Polymerization of synthetic phospholipid monomers has been widely used to enhance the stability of lipid membranes in applications such as membrane-based biosensing, where the inherent instability of fluid-phase lipid bilayers can be problematic. However, lipid polymerization typically decreases membrane fluidity, which may be required to maintain the activity of reconstituted integral proteins and peptides. Prior work has shown that a bilayer composed of binary mixtures of poly(lipid) and fluid lipid exhibits enhanced stability and supports the function of incorporated biomolecules. This work examines the structural basis of these findings using planar supported lipid bilayers (PSLBs) composed of binary mixtures of a polymerizable lipid, 1,2-bis[10-(2′,4′-hexadienoloxy)decanoyl]-sn-glycero-3-phosphocholine (bis-SorbPC), and a nonpolymerizable lipid, 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). Fluorescence recovery after photobleaching (FRAP) measurements showed that long-range lateral diffusion was minimally affected when the poly(lipid) mole ratio was ≤0.7. Atomic force microscopy, used to examine phase segregation in these PSLBs, showed that DPhPC forms a continuous lipid matrix that is 0.2−0.4 nm thicker than the island-like poly(bis-SorbPC) domains, with lateral dimensions of ≤200 nm. The nanoscale phase segregation allows for long-range lateral diffusion of lipid probes in the DPhPC matrix. The combination of fluidity and stability in these materials should make them useful in membrane-based biosensing applications.

Anticoagulant rodenticides (ARs) are used to control rodent populations; however, exposure to nontarget animals occurs. A sensitive and rugged quantitative method was developed, optimized, and validated for eight ARs in liver. Target analytes comprised two chemical classes: hydroxycoumarins (warfarin, coumachlor, dicoumarol, bromadiolone, brodifacoum, and difethialone) and indanediones (diphacinone and chlorophacinone). In this method, liver extracts were cleaned using dispersive solid phase extraction (d-SPE) to remove matrix interferences and analyzed by reverse phase ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Electrospray ionization in negative ion mode combined with multiple reaction monitoring (MRM) using a triple quadrupole mass spectrometer provided simultaneous confirmation and quantitation. Detection limits spanned 0.75-25 ng/g, and lower quantitation limits were established as 50 ng/g. Interassay method accuracy ranged from 92 to 110% across the analytical range (50-2500 ng/g) using matrix-matched calibrants with good repeatability (relative standard deviations 2-16%). Successful method transfer to another laboratory utilizing an Orbitrap mass analyzer, providing high mass accuracy, was assessed by good method reproducibility during blinded study analyses (6-29%; Horwitz ratios (HORRAT) ≤ 1.5).

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF 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-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 based on 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 B as a model receptor-ligand pair, the minimal detectable concentration of toxin was estimated to be 4 nM. On-plate trypsin digestion of bound cholera toxin 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.