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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, 2010Next Generation Peptide MicroarraysF. Ralf Bischoff 1 , Frank Breitling 2 , and Volker Stadler 11 PEPperPRINT GmbH, Heidelberg, 69123, Germany; 2 Karlsruhe Institute of Technology,Institute for Microstructure Technology, Eggenstein-Leopoldshafen, 76344, GermanyIntroductionIn recent years, DNA chips have revolutionized genome research. A similar trend ispredicted for peptide chips in proteome research. Their use ranges from the characterizationof sera, enzymes and antibodies through the screening of new protein biomarkers up to thedevelopment of peptide-based drugs and vaccines. Due to complex manufacturingprocesses, however, available peptide chips fall short of the spot density and thusinformation content of corresponding DNA chips by far. To eliminate these obstacles, wedeveloped a new combinatorial technique with amino acids embedded in solid “amino acidtoner” particles [1]. These toners are printed onto glass slides with micron resolution, usinga custom-made 20-colour laser printer [2]. With all the different amino acid particles finallyprinted, they are melted at once to initiate coupling. Repeated printing and meltingeventually result in custom peptide microarrays with presently 5,440 spots on microscopeslides, and up to 156,000 spots on larger slides.Results and DiscussionSince more than 15 years, peptide arrays were predominantly synthesized by means of thespotting technique developed by Ronald Frank [3]. Thereby activated amino acids aredissolved in a solvent like DMF, and spotted onto cellulose substrates for combinatorialsynthesis of up to 25 peptides per cm 2 . Working with liquid droplets, however, limits thearray resolution due to evaporation effects and spreading on the support. To generatecustom peptide microarrays with higher spot densities and thorough flexibility at asignificantly reduced material need, we developed a new combinatorial technique which isbased on the laser printing of “amino acid toner particles”. Our approach is sketched inFigure 1 (left): First, amino acid toners are printed with high resolution onto a solid support(a), followed by melting of the toner particles to release the embedded amino acids whatinitiates coupling (b). Residual material is then removed by washing (c), before N-terminalFmoc-protecting groups are cleaved from the growing peptide chains (d). Repeating thiscycle of synthesis (a-d) finally results in combinatorially synthesized peptide microarrays.The amino acid toners are manufactured by sophisticated milling and sievingprocesses [1]. They are composed of Fmoc amino acid pentafluorophenyl esters asmonomers, a toner resin, a higher homologue of standard solvents for peptide synthesis(ditolyl sulfoxide and diphenyl formamide instead of DMSO and DMF), and of chargecontrol additives which enable the generation of defined surface charges required for thelaser printing process. Their physical properties have been carefully adjusted to color tonersfrom the OKI C7400 series (Figure 1, middle). The first generation peptide laser printer hasbeen developed in collaboration with Fraunhofer IPA and contains commercially availableprinting units of the same OKI series (Figure 1, right). Instead of the 4 printing units of aconventional color laser printer, the peptide laser printer consists of 20 printing units for the20 most important L-amino acids combined with a linear stage to enable repetitive printingwith micron resolution [2]. Furthermore, the amino acid toners can be printed not only oncellulose sheets and polymer foils, but also on glass slides as peptide chip substrates.Fig. 1. Combinatorial synthesis of peptide microarrays by amino acid toner particles(left); SEM images amino acid toners in comparison with high-end toners of the OKIC7400 series (middle); peptide laser printer with OKI printing drums arranged in a row.18