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Schirwitz C.,German Cancer Research Center | Loeffler F.F.,German Cancer Research Center | Felgenhauer T.,PEPperPRINT | Stadler V.,PEPperPRINT | And 2 more authors.
Biointerphases | Year: 2012

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. © The Author(s) 2012.

Schirwitz C.,German Cancer Research Center | Loeffler F.F.,German Cancer Research Center | Felgenhauer T.,PEPperPRINT | Stadler V.,PEPperPRINT | And 4 more authors.
Advanced Materials | Year: 2013

A method for the one-step purification of high-complexity peptide microarrays is presented. The entire peptide library is transferred from the synthesis support to a gold coated polyvinylidenfluoride (PVDF) membrane, whereby only full-length peptides covalently couple to the receptor membrane via an N-terminally added cysteine. Highly resolved peptide transfer and purification of up to 10 000 features per cm2 is demonstrated. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Agency: European Commission | Branch: FP7 | Program: CP | Phase: ENERGY.2010.10.2-1 | Award Amount: 3.64M | Year: 2011

In a solar cell that converts energy as efficient as the natural plant photosystems, electrons should travel over very short distances, through an extremely ordered structure free of traps. Guided by these principles that were worked out at WIS and CUNY, we want to synthesize many different peptides, and consecutively screen variants thereof for a putative diode function. When linked to light-harvesting building blocks, these should yield a novel type of solar cell that is based on biomimetic principles. To achieve these goals, UPC will chemically synthesize a panel of artificial building blocks that are designed to harvest light and/or tunnel electrons. When assembled into many different peptides, some of these constructs are expected to function as a diode, and, when linked to light-harvesting molecules, as a solar cell. Peptides will be embedded into a self-assembled monolayer of alkanethiols on individual gold pads of a computer chip, which will be designed and manufactured by IMS. Each individual peptide can be addressed by feeding in, and at the same time measuring, the amount of current flow in both directions through the gold pads of the individual pixels. A similar kind of screen should then detect a functional peptide-based solar cell. 10.000 Peptides per cm(\2) will be synthesized by a particle based combinatorial synthesis recently developed by KIT-G and PPP. These peptides will be transferred in the array format to the chip where they couple to the gold pads. Therein, peptides will be coupled through cysteine residues to the flat gold surface, where they are embedded into a membrane-like structure. We expect that our evolution inspired approach may open a novel route to very efficient, and very cheap solar cells. This is due to the small percolation distances, highly ordered modular peptide structures, the large number of generated peptides, the ability to easily combine and modify eventually found peptide-diodes, and the frugal material consumption.

Agency: European Commission | Branch: FP7 | Program: CP-TP | Phase: HEALTH-2007-1.1-4 | Award Amount: 4.03M | Year: 2009

Our particle-based method allows us to synthesise high complexity peptide arrays by combinatorial synthesis and for an unrivalled prize. We plan to further develop this new technology up to the level of robust prototype machines, and mate it to bioinformatics and readout tools. Together, our procedure(s) should boost the field of proteomics in a similar way as the lithographic technologies did with the field of genomics. Central to our novel method are the activated chemical building blocks that are frozen within solid amino acid particles. Thereby, we can use a colour laser printer to send them to defined addresses on a 2D support, where the particles are simply melted to induce a spatially defined coupling reaction of now freed amino acid derivatives. By repeated printing and melting cycles this simple trick yields high complexity peptide arrays. Based on existing pre-prototypes, we will develop a user-friendly peptide laser printer that spatially defined addresses our 20 different amino acid toners in high resolution to a support (WP1), and a scanner that especially fast and sensitive reads out the large formats delivered by the peptide laser printer (WP2). The increased production of amino acid toners and array supports are other bottlenecks in the output of peptide arrays that are tackled in WP3. This should allow us to increase the output of individual peptide spots from currently 0,5 Million to >10 Million peptides per month. Finally, to foster a market for high complexity peptide arrays, we will work out paradigmatic application examples in WP4. These aim to directly screen for antibiotic or apoptosis inducing D-peptides, and for the comprehensive readout of the different antibodies that patrol the serum of autoimmune patients. Based on user-friendly prototype machines, on first paradigmatic application examples for high complexity peptide arrays, and shielded by a strong patent, the participating SMEs will commercialise this new technology.

