Peptide Arrays and Antibody Libraries
Our research is segmented in two different research areas: (1) The combinatorial synthesis of high-density peptide arrays with the help of solid amino acid particles (in cooperation with our spin-off company PEPperPRINT) and (2) the generation of a library of >105 different human hybridomas (more) that should speed up considerably the generation of monoclonal antibodies.
High-density peptide arrays
Peptide arrays were invented by Dr. Ronald Frank  who was the first to parallelize the Nobel prize awarded solid phase peptide synthesis [2; Bruce Merrifield, Nobel prize for chemistry in 1984]. In doing so, he first dissolved the 20 different amino acid derivatives that are needed for peptide synthesis in a solvent, and then spotted these 20 different solutions to defined spots on an amino-terminated solid support. If repeated for several layers, this procedure builds up many different peptides in a combinatorial synthesis, each on a different spot (Fig. 1). One drawback of this “SPOT synthesis” method is the low achievable density of only ~25 peptides per cm2. This is due to the difficulties to deposit very small droplets on a solid support that tend to mix with neighbouring droplets or evaporate before the coupling reaction is finished.
In order to circumvent these technical difficulties, we developed a novel and prize awarded [3, 4] procedure, that first incorporates the amino acid derivatives in solid electrically chargeable “amino acid particles”. The different amino acid particles are then consecutively addressed to very small areas on a two dimensional support, either by a laser printer , or by the electrical fields of a computer chip’s pixel electrodes , where they keep sticking to the surface without intermingling with neighbouring particles. The coupling reaction is then induced simply by melting the whole particle layer, which frees hitherto immobilized amino acid derivatives to diffuse to and react with amino groups on the surface of the support. One technical advantage of the method is the very small spot specific “half domes” created by surface tension that wet very small and discrete areas on the solid support (Fig. 2; see also Fig. 4F in the section Chip printer). (more) Our procedure improves the state-of-the-art ~20-fold in terms of peptides per area and ~100-fold in terms of cost per peptide. A 2nd generation “peptide laser printer” (Fig. 3) is currently being developed within the EU FP7 project PEPLASER (more) by our cooperation partners Fraunhofer IPA and KMS Automation. In summer 2010 we expect this machine to improve the state-of-the-art to then >500.000 peptides per 20x20 cm2. Thereby, and for the first time, completely new applications for peptide arrays should be feasible (more) .
In addition to the xerographic methods (laser printer or CMOS chip with pixel electrode arrays), the combinatorial patterning of the amino acids can be realized with the direct transfer of amino acids due to the laser ablation from the donor slides to the synthesis support (Fig. 5) . With this cLIFT (combinatorial Light Induced Forward Transfer) method, we synthesized, for instance, HA- (Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala) and Flag-peptides (Tyr-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) with a spot pitch of 150 µm. Hereby, activated OPfp amino acids were deposited and coupled to the surface in repetitive manner. Fig. 6 shows the results of the specific fluorescent staining. The significant advantage of the cLIFT-method is the possibility of coupling non-activated amino acids . If two layers with a monomer and an activation reagent are deposited on each other, the molecules can mix with each other by heating the substrate due to the nanometer thickness of those layers. Thus, a non-inactivated amino acid can be deposited upon the activator, and dozens of thousands chemical reactions can starts simultaneously by heating the substrate. We believe that the method of the “stacked nanolayers” is applicable to a wide class of materials and chemical reactions.
The long term goal of this research is a procedure to synthesize freely chosen peptides in good quality on a glass slide, and for a few Euros chemical costs. Thereby, and for the first time, completely new applications for peptide arrays should be feasible. (more)
Fig. 1; Shown is the addition of one further monomeric building block by combinatorial synthesis. A limited number of monomeric building blocks yields a plethora of different oligomers. The 4 nucleotide building blocks of DNA yield 4n different n-meric oligonucleotides. The 20 different amino acid monomers of proteins and peptides yield 20n different n-meric peptides.
