Affinity capture using chimeric membrane proteins bound to magnetic beads for rapid ligand screening by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
The rapid and specific detection of therapeutically important ligands in complex mixtures, that may bind to membrane proteins, remains challenging for many research laboratories and pharmaceutical industries. Through its use in the development of screening assays, mass spectrometry (MS) is currently experiencing a period of tremendous expansion. In the study presented here, we took advantage of the remarkable stability properties of a bacterial membrane protein, the KcsA K+ channel, produced in E. coli and purified as a tetrameric protein in the presence of a detergent. This membrane protein can subserve as a molecular template to display the pore-forming region of human K+ channels, which are considered as targets in the search for inhibitory ligands. The engineered chimeric proteins were linked to metal-bound magnetic beads, for the screening of complex peptide mixtures, such as that of scorpion venoms. The affinity-captured scorpion toxins were eluted prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), and to nano-electrospray ionization tandem mass QqTOF mass spectrometry (MS/MS) analysis. The de novo sequence of the toxins was deduced by combining the MS/MS fragmentation of the reduced form (up to the 33 first residues) and the trypsin digest peptides of the native toxins. This affinity-capture screening assay led to the isolation and characterization of potent and specific ligands of the human K+ channel, Kv1.3. The affinity-capture procedure is fast and reproducible. When linked to magnetic beads, the chimeric membrane protein can be re-used several times without losing any of its selectivity or specificity. This assay also benefits from the fact that it requires minimal amounts of animal venoms or complex mixtures, which can be expensive or difficult to procure.
Legros
Christian
C
Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement (LAMBE), Université d'Evry Val d'Essonne, CNRS UMR 8587, Bd F. Mitterrand, 91025 Evry, France. christian.legros@univ-angers.fr
Guette
Catherine
C
Martin-Eauclaire
Marie-France
MF
Goyffon
Max
M
Tortajada
Jeanine
J
D007118:Immunoassay / Q000379:methods*; D018189:Immunomagnetic Separation / Q000379:methods*; D007553:Isotope Labeling / Q000379:methods*; D008024:Ligands; D011506:Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D008024:Ligands; D011506:Proteins