title: Detection of ligand-receptor binding using microfluidic frontal affinity chromatography on proteoliposomes derived directly from native cell membranes.
abstract: A method for characterization of ligand binding to membrane receptors in their native cell membrane is presented. The methodology is based on microfluidic frontal affinity chromatography coupled to mass spectrometry (FAC-MS). Proteoliposomes with receptor of interest are prepared directly from cell membranes and serve as a stationary phase in a microfluidic flow cell for frontal analysis. The G-Protein-Coupled Receptor (GPCR) Ste2 involved in the pheromone-induced yeast mating pathway is used as a model receptor for proof of principle characterization. The ligand affinity of the natural pheromone peptide, the α-factor, is compared to a set of pheromone analogs having different receptor affinities. With short preparation time, preserved lipid composition and the ability to immobilize proteoliposomes from any cell membrane, we propose that our methodology with immobilized proteoliposomes together with microfluidics FAC-MS can be an important improvement for ligand-receptor studies in native membranes.
authors.affiliation: Nanoxis Consulting AB, Lennart Torstenssongatan 5, Gothenburg SE-400 16, Sweden.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Olesen
authors.forename: Roger
authors.initials: R
authors.lastname: Karlsson
authors.forename: Ulrika
authors.initials: U
authors.lastname: Lind
authors.forename: Max
authors.initials: M
authors.lastname: Davidson
authors.forename: Anders
authors.initials: A
authors.lastname: Blomberg
authors.forename: Anders
authors.initials: A
authors.lastname: Karlsson
mesh_terms: D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D011485:Protein Binding; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism*; D051964:Receptors, Mating Factor / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D012815:Signal Processing, Computer-Assisted; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D011510:Proteolipids; D051964:Receptors, Mating Factor; C498031:STE2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C020485:proteoliposomes