Label-free detection and identification of protein ligands captured by receptors in a polymerized planar lipid bilayer using MALDI-TOF MS.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) coupled with affinity capture is a well-established method to extract biological analytes from complex samples followed by label-free detection and identification. Many bioanalytes of interest bind to membrane-associated receptors; however, the matrices and high-vacuum conditions inherent to MALDI-TOF MS make it largely incompatible with the use of artificial lipid membranes with incorporated receptors as platforms for detection of captured proteins and peptides. Here we show that cross-linking polymerization of a planar supported lipid bilayer (PSLB) provides the stability needed for MALDI-TOF MS analysis of proteins captured by receptors embedded in the membrane. PSLBs composed of poly(bis-sorbylphosphatidylcholine) (poly(bis-SorbPC)) and doped with the ganglioside receptors GM1 and GD1a were used for affinity capture of the B subunits of cholera toxin, heat-labile enterotoxin, and pertussis toxin. The three toxins were captured simultaneously, then detected and identified by MS on the basis of differences in their molecular weights. Poly(bis-SorbPC) PSLBs are inherently resistant to nonspecific protein adsorption, which allowed selective toxin detection to be achieved in complex matrices (bovine serum and shrimp extract). Using GM1-cholera toxin subunit B as a model receptor-ligand pair, we estimated the minimal detectable concentration of toxin to be 4 nM. On-plate tryptic digestion of bound cholera toxin subunit B followed by MS/MS analysis of digested peptides was performed successfully, demonstrating the feasibility of using the PSLB-based affinity capture platform for identification of unknown, membrane-associated proteins. Overall, this work demonstrates that combining a poly(lipid) affinity capture platform with MALDI-TOF MS detection is a viable approach for capture and proteomic characterization of membrane-associated proteins in a label-free manner.

Highly stabilized, polymer-lipid membranes prepared on silica microparticles as stationary phases for capillary chromatography.

The ability to rapidly screen complex libraries of pharmacological modulators is paramount to modern drug discovery efforts. This task is particularly challenging for agents that interact with lipid bilayers or membrane proteins due to the limited chemical, physical, and temporal stability of conventional lipid-based chromatographic stationary phases. Here, we describe the preparation and characterization of a novel stationary phase material composed of highly stable, polymeric-phospholipid bilayers self-assembled onto silica microparticles. Polymer-lipid membranes were prepared by photochemical or redox initiated polymerization of 1,2-bis[10-(2',4'-hexadieoyloxy)decanoyl]-sn-glycero-2-phosphocholine (bis-SorbPC), a synthetic, polymerizable lipid. The resulting polymerized bis-SorbPC (poly(bis-SorbPC)) stationary phases exhibited enhanced stability compared to particles coated with 1,2-dioleoyl-sn-glycero-phosphocholine (unpolymerized) phospholipid bilayers when exposed to chemical (50 mM triton X-100 or 50% acetonitrile) and physical (15 min sonication) insults after 30 days of storage. Further, poly(bis-SorbPC)-coated particles survived slurry packing into fused silica capillaries, compared to unpolymerized lipid membranes, where the lipid bilayer was destroyed during packing. Frontal chromatographic analyses of the lipophilic small molecules acetylsalicylic acid, benzoic acid, and salicylic acid showed >44% increase in retention times (P<0.0001) for all analytes on poly(bis-SorbPC)-functionalized stationary phase compared to bare silica microspheres, suggesting a lipophilic retention mechanism. Phospholipid membrane-functionalized stationary phases that withstand the chemical and physical rigors of capillary LC conditions can substantially increase the efficacy of lipid membrane affinity chromatography, and represents a key advance toward the development of robust membrane protein-functionalized chromatographic stationary phases.

Aptamer-functionalized porous phospholipid nanoshells for direct measurement of Hg(2+) in urine.

A porous phospholipid nanoshell (PPN) sensor functionalized with a specific aptamer sensor agent was prepared for rapid detection of Hg(2+) in human urine with minimal sample preparation. Aptamer sensors provide an important class of optical transducers that can be readily and reproducibly synthesized. A key limitation of aptamer sensors, and many other optical sensors, is the potential of biofouling or biodegradation when used in complex biological matrices such as serum or urine, particularly when high levels of nucleases are present. We prepared Hg(2+)-responsive, PPN-encapsulated aptamer sensors that overcome these limitations. PPNs provide a protective barrier to encapsulate the aptamer sensor in an aqueous environment free of diffusional restrictions encountered with many polymer nanomaterials. The unique porous properties of the PPN membrane enable ready and rapid transfer of small molecular weight ions and molecules into the sensor interior while minimizing the macromolecular interactions between the transducer and degradants or interferents in the exterior milieu. Using Hg(2+)-responsive, PPN-encapsulated aptamer sensors, we were able to detect sub-100 ppb (chronic threshold limit from urine test) Hg(2+) in human urine with no sample preparation, whereas free aptamer sensors yielded inaccurate results due to interferences from the matrix. The PPN architecture provides a new platform for construction of aptamer-functionalized sensors that target low molecular weight species in complex matrices, beyond the Hg(2+) demonstrated here.

