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.
An essential constituent of a Nanodisc are a pair of membrane scaffold proteins (MSPs), which Dr. Sligar created by reengineering human apolipoprotein A-1. The University of Illinois and Sigma Life Science partnered to make MSPs broadly accessible to researchers. Under defined conditions, MSPs can sequester phospholipids along with targeted membrane proteins to yield soluble nanoscale discoidal bilayers. A typical Nanodisc contains two stacked MSPs that encircle approximately 130 phospholipids and one membrane protein (Figure 1).
Methods: Constructing Nanodiscs
Purified polyhistidine-labeled Cytochrome P450 3A4 (CYP 3A4), cytochrome P450 reductase (CPR), and rhodopsin were individually incorporated into Nanodiscs along with palmitoyl-oleoyl-phosphatidylcholine (POPC) and MSP1D1 constructs.
For rhodopsin, an MSP1D1 construct containing a polyhistidine tag was used. CYP 3A4, CPR, and rhodopsin were initially solubilized in 0.1% Emulgen, 0.1% Triton X-100, and 90 mM octyl glucoside, respectively.
Membrane proteins were concentrated to 100 μM and MSPs to 200 μM. Solid POPC was dissolved in chloroform, dried under nitrogen, and placed overnight in a vacuum desiccator. POPC was then reconstituted to 50 mM in buffer containing 100 mM cholate and sonicated to ensure complete dissolution.
Solubilized POPC, membrane protein, and MSP were combined at a molar ratio of 1:65:130:0.1 (MSP/POPC/cholate/membrane protein). Excess MSP helps to insure that only one membrane protein is incorporated into each Nanodisc. This assembly mixture was incubated on ice for one hour.
Nanodiscs were then allowed to undergo spontaneous assembly upon removal of cholate by the addition of 0.5–0.8 g of Amberlite XAD-2 per mL of assembly solution, and placing them on an orbital shaker for four hours. After removal of Amberlite XAD-2 by centrifugation, intact Nanodiscs were purified by size-exclusion chromatography on a Superdex HR200 10/30 column. Nanodiscs with CYP3A4 were further purified on a nickel nitriloacetic chelation column. Nanodiscs were finally concentrated to a concentration of ~10 μM of membrane protein.
Methods: Ultrathin-Layer MALDI-MS
For the ultrathin MALDI-MS, a stainless steel 100-spot Voyager sample plate was washed with multiple alternating methanol and water rinses. Either α-cyano-4-hydroxycinnamic acid (4HCCA) or sinapinic acid (SA) matrices can be used. SA was used in the majority of studies since it yielded superior membrane protein ion peaks.
For studies where high mass accuracy is desired, 4HCCA exhibits more polycationic species than SA. In contrast, SA proved more useful for applications where simpler spectra or singly charged species is desirable.
For the ultrathin-layer solution (TWA), 150 μL of (1:500:500 v/v/v TFA/water/acetonitrile) was added to 10 mg solid matrix and thoroughly mixed by repeated aspiration into a pipet tip and vortexing. Undissolved matrix was removed by centrifugation at 14,000 rpm for five minutes. The supernatant was removed and diluted with 450 μL of isopropanol.
Typically, 25 μL of this final TWA solution is allowed to dry. A lint-free wipe was used to gently wipe off the plate, leaving only a barely visible matrix layer.
The matrix solution containing the Nanodiscs was similarly prepared by allowing a 2:1:3 v/v/v formic acid/water/isopropanol solution (FWI) to sit overnight. Then 150 μL of the FWI solution was added to 10 mg of solid matrix, thoroughly mixed, centrifuged, and the supernatant removed in the same manner as described for the TWA solution.
The Nanodisc samples were diluted 1:20 with this FWI-matrix solution and 0.3 μL was spotted on the ultrathin-layer plate. After 10–15 seconds the remaining solution was removed from the plate by aspiration, leaving a homogenous polycrystalline layer across the spot. Cold 0.1% TFA in water was used to wash each spot for several seconds.
MALDI-TOF analysis was performed on an Applied Biosystems Voyager-DE STR MALDI-TOF (Life Technologies) at 25 kV accelerating voltage in linear positive-ion mode with delayed extraction and a 337 nm nitrogen laser with pulses 3 nanoseconds in duration. Laser power was optimized for each matrix. Spectra were averaged over 1,000 shots, analyzed, smoothed, and baseline-corrected in Voyager Data Explorer 18.104.22.168 software.
To demonstrate the versatility and reproducibility of the MSP signal elimination, analysis was performed on three different membrane proteins: CPR, a redox protein that contains a single helical transmembrane anchor; CYP450; and rhodopsin, a more completely embedded G-protein coupled receptor also shown to exist as a dimer within the Nanodiscs. In each case, a greatly enhanced signal from membrane protein and nearly complete elimination of MSP signal was observed.
A significant factor of MS signal interference from MSPs was the duration of crystallization time allowed prior to aspiration of the spot containing the Nanodisc matrix. If the solution was aspirated immediately, a coffee-ring drying pattern formed. A strong MSP signal was observed in the thinner center deposits, while minimal MSP signal was observed in the surrounding thick-layered ring (Figure 2).
When the spots were allowed to dry for 10 seconds or more before aspiration, the crystal layer formed a highly homogenous structure and showed nearly no interfering MSP signal. The extended drying time enabled the MSP to partition into the solvent phase and the membrane protein to partition onto the polycrystalline film. As the solvent evaporates, the membrane protein is homogeneously co-incorporated into the growing crystal layer. The TFA rinse then washes away any residual MSP.
Ultrathin MALDI-MS analysis of Nanodisc-incorporated membrane proteins has immediate utility for the identification and characterization of protein-protein interactions.
Further elucidation of cellular processes and signaling events will rely heavily on analysis of complex protein interaction pathways separated by plasma and organelle lipid bilayer partitions. Since membrane proteins play a critical role in the control of these partitions and are often the most important functional component of the pathway, effective analytical tools such as MS are essential. The incorporation of previously recalcitrant membrane proteins into Nanodiscs and the analysis of full-length membrane proteins by ultrathin-layer MALDI-MS provides a method to overcome this barrier.