title: SPOT synthesis: reliability of array-based measurement of peptide binding affinity.
abstract: Peptide arrays prepared by the SPOT synthesis technology have emerged as a proteomic tool to study molecular recognition and identify biologically active peptides. However, it was previously not clear how accurately signal intensities obtained by probing peptide arrays for protein binding really reflect the dissociation constants of the protein-peptide complexes. Using the monoclonal antibody CB4-1 as a model system, we systematically compared dissociation constants of antibody-peptide complexes with signal intensities obtained using the SPOT technology. By analyzing a set of peptides possessing different affinities to the antibody, we determined the strengths of the SPOT screening method. The accuracy of the measured results was improved by taking regional trends in the membrane surface into account. A model based on the mass action law compares well with the experimental results. Interestingly, the applied concentrations of the binding partners do not directly correspond to the effective concentrations in the assay. We show that the SPOT technology is an accurate method for assigning the spots' measured signal intensities to three different binding affinity classes. The dissociation constants of the intermediate region were found to be between pK(dis)=5 and pK(dis)=7. Altering the experimental parameters causes a directed change of this region.
authors.affiliation: Institute of Biochemistry, Charité, Universitätsmedizin Berlin, Monbijoustr. 2, 10117 Berlin, Germany.
authors.forename: Armin A
authors.initials: AA
authors.lastname: Weiser
authors.forename: Michal
authors.initials: M
authors.lastname: Or-Guil
authors.forename: Victor
authors.initials: V
authors.lastname: Tapia
authors.forename: Astrid
authors.initials: A
authors.lastname: Leichsenring
authors.forename: Johannes
authors.initials: J
authors.lastname: Schuchhardt
authors.forename: Cornelius
authors.initials: C
authors.lastname: Frömmel
authors.forename: Rudolf
authors.initials: R
authors.lastname: Volkmer-Engert
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002482:Cellulose / Q000737:chemistry; D020650:Combinatorial Chemistry Techniques; D008567:Membranes, Artificial; D010452:Peptide Biosynthesis / Q000502:physiology*; D020349:Surface Plasmon Resonance
chemical_list: D000911:Antibodies, Monoclonal; D008567:Membranes, Artificial; D002482:Cellulose