Detailed elucidation of protein structure and function typically requires removal of the protein from its in vivo environment. However, applying traditional and detergent-based protein extraction and purification workflows to membrane proteins typically produces insoluble, inactive, and heterogeneous fractions despite various attempts to conserve the surrounding lipid bilayer environment.
As a result, membrane proteins are considered recalcitrant research targets and insufficiently investigated. Yet membrane proteins are among the most important members of the proteome. As much as half of all protein drug targets are membrane-bound, and an estimated 29% of all proteins possess regions that share homology with known transmembrane domains.
Nanodiscs have recently emerged as a useful tool for producing water-soluble active-membrane proteins. In the past few years, an accelerating number of membrane proteins have been successfully studied using the Nanodisc platform, including multiple GPCRs, coagulation factors, toxins, Cytochrome P450s, bacteriorhodopsin, TAR receptors, ABC transporters, glucose transporters, and the SecYEG assembly.
Analytical tools utilized to study proteins incorporated into Nanodiscs include nuclear magnetic resonance spectroscopy, electron microscopy, optical spectroscopy, refractive index-based plasmonic sensing, and several types of mass spectroscopy.
Analysis of full-length proteins in Nanodiscs by mass spectroscopy has been limited, however, by interference from the high proportion of lipids and essential nonmembrane proteins within the Nanodisc.
Elimination of this interference was achieved this year using an ultrathin-layer matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS) method optimized for proteins incorporated into Nanodiscs, as reported by University of Illinois professor Stephen Sligar, Ph.D., in Analytical and Bioanalytical Chemistry.
The method utilizes a sample plate surface prepared with a thin layer of matrix that provides seed crystals, enabling nucleation of a homologous polycrystalline sample-matrix layer. The simplicity of this process improvement has resulted in broad acceptance for detergent-based systems, particularly in conjunction with crystallography, and is well suited for optimization for experiments with Nanodiscs.