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Discover how PEPperPRINT Peptide Microarray products have been used in different fields of research.

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Identification of novel antigens contributing to autoimmunity in cardiovascular diseases

Müller, Anna-Maria; Bockstahler, Mariella; Hristov, Georgi; Weiß, Christel; Fischer, Andrea; Korkmaz-Icöz, Sevil; Giannitsis, Evangelos; Poller, Wolfgang; Schultheiss, Heinz-Peter; Katus, Hugo A.; Kaya, Ziya
Clinical Immunology.
Dec 2016
10.1016/j.clim.2016.09.003
In myocarditis and dilated cardiomyopathy (DCM) patients the immune system may play an important role in disease progression. In this study, we aimed to identify new antigens as a target for autoimmune response that might play a crucial role in these diseases. Therefore, a peptide-array was used to investigate antibody binding profiles in patients with autoimmune myocarditis or DCM compared to healthy controls and thus to identify disease relevant antigens. To analyze the pathogenicity of the identified antigens, an experimental autoimmune myocarditis (EAM) model was used. Hereby, 3 peptide sequences, derived from myosin-binding-protein-C (MYBPC) fast-type, RNA-binding-protein 20 (RBM20), and dystrophin, showed pathogenic effects on the myocardium of mice. In summary, 3 potentially cardiopathogenic peptides (MYBPC fast-type, RBM20, dystrophin) were identified. Thus, this study could serve as a basis for future investigations aimed at determining further antigens leading to pathogenic effects on the myocardium of DCM as well as myocarditis patients.

Serum peptide reactivities may distinguish neuromyelitis optica subgroups and multiple sclerosis

Metz, Imke; Beißbarth, Tim; Ellenberger, David; Pache, Florence; Stork, Lidia; Ringelstein, Marius; Aktas, Orhan; Jarius, Sven; Wildemann, Brigitte; Dihazi, Hassan; Friede, Tim; Brück, Wolfgang; Ruprecht, Klemens; Paul, Friedemann
Neurol Neuroimmunol Neuroinflamm.
Apr 2016
10.1212/NXI.0000000000000204
Objective: To assess in an observational study whether serum peptide antibody reactivities may distinguish aquaporin-4 (AQP4) antibody (Ab)–positive and -negative neuromyelitis optica spectrum disorders (NMOSD) and relapsing-remitting multiple sclerosis (RRMS). Methods: We screened 8,700 peptides that included human and viral antigens of potential relevance for inflammatory demyelinating diseases and random peptides with pooled sera from different patient groups and healthy controls to set up a customized microarray with 700 peptides. With this microarray, we tested sera from 66 patients with AQP4-Ab-positive (n = 16) and AQP4-Ab-negative (n = 19) NMOSD, RRMS (n = 11), and healthy controls (n = 20). Results: Differential peptide reactivities distinguished NMOSD subgroups from RRMS in 80% of patients. However, the 2 NMOSD subgroups were not well-discriminated, although those patients are clearly separated by their antibody reactivities against AQP4 in cell-based assays. Elevated reactivities to myelin and Epstein-Barr virus peptides were present in RRMS and to AQP4 and AQP1 peptides in AQP4-Ab-positive NMOSD. Conclusions: While AQP4-Ab-positive and -negative NMOSD subgroups are not well-discriminated by peptide antibody reactivities, our findings suggest that peptide antibody reactivities may have the potential to distinguish between both NMOSD subgroups and MS. Future studies should thus concentrate on evaluating peptide antibody reactivities for the differentiation of AQP4-Ab-negative NMOSD and MS.

