title: Multiplex localization of sequential peptide epitopes by use of a planar microbead chip.
abstract: Epitope mapping is crucial for the characterization of protein-specific antibodies. Commonly, small overlapping peptides are chemically synthesized and immobilized to determine the specific peptide sequence. In this study, we report the use of a fast and inexpensive planar microbead chip for epitope mapping. We developed a generic strategy for expressing recombinant peptide libraries instead of using expensive synthetic peptide libraries. A biotin moiety was introduced in vivo at a defined peptide position using biotin ligase. Peptides in crude Escherichia coli lysate were coupled onto streptavidin-coated microbeads by incubation, thereby avoiding tedious purification procedures. For read-out we used a multiplex planar microbead chip with size- and fluorescence-encoded microbead populations. For epitope mapping, up to 18 populations of peptide-loaded microbeads (at least 20 microbeads per peptide) displaying the primary sequence of a protein were analyzed simultaneously. If an epitope was recognized by an antibody, a secondary fluorescence-labeled antibody generated a signal that was quantified, and the mean value of all microbeads in the population was calculated. We mapped the epitopes for rabbit anti-PA28γ (proteasome activator 28γ) polyclonal serum, for a murine monoclonal antibody against PA28γ, and for a murine monoclonal antibody against the hamster polyoma virus major capsid protein VP1 as models. In each case, the identification of one distinct peptide sequence out of up to 18 sequences was possible. Using this approach, an epitope can be mapped multiparametrically within three weeks.
authors.affiliation: Brandenburg Technical University Cottbus - Senftenberg, Faculty of Natural Sciences, Großenhainer Straße 57, D-01968 Senftenberg, Germany. Electronic address: carsten.schmidt@b-tu.de.
authors.forename: Carsten
authors.initials: C
authors.lastname: Schmidt
authors.affiliation: Brandenburg Technical University Cottbus - Senftenberg, Faculty of Natural Sciences, Großenhainer Straße 57, D-01968 Senftenberg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Rödiger
authors.affiliation: Brandenburg Technical University Cottbus - Senftenberg, Faculty of Natural Sciences, Großenhainer Straße 57, D-01968 Senftenberg, Germany; Department of Rheumatology and Clinical Immunology and Autoinflammatory Reference Centre at Charité, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Gruner
authors.affiliation: Brandenburg Technical University Cottbus - Senftenberg, Faculty of Natural Sciences, Großenhainer Straße 57, D-01968 Senftenberg, Germany; Institute for Biochemistry, University Medicine Berlin, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Moncsek
authors.affiliation: Brandenburg Technical University Cottbus - Senftenberg, Faculty of Natural Sciences, Großenhainer Straße 57, D-01968 Senftenberg, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Stohwasser
authors.affiliation: University of Potsdam, Chair Immunotechnology, Karl-Liebknecht-Str. 24-25, D-14476 Potsdam - Golm, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Hanack
authors.affiliation: Brandenburg Technical University Cottbus - Senftenberg, Faculty of Natural Sciences, Großenhainer Straße 57, D-01968 Senftenberg, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Schierack
authors.affiliation: Brandenburg Technical University Cottbus - Senftenberg, Faculty of Natural Sciences, Großenhainer Straße 57, D-01968 Senftenberg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Schröder
mesh_terms: D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry*; D056656:Lab-On-A-Chip Devices; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D010455:Peptides / Q000737:chemistry*
chemical_list: D000939:Epitopes; D009841:Oligonucleotides; D010455:Peptides
keywords: Epitope mapping; In vivo biotinylation; Microbeads; Multiplexed assays; VideoScan technology