Detection systems cross reacting with peptides – Analysis at the amino acid level

Abstract

There are several methods commonly used to measure protein-protein interactions and binding affinities. Quite contrary to most of these methods, protein- and peptide arrays on cellulose membranes or glass slides are suitable for high-throughput measurement, as they provide a higher density of probes and a multitude of peptide-protein interactions can be measured in parallel [1]. The most important application of the SPOT synthesis technique is to simultaneously detect a high number of peptides that have a strong binding affinity to defined targets. The validity of the results, however, depends on the ability of the detection system to indicate binding events whilst not interfering with the experiment itself through cross reaction. We tested three common peptide detection systems (TAMRA, FITC, Biotin/ Streptavidine) for their ability to interact with cellulose bound peptides at the amino acid level. Peptides with different amino acid cores were synthesized and tested for interaction with common dyes and detection systems. Our goal was to discover the potential of 5-(and 6)-carboxytetramethylrhodamine [5(6)-TAMRA], fluoresceinisothiocyanate [FITC], biotin and streptavidine to crossreact with individual amino acids. To this end we designed 20 peptides of the sequence XXX[aa]5XXX, where [aa]5 denotes five repeats of one of the 20 amino acids, and prepared them via SPOT synthesis [2]. Glycine was chosen as the flanking residue X to act as a spacer molecule. As analytes small peptides (gly-gly-gly) were solid phase synthesized and afterwards labelled with the detection compound of interest. The resulting amino acid library was then incubated with the glycine labelled detection system and evaluated via optical and fluorescent methods.Our approach identified several amino acids interacting with different detection systems. These results will strengthen the reliability of the analysis of SPOT synthesis generated data in the future. 1.Andresen, H. et al. Functional peptide microarrays for specific and sensitive antibody diagnostics. Proteomics, 6(5):1376– 1384, Mar 2006.2.Frank, R. The spot-synthesis technique. Synthetic peptide arrays on membrane supports – principles and applications. J Immunol Methods, 267(1):13–26, 2002.