title: Micro-structured peptide surfaces for the detection of high-affinity peptide-receptor interactions in living cells.
abstract: Peptide ligands have great potential as selective agents for diagnostic imaging and therapeutic targeting of human cancers. A number of high-throughput assays for screening potential candidate peptides have been developed. Although these screening assays are indispensable for the identification of peptide ligands at a large scale, it is crucial to validate peptide binding and selectivity for targeted receptors in a live-cell context. For testing high-affinity peptide-receptor interactions in the plasma membrane of living cells, we developed cell-resistant, micro-structured glass surfaces with high-density and high-contrast peptide features. Cell adhesion and recruitment of fluorescent receptors to micro-patterned peptides in the live-cell membrane were evaluated by reflection interference contrast (RIC) and total internal reflection (TIRF) microscopy, respectively. To demonstrate both the specificity and modularity of the assay, co-patterning of fluorescent receptors with three different immobilized micro-structured ligands was shown: first, interaction of green fluorescent protein (GFP)-tagged epidermal growth factor (EGF) receptor expressed in Jurkat cells with immobilized EGF was detected and quantified. Second, using Jurkat cells, we demonstrated specific interaction of yellow fluorescent protein (YFP)-tagged β3 integrin with c(RGDfK) peptide. Third, we identified indirect recruitment of GFP-tagged α5 integrin to an 11-mer peptide. In summary, our results show that the developed micro-structured surfaces are a useful tool for the validation and quantification of peptide-receptor interactions in their natural cellular environment.
authors.affiliation: Center for Advanced Bioanalysis GmbH, Gruberstrasse 40, 4020 Linz, Austria. Electronic address: anna-maria.lipp@cbl.at.
authors.forename: Anna-Maria
authors.initials: AM
authors.lastname: Lipp
authors.affiliation: Center for Advanced Bioanalysis GmbH, Gruberstrasse 40, 4020 Linz, Austria. Electronic address: bozhi.ji@cbl.at.
authors.forename: Bozhi
authors.initials: B
authors.lastname: Ji
authors.affiliation: Center for Advanced Bioanalysis GmbH, Gruberstrasse 40, 4020 Linz, Austria. Electronic address: roland.hager@cbl.at.
authors.forename: Roland
authors.initials: R
authors.lastname: Hager
authors.affiliation: Center for Advanced Bioanalysis GmbH, Gruberstrasse 40, 4020 Linz, Austria. Electronic address: sandra.haas@cbl.at.
authors.forename: Sandra
authors.initials: S
authors.lastname: Haas
authors.affiliation: Center for Advanced Bioanalysis GmbH, Gruberstrasse 40, 4020 Linz, Austria. Electronic address: simone.schweiggl@cbl.at.
authors.forename: Simone
authors.initials: S
authors.lastname: Schweiggl
authors.affiliation: Center for Advanced Bioanalysis GmbH, Gruberstrasse 40, 4020 Linz, Austria. Electronic address: alois.sonnleitner@cbl.at.
authors.forename: Alois
authors.initials: A
authors.lastname: Sonnleitner
authors.affiliation: Center for Advanced Bioanalysis GmbH, Gruberstrasse 40, 4020 Linz, Austria. Electronic address: thomas.haselgruebler@cbl.at.
authors.forename: Thomas
authors.initials: T
authors.lastname: Haselgrübler
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D015374:Biosensing Techniques; D002448:Cell Adhesion / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008024:Ligands; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000737:chemistry*; D018000:Receptors, Peptide / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D008024:Ligands; D010455:Peptides; D018000:Receptors, Peptide
keywords: Cell adhesion; Live-cell assay; Micro-patterning; Micro-structured streptavidin; Peptide–receptor interaction; Photolithography