Sensing Immune Responses with Customized Peptide Microarrays

Schirwitz, Christopher; Loeffler, Felix F.; Felgenhauer, Thomas; Stadler, Volker; Breitling, Frank; Bischoff, F. Ralf
Biointerphases.
Aug 2012
10.1007/s13758-012-0047-5
The intent to solve biological and biomedical questions in high-throughput led to an immense interest in microarray technologies. Nowadays, DNA microarrays are routinely used to screen for oligonucleotide interactions within a large variety of potential interaction partners. To study interactions on the protein level with the same efficiency, protein and peptide microarrays offer similar advantages, but their production is more demanding. A new technology to produce peptide microarrays with a laser printer provides access to affordable and highly complex peptide microarrays. Such a peptide microarray can contain up to 775 peptide spots per cm², whereby the position of each peptide spot and, thus, the amino acid sequence of the corresponding peptide, is exactly known. Compared to other techniques, such as the SPOT synthesis, more features per cm² at lower costs can be synthesized which paves the way for laser printed peptide microarrays to take on roles as efficient and affordable biomedical sensors. Here, we describe the laser printer-based synthesis of peptide microarrays and focus on an application involving the blood sera of tetanus immunized individuals, indicating the potential of peptide arrays to sense immune responses.

Physical Characterization of the “Immunosignaturing Effect”

Stafford, Phillip; Halperin, Rebecca; Legutki, Joseph Bart; Magee, Dewey Mitchell; Galgiani, John; Johnston, Stephen Albert
Mol Cell Proteomics.
Apr 2012
10.1074/mcp.M111.011593
Identifying new, effective biomarkers for diseases is proving to be a challenging problem. We have proposed that antibodies may offer a solution to this problem. The physical features and abundance of antibodies make them ideal biomarkers. Additionally, antibodies are often elicited early in the ontogeny of different chronic and infectious diseases. We previously reported that antibodies from patients with infectious disease and separately those with Alzheimer’s disease display a characteristic and reproducible immunosignature on a microarray of 10,000 random sequence peptides. Here we investigate the physical and chemical parameters underlying how immunosignaturing works. We first show that a variety of monoclonal and polyclonal antibodies raised against different classes of antigens produce distinct profiles on this microarray and the relative affinities are determined. A proposal for how antibodies bind the random sequences is tested. Sera from vaccinated mice and people suffering from a fugal infection are individually assayed to determine the complexity of signals that can be distinguished. Based on these results, we propose that this simple, general and inexpensive system could be optimized to generate a new class of antibody biomarkers for a wide variety of diseases.

Single-Molecule Detection on a Protein-Array Assay Platform for the Exposure of a Tuberculosis Antigen

Schmidt, Ronny; Jacak, Jaroslaw; Schirwitz, Christopher; Stadler, Volker; Michel, Gerd; Marmé, Nicole; Schütz, Gerhard J.; Hoheisel, Jörg D.; Knemeyer, Jens-Peter
J. Proteome Res..
Jan 2011
10.1021/pr101070j

Combinatorial Peptide Synthesis on a Microchip

Schirwitz, Christopher; Block, Ines; König, Kai; Nesterov, Alexander; Fernandez, Simon; Felgenhauer, Thomas; Leibe, Klaus; Torralba, Gloria; Hausmann, Michael; Lindenstruth, Volker; Stadler, Volker; Breitling, Frank; Bischoff, F. Ralf
Current Protocols in Protein Science.
Aug 2009
10.1002/0471140864.ps1802s57
Microchips are used in the combinatorial synthesis of peptide arrays by means of amino acid microparticle deposition. The surface of custom-built microchips can be equipped with an amino-modified poly(ethylene glycol)methacrylate (PEGMA) graft polymer coating, which permits high loading of functional groups and resists nonspecific protein adsorption. Specific microparticles that are addressed to the polymer-coated microchip surface in a well defined pattern release preactivated amino acids upon melting, and thus allow combinatorial synthesis of high-complexity peptide arrays directly on the chip surface. Currently, arrays with densities of up to 40,000 peptide spots/cm2 can be generated in this way, with a minimum of coupling cycles required for full combinatorial synthesis. Without using any additional blocking agent, specific peptide recognition has been verified by background-free immunostaining on the chip-based array. This unit describes microchip surface modification, combinatorial peptide array synthesis on the chip, and a typical immunoassay employing the resulting high-density peptide arrays.