Fig. 2; Combinatorial synthesis of a peptide array with a laser printer
Fig. 3; Peptide laser printer with a pattern of amino acids that were printed and coupled to a glass support.
Fig. 4; Particle based combinatorial synthesis of peptide arrays. A, E: charged amino acid particles are directed to individually chosen pixel electrodes B: spatially defined deposition of 20 different amino acid particles C, F: within melted amino acid particles Fmoc-amino acid OPfp esters diffuse to the surface of the solid support where they couple to free amino groups of the support F, G: consecutive coupling cycles yield a peptide array.
Fig. 5; (a-h) The combinatorial laser induced transfer of synthesis building blocks. Donor-slide has different synthesis building blocks embedded into polymer matrix (labeled with different colors in (b) and (c)). The donor slides are positioned on the acceptor slide (a) with the transfer material downwards. A laser transfers small amounts of the material onto the acceptor slide. Patterns with different monomers are generated with laser transfer with different donor slides. The coupling of the monomers (e) begins with the heating of the acceptor slide. Subsequently, the unreacted monomers are washed away (f) and the usual steps for the peptide chemistry, such as acetylation of unreacted amino groups and the removal of the protective groups, are carried out (g). The repetition of the cycle (a-g) leads to the combinatorial peptide synthesis in the array format (h). (I) cLIFT synthesizer. The laser radiation is modulated with an acousto-optic modulator (AOM) and directed to the scanning system. Donor and acceptor slides are automatically exchanged by a slide loader.
Fig. 6; Fluorescence images of the cLIFT-synthesized patterns of Flag and HA peptides stained with specific anti-Flag and anti-HA antibodies. Scale: (a) 2 mm, (b) 1 mm, (c) 250 μm.
 Frank R. (1992) Spot synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron 48, 9217-9232.
 Felgenhauer T, Schirwitz C und Breitling F. (2010) Peptide synthesis. Ullmann's Encyclopedia of Industrial Chemistry (Last updated: 29 Apr 2010) published online 10.1002/14356007.a19_157.pub2
 Stadler V, Bischoff FR, Felgenhauer T, Kring M und Breitling F. Winner of the business plan competition Science4Life (awarded with 30.000 €) with the project Fertigung und Vertrieb von Biochips zur Parallel-Synthese unterschiedlicher Peptide (Juni 2009).
 Breitling F, Bischoff FR, Stadler V, Felgenhauer T, Leibe K, Fernandez S (all from DKFZ or KIT) and Güttler S, Gröning M, Willems P, Biesinger B (Fraunhofer IPA). Winner of the „Wissenschaftspreis des Stifterverbande“ (awarded with 50.000 € ) with the project Peptide laser printer (May 2008).
 Stadler V, Felgenhauer T, Beyer M, Fernandez S, Leibe K, Güttler S, Gröning M, Torralba G, Lindenstruth V, Nesterov A, Block I, Pipkorn R, Poustka A, Bischoff FR und Breitling F. (2008) Combinatorial synthesis of peptide arrays with a laser printer Angew. Chem. Int. Ed. 47, 7132–7135.
 Beyer M, Nesterov A, Block I, König K, Felgenhauer T, Fernandez S, Leibe K, Torralba G, Hausmann M, Trunk U, Lindenstruth V, Bischoff FR, Stadler V und Breitling F. (2007) Combinatorial synthesis of peptide arrays onto a computer chip’s surface. Science 318, 1888.
 Breitling F, Nesterov A, Stadler V, Felgenhauer T und Bischoff FR. (2010) High-density peptide arrays. Molecular BioSystems 5, 224-234. Epub 2009 Jan 16. Review
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 Loeffler F F, Breitling F, Nesterov-Mueller A, Kombinatorische Chemie im hochdichten Arrayformat, BIOspectrum, 2016, 5: 532-534; DOI:10.1007/s12268-016-0720-1