Stabilized porous liposomes with encapsulated Gd-labeled dextran as a highly efficient MRI contrast agent.

A highly efficient contrast agent for magnetic resonance imaging was developed by encapsulating gadolinium within a stabilized porous liposome. The highly porous membrane leads to a high relaxivity of the encapsulated Gd. The stability of the liposome was improved by forming a polymer network within the bilayer membrane.

Practical considerations for preparing polymerized phospholipid bilayer capillary coatings for protein separations.

Phosphorylcholine (PC) based phospholipid bilayers have proven useful as capillary coating materials due to their inherent resistance to non-specific protein adsorption. The primary limitation of this important class of capillary coatings remains the limited long-term chemical and physical stability of the coatings. Recently, a method for increasing phospholipid coating stability in fused silica capillaries via utilization of polymerized, synthetic phospholipids was reported. Here, we expand upon these studies by investigating polymerized lipid bilayer capillary coatings with respect to separation performance including run-to-run, day-to-day and column-to-column reproducibility and long-term stability. In addition, the effects of pH and capillary inner diameter on polymerized phospholipid coated capillaries were investigated to identify optimized coating conditions. The coatings are stabilized for protein separations across a wide range of pH values (4.0-9.3), a unique property for capillary coating materials. Additionally, smaller inner diameter capillaries (≤50 µm) were found to yield marked enhancements in coating stability and reproducibility compared to wider bore capillaries, demonstrating the importance of capillary size for separations employing polymerized phospholipid coatings.

Studies of mixed liposomes with novel sorbyl functionalized head group lipids.

Three novel polymerizable amphiphiles with a sorbyl-substituted head group were synthesized and systematically characterized. These amphiphiles are neutral in charge. None of these molecules forms vesicles by itself, presumably due to lack of amphiphilicity and/or extensive head group interaction. Therefore, mixed vesicles were formed with other fluid lipids such as DPenPC, eggPC, or DOPC. We investigated the properties of these mixtures in both vesicles and Langmuir films. The Langmuir isotherms show formation of monolayers by all three molecules. However, the isotherms for mixed monolayers suggest that two components are largely immiscible to the mixing lipid. Under polymerization conditions, mixed vesicles of these amphiphiles form oligomers, suggesting that in spite of a larger head group, they form mesophases.

Determination of pore sizes and relative porosity in porous nanoshell architectures using dextran retention with single monomer resolution and proton permeation.

Unilamellar phospholipid vesicles prepared using the polymerizable lipid bis-sorbylphosphatidylcholine (bis-SorbPC) yield three-dimensional nanoarchitectures that are highly permeable to small molecules. The resulting porous phospholipid nanoshells (PPNs) are potentially useful for a range of biomedical applications including nanosensors and nanodelivery vehicles for cellular assays and manipulations. The uniformity and size distribution of the pores, key properties for sensor design and utilization, have not previously been reported. Fluorophore-assisted carbohydrate electrophoresis (FACE) was utilized to assess the nominal molecular weight cutoff limit (NMCL) of the PPN via analysis of retained dextran with single monomer resolution. The NMCL of PPNs prepared from pure bis-SorbPC was equivalent to a 1800 Da linear dextran, corresponding to a maximum pore diameter of 2.6 nm. Further investigation of PPNs prepared using binary mixtures of bis-SorbPC and dioleoylphosphatidylcholine (DOPC) revealed a similar NMCL when the bis-SorbPC content exceeded 30 mol %, whereas different size-dependent permeation was observed below this composition. Below 30 mol % bis-SorbPC, dextran retention provided insufficient mass resolution (162 Da) to observe porosity on the experimental time scale; however, proton permeability showed a marked enhancement for bis-SorbPC ≥ 10 mol %. Combined, these data suggest that the NMCL for native pores in bis-SorbPC PPNs results from an inherent property within the lipid assembly that can be partially disrupted by dilution of bis-SorbPC below a critical value for domain formation. Additionally, the analytical method described herein should prove useful for the challenging task of elucidating porosity in a range of three-dimensional nanomaterials.

Polymerized planar suspended lipid bilayers for single ion channel recordings: comparison of several dienoyl lipids.

