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

Abstract

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.

Rapid high mass resolution mass spectrometry using matrix-assisted ionization

“…All of the species exist predominantly as multiply protonated ions, although some additional lower abundant peaks are observed due to the presence of sodium-adducted ions as well. Analysis of the B-subunit of pertussis toxin acquired using a MALDI-TOF mass spectrometer [23] resulted in singly charged ions of the S2 and S3 subunits (MW ca. 21.9 kDa), differing in mass by ca.…”

Photopolymerization of Dienoyl Lipids Creates Planar Supported Poly(lipid) Membranes with Retained Fluidity

“…(Note: X n is problematic to determine for a PSLB with an area of a few cm 2 because it contains a small number of molecules and their ionization efficiency is very low. 39 )…”

“…bis-SorbPC and mono-SorbPC were prepared via a modified version of that described by Lamparski et al 45,46 Preparatory scale purification of bis-SorbPC was performed by reverse phase HPLC. 39 Synthesis of bis-DenPC and mono-DenPC was performed using the methods reported by Jones et al 47 and Liu et al 48 respectively. Lipids were kept at −80 °C for long-term storage and −20 °C for short-term storage.…”

“…3,19,26–39 Numerous parameters, such as the chemical structure of the lipid, the polymerization method, and the type of supramolecular assembly, influence the degree to which polymerization alters bilayer properties. Remarkably enhanced membrane stability, altered permeability to ions and small molecules, changes in thermotropic behavior, and other important properties have been observed.…”

“…30–32,36–39 Here lateral diffusion in unpolymerized and UV polymerized PSLBs formed from dienoyl lipids was characterized using FRAP. The variables examined included the location and number of polymerizable groups in the lipid tails and the effect of polymerization above and below the main phase transition temperature ( T m ).…”