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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010A Study to Assess the Cross-Reactivity of CelluloseMembrane Bound Peptides with Detection Systems:An Analysis at the Amino Acid LevelCarsten C. Mahrenholz, Victor Tapia, and Rudolf VolkmerInstitute of Medical Immunology, Charité Medical School, Berlin, GermanyContact: rve@charite.deIntroductionThe growing demand for binding assays to study protein-protein interaction can beaddressed by peptide array-based methods [1]. The SPOT technique is a widespreadpeptide-array technology, which is able to distinguish semi-quantitatively the bindingaffinities of peptides to defined protein targets within one array. The quality of an assaysystem used for probing peptide arrays depends on the well-balanced combination ofscreening and read-out methods. The former address the steady-state of analyte capture,whereas the latter provide the means of detecting captured analyte. In all cases, however,false positive results can occur when challenging a peptide array with analyte or detectingcaptured analyte with label conjugates. Little is known about the cross-reactivity ofpeptides with the detection agents. Here we describe at the amino acid level the potential of5-(and 6)-carboxytetramethylrhodamine [5(6)-TAMRA] to cross-react with individualamino acids in a peptide sequence. Peptides with different amino acid cores weresynthesized and tested for interaction with common dyes and detection systems. Furtherstudies including random peptide libraries and other influential factors, as well asadditional detection systems (fluoresceinisothiocyanate [FITC], and biotin/streptavidin) canbe found in our corresponding manuscript [2].Experimental SetupTo investigate the potential interaction of this detection system with individual aminoacids, 20 peptides of the sequence GGG[B] 5 GGG were designed. Herein, [B] 5 denotes fiverepeats of one of the 20 amino acids (for a schematic overview see Figure 1). Glycine wasused to create nonreactive regions flanking the functional core at the N- and C-termini. Thisapproach generates peptides of reasonable length for the homogeneous display of thedefined cores. The peptides were prepared via SPOT synthesis [3], with eachGGG[B] 5 GGG sequence repeated three times in columns on the peptide array.As soluble interaction partner, a peptide of the sequence Gly-Gly-Gly wassynthesized, N-terminally modified with TAMRA (label-GGG), and finally purified byHPLC. This tripeptide was usedto better meet the assay conditions,because labels are usuallychemically coupled to an analyteor a detection antibody.Peptide arrays containing thecore-motifs were incubated in situwith a label-GGG and evaluatedusing optical and fluorescentmethods. Strict conditionsincluding short incubation periodsand long-time washing procedureswere applied to ensurestringency of binding. Bindingexperiments resulted in measurablespot signal intensitiessignifying directly or indirectlycaptured label conjugate.Fig. 1. Peptide and analyte composition.166