The stabilization of suspended planar lipid membranes, or black lipid membranes (BLMs), through polymerization of mono- and bis-functionalized dienoyl lipids was investigated. Electrical properties, including capacitance, conductance, and dielectric breakdown voltage, were determined for BLMs composed of mono-DenPC, bis-DenPC, mono-SorbPC, and bis-SorbPC both prior to and following photopolymerization, with diphytanoyl phosphocholine (DPhPC) serving as a control. Poly(lipid) BLMs exhibited significantly longer lifetimes and increased the stability of air-water transfers. BLM stability followed the order bis-DenPC > mono-DenPC ≈ mono-SorbPC > bis-SorbPC. The conductance of bis-SorbPC BLMs was significantly higher than that of the other lipids, which is attributed to a high density of hydrophilic pores, resulting in relatively unstable membranes. The use of poly(lipid) BLMs as matrices for supporting the activity of an ion channel protein (IC) was explored using α-hemolysin (α-HL), a model IC. Characteristic i-V plots of α-HL were maintained following photopolymerization of bis-DenPC, mono-DenPC, and mono-SorbPC, demonstrating the utility of these materials for preparing more durable BLMs for single-channel recordings of reconstituted ICs.

Fractional polymerization of a suspended planar bilayer creates a fluid, highly stable membrane for ion channel recordings.

Suspended planar lipid membranes (or black lipid membranes (BLMs)) are widely used for studying reconstituted ion channels, although they lack the chemical and mechanical stability needed for incorporation into high-throughput biosensors and biochips. Lipid polymerization enhances BLM stability but is incompatible with ion channel function when membrane fluidity is required. Here, we demonstrate the preparation of a highly stable BLM that retains significant fluidity by using a mixture of polymerizable and nonpolymerizable phospholipids. Alamethicin, a voltage-gated peptide channel for which membrane fluidity is required for activity, was reconstituted into mixed BLMs prepared using bis-dienoyl phosphatidylcholine (bis-DenPC) and diphytanoyl phosphatidylcholine (DPhPC). Polymerization yielded BLMs that retain the fluidity required for alamethicin activity yet are stable for several days as compared to a few hours prior to polymerization. Thus, these polymerized, binary composition BLMs feature both fluidity and long-term stability.

Enhanced long-term stability for single ion channel recordings using suspended poly(lipid) bilayers.

Black lipid membranes (BLMs) are widely used for recording the activity of incorporated ion channel proteins. However, BLMs are inherently unstable structures that typically rupture within a few hours after formation. Here, stabilized BLMs were formed using the polymerizable lipid bis-dienoyl phosphatidylcholine (bis-DenPC) on glass pipettes of approximately 10 microm (I.D.). After polymerization, these BLMs maintained steady conductance values for several weeks, as compared to a few hours for unpolymerized membranes. The activity of an ion channel, alpha-hemolysin, incorporated into bis-DenPC BLMs prior to polymerization, was maintained for 1 week after BLM formation and polymerization. These lifetimes are a substantial improvement over those achievable with conventional BLM technologies. Polymerized BLMs containing functional ion channels may represent an enabling technology for development of robust biosensors and drug screening devices.

Ultra-high vacuum surface analysis study of rhodopsin incorporation into supported lipid bilayers.

Planar supported lipid bilayers that are stable under ambient atmospheric and ultra-high-vacuum conditions were prepared by cross-linking polymerization of bis-sorbylphosphatidylcholine (bis-SorbPC). X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were employed to investigate bilayers that were cross-linked using either redox-initiated radical polymerization or ultraviolet photopolymerization. The redox method yields a more structurally intact bilayer; however, the UV method is more compatible with incorporation of transmembrane proteins. UV polymerization was therefore used to prepare cross-linked bilayers with incorporated bovine rhodopsin, a light-activated, G-protein-coupled receptor (GPCR). A previous study (Subramaniam, V.; Alves, I. D.; Salgado, G. F. J.; Lau, P. W.; Wysocki, R. J.; Salamon, Z.; Tollin, G.; Hruby, V. J.; Brown, M. F.; Saavedra, S. S. J. Am. Chem. Soc. 2005, 127, 5320-5321) showed that rhodopsin retains photoactivity after incorporation into UV-polymerized bis-SorbPC, but did not address how the protein is associated with the bilayer. In this study, we show that rhodopsin is retained in supported bilayers of poly(bis-SorbPC) under ultra-high-vacuum conditions, on the basis of the increase in the XPS nitrogen concentration and the presence of characteristic amino acid peaks in the ToF-SIMS data. Angle-resolved XPS data show that the protein is inserted into the bilayer, rather than adsorbed on the bilayer surface. This is the first study to demonstrate the use of ultra-high-vacuum techniques for structural studies of supported proteolipid bilayers.