From a technical point of view there is one especially prominent feature of our particle based combinatorial synthesis of peptide arrays: We can synthesize for the first time affordable very high-density peptide arrays in good quality and with freely chosen sequences [16, 17, 18]. A similar technical development in the synthesis of oligonucleotide arrays revolutionized the whole field of genomics . However, this technical breakthrough was achieved with a lithographic synthesis method  that doesn’t function for the synthesis of peptide arrays. This is due to the peptide-specific (too) many coupling cycles that are needed to synthesize a peptide array with lithographic methods where always only one monomer couples to the support. Therefore, 20 x 10 coupling cycles are needed for the synthesis of a decameric peptide array (20 different amino acid monomers) , while only 4 x 10 coupling cycles are needed for the corresponding oligonucleotide arrays (4 different nucleotide monomers). Basically, this is the reason why lithographic methods revolutionized the manufacturing of oligonucleotide arrays, but not the corresponding peptide arrays. Please note, that all the printing procedures including our particle based method address all the 20 different monomers in parallel to the array support (only one coupling cycle per layer).
Until very recently peptide arrays cost more then 1 € per peptide (www.jpt.com). Currently, such arrays cost 13 cent per peptide at PEPperPRINT (year 2010) – a price that should continuously fall in the forthcoming years concomitantly with increasing customers demand and a more matured technology (www.PEPperPRINT.com). Especially, the chip printer that will be advanced at the KIT might help to bring down the cost per peptide considerably in the forthcoming years.
This raises the next question: What applications could we foresee for peptide arrays that for the first time assemble thousands of peptides in an affordable array? They will certainly have an impact in the life sciences, as a complex diagnostics that assembles many different antigens, as a tool for search for peptide based therapeutic, and especially also as a novel research tool . Initially, however, we expect that several less ambitious applications might become a wide spread routine. We expect that cheap peptide arrays, e.g. will be routinely used to answer the question against exactly which linear epitopes a mouse or a rabbit produced antibodies when immunized with a protein. The answer to this question is especially important when screening for an antibody that discriminates closely related proteins. Fig. 8 shows the results of such an experiment.
Fig. 8; Peptides with overlap-ping sequences derived from protein #A stain specifically with anti-A-Serum 1.
The continuing links describe some selected application examples that will be advanced at KIT in the forthcoming years. However, many of these applications require an even further reduced cost per peptide. In the next 2-4 years KIT will therefore focus on advancing our particle based peptide synthesis (smaller synthesis sites, better repetitive yield, combination with labelling free readout technologies). Certainly, these future plans also depend on our success in the recruitment of third-party funds.
Epitope mapping and fingerprint analysis of antibodies elicited by vaccinations with high density peptide arrays
Vaccinations are one of the most potent tools to fight infectious diseases. However, cross-reactions are an ongoing problem and there is an urgent need to fully understand the mechanisms of the immune response, especially regarding vaccinations. Here, we present a method, which employs peptide arrays, to determine linear immunodominant epitopes and to identify in a second step the amino acids that are essential for the binding of an antibody to the identified epitope – the antibody fingerprint. This approach allows us to clarify if the antibody repertoires of different individuals target the same epitopes down to the single amino acid level. The identification of immunodominant epitopes and the elucidation of the combination of those epitopes responsible for a successful neutralization of toxins in different patients could be the first step towards a peptide-based vaccine.
As a model system (Fig. 9), we investigated linear epitopes in the immune response to the tetanus and diphtheria toxins in sera of 19 vaccinated Europeans. The most prominent epitope, appearing in 8 different sera, is located on one of the binding domains of the heavy chain and was investigated in a substitution analysis. Therefore, every amino acid in the original sequence of the antigenic peptide was one by one exchanged by all other 19 amino acids, while the rest of the sequence remained conserved. The 8 identified fingerprints were strongly conserved and consisted of 5 to 6 essential amino acids over an epitope length of 9 amino acids. These similarities in the antibody species are astonishing considering the randomness of the development of antibodies. This approach of studying antibody fingerprints using peptide arrays should be transferable to any kind of humoral immune response to protein antigens, which allows for new insights in antibody repertoire development.
Fig. 9; A: The linear amino acid sequence of a protein is cut in silico into peptides with overlapping sequences. B: The resulting sequences are synthesized onto a peptide array. C: The peptide array is incubated with human serum and subsequently with fluorescently labeled secondary antibodies. A fluorescence scan reveals the information which peptides are bound by serum antibodies. D: The peptide binding is mapped onto the 3D protein structure. E: In a substitution analysis, every amino acid is systematically exchanged by all other amino acids to assess the antibody binding fingerprint.
Single amino acid fingerprinting of the human antibody repertoire with high density peptide arrays
The antibody species that patrol in a patient’s blood are an invaluable part of the immune system. While most of them shield us from life-threatening infections, some of them do harm in autoimmune diseases. If we knew exactly all the antigens that elicited all the antibody species within a group of patients, we could learn which ones correlate with immune protection, are irrelevant, or do harm. Here, we demonstrate an approach to this question (Fig. 10): First, we use a plethora of phage-displayed peptides to identify many different serum antibody binding peptides. Next, we synthesize identified peptides in the array format and rescreen the serum used for phage panning to validate antibody binding peptides. Finally, we systematically vary the sequence of validated antibody binding peptides to identify those amino acids within the peptides that are crucial for binding “their” antibody species. The resulting immune fingerprints can then be used to trace them back to potential antigens. The investigation of the antibodies of an individual in this manner led to the identification of 73 fingerprints, of which some are closely related. One motif could be traced back to the immunodominant capsid protein VP1 of the polio virus. Due to common vaccinations against this virus, it is very likely for our individual to have such an antibody species patrolling in his serum. Without any pre-information, it should become possible with our approach, to pinpoint those antibody species that correlate with a hitherto enigmatic disease.
Fig. 10; In an initial pre-screen, up to 109 random peptides displayed on phage were screened for their binding to serum antibodies, immobilized on beads. Next, the identified epitope peptides were validated with solid material-based peptide microarray technology. Finally, the validated epitopes were fine mapped by comprehensive substitution analysis. The resulting “epitope fingerprints” enable the identification of those proteins that match the antibody specificity, and, eventually, the correlation to disease causing agents.
 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
 Fodor SP, Read JL, Pirrung MC, Stryer L, Lu AT und Solas D. (1991) Light-directed, spatially addressable parallel chemical synthesis. Science 251, 767-773.
 Pellois JP, Zhou XC, Srivannavit O, Zhou TC, Gulari E und Gao XL. (2002) Individually addressable parallel peptide synthesis on microchips. Nature Biotechnology 20, 922-926.