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Fig. 2. TAMRA cross-reaction. (A)Fluorescence emission of eachcorresponding spot measured at645 nm is calculated from acircular region around the spotA center detected in the image. Allsignals below an SI of 500 are atthe background level and shouldtherefore not be consideredinteractions between the amino acidcore and the detection system.(B) Fluorescent and (C) densitometricread-out. Each spot representsa cellulose membrane-boundB peptide of the sequenceGGG[B] 5 GGG, where [B] 5 denotesfive repeats of one of the 20 aminoC acids. Contrast was adjusted toensure better visibility. The negativecontrol without analyte shows nosignal. Error bars represent thestandard deviation of three spots.Results and DiscussionThe results draw a clear picture of the cross-reactivity of amino acid cores with the peptideTAMRA-GGG. As shown in Figure 2, significant spot signal intensities at 645 nm wereobserved for Phe, Tyr, and Trp cores. The strength of cross-reactivity between these aminoacids and TAMRA follows the order Phe < Tyr < Trp. Additionally, densitometry was usedto read the capturing of TAMRA-GGG via staining. As shown in Figure 2B and 2C, theresults are in accordance with the fluorescence read-out approach. The aromatic TAMRAmoiety interacts exclusively with aromatic amino acid cores (Figure 2A). Therefore,aromatic stacking is most likely the common driving force for the interaction betweenamino acid and TAMRA. Stacking is a widespread mechanism for stabilizing organicmoieties. It is accomplished by the favorable interaction of -electrons of aromatic systems[4]. In this case, the -electron systems of TAMRA and the side group of Trp may interactin an energetically favorable manner via stacking interactions, which the smaller aromaticsystems of Tyr and Phe possibly cannot provide to the same extent.Further studies including random peptide libraries and other influential factors, as wellas additional detection systems (fluoresceinisothiocyanate [FITC], and biotin/streptavidin)can be found in our corresponding manuscript [2].One has to bear in mind that a method is always limited by the effectiveness andvalidity of the read-out system. To prevent or identify false positives, factoring in theseresults is highly recommended when analyzing measurements. Taking these new resultsinto consideration will, in future, strengthen the reliability of the analysis ofSPOT-synthesis-generated data.AcknowledgmentsThis work was generously funded by the Manchot Foundation (Henkel KGaA) and supported byscholar- and fellowships from the Federation of the Societies of Biochemistry and Molecular Biology,the Charité Medical School, and GlaxoSmithKline.References1. Andresen, H., et al. Proteomics 6(5), 1376-1384 (2006).2. Mahrenholz, C.C., et al. J. Pep. Sci. 16, 297-302 (2010).3. Frank, R. J. Immunol. Methods 267(1), 13-26 (2002).4. Sygula, A., et al. J. Am. Chem. Soc. 129(13), 3842-3843 (2007).167