Agency: European Commission | Branch: FP7 | Program: CP-FP | Phase: HEALTH.2011.1.1-1 | Award Amount: 5.55M | Year: 2012

We want to further develop our tools and technologies for high-throughput research, with the final goals being (I.) the particle-based combinatorial synthesis of 1 Mio different peptides on a glass slide for chemical costs of ~50 (KIT, CBL, MS, TUV), and (II.) the labelling-free parallel readout of binding affinities by a variant reflectometric interference spectroscopy method for ~10.000 peptide spots per cm(\2) when staining the array with an unlabeled protein (BIA, KIT). These tools provide the basis (III.) for a standardized, fast, and reliable high-throughput procedure that we want to develop in order to find high-affinity peptide binders against any pharmaceutically interesting target protein. Such a procedure might have an important impact in medicine and in the biotechnology industry. In order to achieve this goal, we will use display techniques that in combination with high-throughput sequencing typically will identify ~100.000 putative peptide binders per target protein (ISO). These will be synthesized in array format to validate binding to the target protein by an independent method (PPP, DKFZ). Next, based on binders from initial screens, many variant peptides are synthesized in high-density array format for iterative screens (PPP, DKFZ, KIT), whereby massive parallel labelling-free detection of binders pinpoints higher-affinity binders (BIA). In order to validate our novel high-throughput procedure, (IV.) we want to find high-affinity peptide binders against relevant target proteins (delivered by APO and OXF), and test these binders in biological assays (OXF, APO).

Schmidt R.,German Cancer Research Center | Jacak J.,Johannes Kepler University | Schirwitz C.,German Cancer Research Center | Stadler V.,PEPperPRINT | And 5 more authors.
Journal of Proteome Research | Year: 2011

Based on a single-molecule sensitive fluorescence-linked immunosorbent assay, an analytical platform for the detection of lipoarabinomannan (LAM), a lipopolysaccharide marker of tuberculosis, was established that is about 3 orders of magnitude more sensitive than comparable current ELISA assays. No amplification step was required. Also, no particular sample preparation had to be done. Since individual binding events are detected, true quantification was possible simply by counting individual signals. Utilizing a total internal reflection configuration, unprocessed biological samples (human urine and plasma) to which LAM was added could be analyzed without the requirement of sample purification or washing steps during analysis. Samples containing about 600 antigen molecules per microliter produced a distinct signal. The methodology developed can be employed for any set of target molecules for which appropriate antibodies exist. © 2011 American Chemical Society.

Mock A.,University of Heidelberg | Warta R.,University of Heidelberg | Geisenberger C.,University of Heidelberg | Bischoff R.,PEPperPRINT | And 23 more authors.
Oncotarget | Year: 2015

Liquid biopsies come of age offering unexploited potential to monitor and react to tumor evolution. We developed a cost-effective assay to non-invasively determine the immune status of glioblastoma (GBM) patients. Employing newly developed printed peptide microarrays we assessed the B-cell response against tumor-associated antigens (TAAs) in 214 patients. Firstly, sera of long-term (36+ months, LTS, n=10) and short-term (6-10 months, STS, n=14) surviving patients were screened for prognostic antibodies against 1745 13-mer peptides covering known TAAs (TNC, EGFR, GLEA2, PHF3, FABP5, MAGEA3). Next, survival associations were investigated in two retrospective independent multicenter validation sets (n=61, n=129, all IDH1- wildtype). Reliability of measurements was tested using a second array technology (spotted arrays). LTS/STS screening analyses identified 106 differential antibody responses. Evaluating the Top30 peptides in validation set 1 revealed three prognostic peptides. Prediction of TNC peptide VCEDGFTGPDCAE was confirmed in a second set (p=0.043, HR=0.66 [0.44-0.99]) and was unrelated to TNC protein expression. Median signals of printed arrays correlated with pre-synthesized spotted microarrays (p<0.0002, R=0.33). Multiple survival analysis revealed independence of age, gender, KPI and MGMT status. We present a novel peptide microarray immune assay that identified increased anti-TNC VCEDGFTGPDCAE serum antibody titer as a promising non-invasive biomarker for prolonged survival.

Felgenhauer T.,PEPperPRINT
Chimica Oggi | Year: 2010

Microarrays promise to advance biology through the parallel screening for many different binding partners. In the field of DNA microarrays lithographic techniques enables the combinatorial synthesis in densities of several ten thousand oligonucleotides per cm2. The availability of such high dense screening tools revolutionized the field of genomics. A similar development is expected for the field of proteomics, but was hindered in past times by low densities and the lack of an adequate diversity in peptide array production. This article describes recent developments in this field with an emphasis on methods that have the potency to overcome the obstacles in peptide synthesis and lead to affordable very high density peptide arrays.

PEPperPRINT | Entity website

PEPperCHIP Human Epitome Microarray For the very first time, the new PEPperCHIP Human Epitome Microarray covers all linear human B-cell epitopes of the Immune Epitope Database. The 28,895 different peptides of the PEPperCHIP Human Epitome Microarray are based on23,163 database epitopes of 2,542 different proteins and 468 different organisms ...

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