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

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Reductionist Approach in Peptide-Based Nanotechnology

Gazit, Ehud
Annu. Rev. Biochem..
Jun 2018
10.1146/annurev-biochem-062917-012541
The formation of ordered nanostructures by molecular self-assembly of proteins and peptides represents one of the principal directions in nanotechnology. Indeed, polyamides provide superior features as materials with diverse physical properties. A reductionist approach allowed the identification of extremely short peptide sequences, as short as dipeptides, which could form well-ordered amyloid-like β-sheet-rich assemblies comparable to supramolecular structures made of much larger proteins. Some of the peptide assemblies show remarkable mechanical, optical, and electrical characteristics. Another direction of reductionism utilized a natural noncoded amino acid, α-aminoisobutryic acid, to form short superhelical assemblies. The use of this exceptional helix inducer motif allowed the fabrication of single heptad repeats used in various biointerfaces, including their use as surfactants and DNA-binding agents. Two additional directions of the reductionist approach include the use of peptide nucleic acids (PNAs) and coassembly techniques. The diversified accomplishments of the reductionist approach, as well as the exciting future advances it bears, are discussed.

A public antibody lineage that potently inhibits malaria infection through dual binding to the circumsporozoite protein

Tan, Joshua; Sack, Brandon K; Oyen, David; Zenklusen, Isabelle; Piccoli, Luca; Barbieri, Sonia; Foglierini, Mathilde; Fregni, Chiara Silacci; Marcandalli, Jessica; Jongo, Said; Abdulla, Salim; Perez, Laurent; Corradin, Giampietro; Varani, Luca; Sallusto, Federica; Sim, Betty Kim Lee; Hoffman, Stephen L; Kappe, Stefan H I; Daubenberger, Claudia; Wilson, Ian A; Lanzavecchia, Antonio
Nat Med.
Mar 2018
10.1038/nm.4513
Immunization with attenuated Plasmodium falciparum sporozoites (PfSPZs) has been shown to be protective against malaria, but the features of the antibody response induced by this treatment remain unclear. To investigate this response in detail, we isolated IgM and IgG monoclonal antibodies from Tanzanian volunteers who were immunized with repeated injection of Sanaria PfSPZ Vaccine and who were found to be protected from controlled human malaria infection with infectious homologous PfSPZs. All isolated IgG monoclonal antibodies bound to P. falciparum circumsporozoite protein (PfCSP) and recognized distinct epitopes in its N terminus, NANP-repeat region, and C terminus. Strikingly, the most effective antibodies, as determined in a humanized mouse model, bound not only to the repeat region, but also to a minimal peptide at the PfCSP N-terminal junction that is not in the RTS,S vaccine. These dual-specific antibodies were isolated from different donors and were encoded by VH3-30 or VH3-33 alleles that encode tryptophan or arginine at position 52. Using structural and mutational data, we describe the elements required for germline recognition and affinity maturation. Our study provides potent neutralizing antibodies and relevant information for lineage-targeted vaccine design and immunization strategies.

A Trifunctional Linker for Purified 3D Assembled Peptide Structure Arrays

Mattes, Daniela S.; Rentschler, Simone; Foertsch, Tobias C.; Münch, Stephan W.; Loeffler, Felix F.; Nesterov-Mueller, Alexander; Bräse, Stefan; Breitling, Frank
Small Methods.
Feb 2018
10.1002/smtd.201700205
Microarrays are an important tool in modern research that allow the rapid screening of many different interactions simultaneously. Peptide arrays, which bear different peptides arranged in separate spots, permit high-throughput screening to investigate linear and cyclic binding sites. To study conformational or discontinuous binding sites, protein arrays are the major choice. However, the tremendous costs for the generation of high-density protein arrays of high purity restrict progress in protein research. Therefore, peptide-based arrays, which can mimic assembled peptide structures, have an enormous potential. Here, a method is presented to create such structures in the array format as an alternative to protein arrays. A trifunctional linker is developed with an azide, a protected alkyne, and a carboxyl group, which can react with two or three different peptides. Due to the spatial proximity, the peptides interact and can form an assembled peptide structure. As a proof of concept, assembled peptide structures are demonstrated on beads and on a polymer surface and the approach can be validated via matrix-assisted laser desorption/ionization spectrometry. Furthermore, a multistep transfer of peptide arrays is shown, generating purified assembled peptide structure arrays in high density.

Development and characterization of a human monoclonal antibody targeting the N-terminal region of hepatitis C virus envelope glycoprotein E1

Mesalam, Ahmed Atef; Desombere, Isabelle; Farhoudi, Ali; Van Houtte, Freya; Verhoye, Lieven; Ball, Jonathan; Dubuisson, Jean; Foung, Steven K.H.; Patel, Arvind H.; Persson, Mats A.A.; Leroux-Roels, Geert; Meuleman, Philip
Virology.
Jan 2018
10.1016/j.virol.2017.10.019
Monoclonal antibodies (mAbs) targeting the hepatitis C virus (HCV) envelope have been raised mainly against envelope protein 2 (E2), while the antigenic epitopes of envelope protein 1 (E1) are not fully identified. Here we describe the detailed characterization of a human mAb, designated A6, generated from an HCV genotype 1b infected patient. ELISA results showed reactivity of mAb A6 to full-length HCV E1E2 of genotypes 1a, 1b and 2a. Epitope mapping identified a region spanning amino acids 230–239 within the N-terminal region of E1 as critical for binding. Antibody binding to this epitope was not conformation dependent. Neutralization assays showed that mAb A6 lacks neutralizing capacity and does not interfere with the activity of known neutralizing antibodies. In summary, mAb A6 is an important tool to study the structure and function of E1 within the viral envelope, a crucial step in the development of an effective prophylactic HCV vaccine.

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.

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