresis or matrix‐assisted laser desorption/ionization mass spectrometric analysis. Recent progress and future perspectives are discussed.

Several members of Enterobacteriaceas were analyzed by matrix‐assisted laser desorption ionization (MALDI) time‐of‐flight mass spectrometry (TOFMS). Characteristic mass spectral peaks and patterns were observed in the mass range of 2 to 20 kDa. The mass peaks reported to be reproducibly observed by previous researchers, which were claimed to serve as species/strain‐specific biomarkers, are consistently observed in our current study. Despite the high degree of similarity found in the MALDI mass spectra within the enteric bacteria, minor yet notable differences existed to allow their differentiation. Five spectral peaks at m/z 4364, 5380, 6384, 6856, and 9540, generated reproducibly for each genus studied here, are assigned as family‐specific biomarkers for the Family Enterobacteriaceae. The mass peaks at m/z 7324, 7724, 9136, and 9253 are assigned as genus‐specific biomarkers for Salmonella. Some unique biomarkers characterizing the species and strains of E. coli are also presented. Copyright © 1999 John Wiley & Sons, Ltd.