Particle-Based Synthesis of Peptide Arrays

Breitling, Frank; Felgenhauer, Thomas; Nesterov, Alexander; Lindenstruth, Volker; Stadler, Volker; Bischoff, F. Ralf
ChemBioChem.
Mar 2009
10.1002/cbic.200800735
Lithographic methods allow for the combinatorial synthesis of >50,000 oligonucleotides per cm(2), and this has revolutionized the field of genomics. High-density peptide arrays promise to advance the field of proteomics in a similar way, but currently lag behind. This is mainly due to the monomer-by-monomer repeated consecutive coupling of 20 different amino acids associated with lithography, which adds up to an excessive number of coupling cycles. Combinatorial synthesis based on electrically charged solid amino acid particles resolves this problem. A color laser printer or a chip addresses the different charged particles consecutively to a solid support, where, when completed, the whole layer of solid amino acid particles is melted at once. This frees hitherto immobilized amino acids to couple all 20 different amino acids to the support in one single coupling reaction. The method should allow for the translation of entire genomes into sets of overlapping peptides to be used in proteome research.

High-density peptide arrays

Breitling, Frank; Nesterov, Alexander; Stadler, Volker; Felgenhauer, Thomas; Bischoff, F. Ralf
Mol. BioSyst..
Jan 2009
10.1039/b819850k
Arrays promise to advance biology by allowing parallel screening for many different binding partners. Meanwhile, lithographic methods enable combinatorial synthesis of >50 000 oligonucleotides per cm2, an advance that has revolutionized the whole field of genomics. A similar development is expected for the field of proteomics, provided that affordable, very high-density peptide arrays are available. However, peptide arrays lag behind oligonucleotide arrays. This review discusses recent developments in the field with an emphasis on methods that lead to very high-density peptide arrays.

A Novel Combinatorial Approach to High-Density Peptide Arrays

Beyer, Mario; Block, Ines; König, Kai; Nesterov, Alexander; Fernandez, Simon; Felgenhauer, Thomas; Schirwitz, Christopher; Leibe, Klaus; Bischoff, Ralf F.; Breitling, Frank; Stadler, Volker
Jan 2009
10.1007/978-1-60327-394-7_16
Combinatorial synthesis of peptides on solid supports (1), either as spots on cellulose membranes (2) or with split-pool-libraries on polymer beads (3), substantially forwarded research in the field of peptide-protein interactions. Admittedly, these concepts have specific limitations, on one hand the number of synthesizable peptide sequences per area, on the other hand elaborate decoding/encoding strategies, false-positive results and sequence limitations. We recently established a method to produce high-density peptide arrays on microelectronic chips (4). Solid amino acid microparticles were charged by friction and transferred to defined pixel electrodes onto the chip’s surface, where they couple to a functional polymer coating simply upon melting (Fig. 16.1 A-D,F). By applying standard Fmoc chemistry according to Merrifield, peptide array densities of up to 40,000 spots per square centimetre were achieved (Fig. 16.1G). The term Merrifield synthesis describes the consecutive linear coupling and deprotecting of L-amino acids modified with base-labile fluorenylmethoxy (Fmoc) groups at the N-terminus and different acid-sensitive protecting groups at their side chains. Removing side chain protecting groups takes place only once at the very end of each synthesis and generates the natural peptide sequence thereby.

Combinatorial Synthesis of Peptide Arrays with a Laser Printer

Stadler, Volker; Felgenhauer, Thomas; Beyer, Mario; Fernandez, Simon; Leibe, Klaus; Güttler, Stefan; Gröning, Martin; König, Kai; Torralba, Gloria; Hausmann, Michael; Lindenstruth, Volker; Nesterov, Alexander; Block, Ines; Pipkorn, Rüdiger; Poustka, Annemarie; Bischoff, F. Ralf; Breitling, Frank
Angew. Chem. Int. Ed..
Sep 2008
10.1002/anie.200801616
Special delivery: The “freezing” of activated amino acid derivatives within solid particles enables a laser printer to deliver these “postal packages” to defined locations on a solid support with high resolution. Subsequent parallel coupling is initiated simply by melting a whole layer of 20 different amino acid particles (see schematic representation; Fmoc=9-fluorenylmethoxycarbonyl).

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