title: 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 (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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, 1306 East University Boulevard, Tucson, AZ, 85721, USA.
authors.forename: Boying
authors.initials: B
authors.lastname: Liang
authors.forename: Yue
authors.initials: Y
authors.lastname: Ju
authors.forename: James R
authors.initials: JR
authors.lastname: Joubert
authors.forename: Erin J
authors.initials: EJ
authors.lastname: Kaleta
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Lopez
authors.forename: Ian W
authors.initials: IW
authors.lastname: Jones
authors.forename: Henry K
authors.initials: HK
authors.lastname: Hall
authors.forename: Saliya N
authors.initials: SN
authors.lastname: Ratnayaka
authors.forename: Vicki H
authors.initials: VH
authors.lastname: Wysocki
authors.forename: S Scott
authors.initials: SS
authors.lastname: Saavedra
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000032:analysis; D002772:Cholera Toxin / Q000032:analysis / Q000378:metabolism; D004768:Enterotoxins / Q000032:analysis; D029968:Escherichia coli Proteins / Q000032:analysis; D005677:G(M1) Ganglioside / Q000031:analogs & derivatives / Q000737:chemistry; D008024:Ligands; D057230:Limit of Detection; D008051:Lipid Bilayers / Q000737:chemistry*; D008969:Molecular Sequence Data; D037342:Pertussis Toxin / Q000032:analysis; D010713:Phosphatidylcholines / Q000737:chemistry; D058105:Polymerization; D011108:Polymers / Q000737:chemistry; D011506:Proteins / Q000032:analysis*; D011956:Receptors, Cell Surface / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D008024:Ligands; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D011108:Polymers; D011506:Proteins; D011956:Receptors, Cell Surface; C034993:ganglioside receptor; C000607174:poly(bis-sorbyl phosphatidylcholine); C064020:ganglioside GD1alpha; D005677:G(M1) Ganglioside; D002772:Cholera Toxin; C027538:heat-labile enterotoxin, E coli; D037342:Pertussis Toxin