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Once printed, the amino acid particles are melted at 90°C to liquefy the solid toner matrixand to induce combinatorial peptide synthesis. Due to these unusual reaction conditions interms of solvent and temperature, we also investigated couplings yields and unwanted sidereactionslike racemization and aspartimide formation that might have been induced byelevated temperatures [2]. Stepwise coupling yields were determined by Fmoc deprotectionand UV/Vis spectrometry, and compared to peptide synthesis in DMF. On two-dimensionalglass slides coated with a thin polymer film [4], and without pre-swelling, repetitivecoupling yields were 90% on average for our toner-based approach which was equivalent tocoupling in DMF on the same slides. To get close to maximum yields even with 15-20merpeptides, we thus typically carry out double couplings and acetylate residual amino groupsafter each coupling step to truncate unreacted sequences. Furthermore, the peptide integritycould be verified by mass spectrometry and HPLC. These methods did not reveal any majorsignals besides the full-length peptides, which indicates that heat-induced side reaction didnot occur to a measurable extent.The peptide laser printer enables the combinatorial synthesis of peptide microarrayswith 5,440 peptides per microscope slide and up to 156,000 peptides on larger slideformats. We meanwhile applied these peptide microarrays for epitope mapping ofantibodies with amino acid resolution(Figure 2a), the screening of peptidetarget binders, the characterization ofkinases and proteases, the analysis ofserum responses upon immunization(Figure 2b), and the screening ofserum biomarkers. The mapping ofantibody epitopes and serum analysisafter immunization is done bytranslation of the target protein into anarray of overlapping peptides. Thischip is incubated with the antibody orserum sample, followed by stainingwith secondary and control antibodiesFig.2. Epitope mapping of a monoclonal antibodywith amino acid resolution (a); analysis ofserum responses after immunization against twohomologue protein antigens (b).(Figure 2, spot frames). Due to thehigh spot densities, we can reducesample consumption to 5-10 µL perantibody or serum. In otherapproaches, we used libraries ofstochastic peptides, i.e. non-natural sequences which are generated by a computeralgorithm. Incubated with a monoclonal Flag antibody, we were e.g. able to identifypeptide binders with a certain similarity to the wild-type epitope (DYKDDDDK) from alibrary of 9,000 different 15mer peptides. These binders were then permuted in aconsecutively synthesized chip and again stained with the Flag antibody to finally revealthe consensus motif DYK from an initially random library.A second generation of the peptide laser printer is presently put into operation and willbe able to increase chip content to ~ 20,000 peptides per microscope slide and ~500,000peptides on larger slides. Custom peptide microarrays are made available byPEPperPRINT, a spin-off of the German Cancer Research Center in Heidelberg.PEPperPRINT also provides comprehensive epitope mapping and R&D services forpeptide discovery and development.AcknowledgmentsWe thank our co-workers at the research group “Chip-Based Peptide Libraries at the German CancerResearch Center and at PEPperPRINT and the Fraunhofer IPA for their contribution. This work wassupported by grants from the Federal Ministry of Education and Research (0315642A) and theEuropean Union (Grant Agreement Number 223243)References1. Beyer, M., et al. Science 318, 1888 (2007).2. Stadler, V., et al. Angew. Chem. Inter. Ed. 47, 7132 (2008).3. Frank, R., Tetrahedron 48, 9217 (1992).4. Beyer, M., et al. Biomaterials 27, 3505 (2006).19