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

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Bacterial inhibitors

Xie, Hua
Jul 2019
Peptides related to certain portions of the arginine deiminase enzyme from the bacterium Streptococcus cristatus are provided that disrupt the formation and composition of biofilms containing the oral pathogen Porphyromonas gingivalis, and also modulate the virulence of P. gingivalis. Pharmaceutical compositions containing such peptides and method of using the same are disclosed.

Miniaturized and Automated Synthesis of Biomolecules—Overview and Perspectives

Mattes, Daniela S.; Jung, Nicole; Weber, Laura K.; Bräse, Stefan; Breitling, Frank
Adv. Mater..
Jun 2019
10.1002/adma.201806656
Chemical synthesis is performed by reacting different chemical building blocks with defined stoichiometry, while meeting additional conditions, such as temperature and reaction time. Such a procedure is especially suited for automation and miniaturization. Life sciences lead the way to synthesizing millions of different oligonucleotides in extremely miniaturized reaction sites, e.g., pinpointing active genes in whole genomes, while chemistry advances different types of automation. Recent progress in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging could match miniaturized chemical synthesis with a powerful analytical tool to validate the outcome of many different synthesis pathways beyond applications in the life sciences. Thereby, due to the radical miniaturization of chemical synthesis, thousands of molecules can be synthesized. This in turn should allow ambitious research, e.g., finding novel synthesis routes or directly screening for photocatalysts. Herein, different technologies are discussed that might be involved in this endeavor. A special emphasis is given to the obstacles that need to be tackled when depositing tiny amounts of materials to many different extremely miniaturized reaction sites.

A high-sensitivity enzyme immunoassay for the quantification of soluble human semaphorin 4D in plasma

Laber, Anna; Gadermaier, Elisabeth; Wallwitz, Jacqueline; Berg, Gabriela; Himmler, Gottfried
Analytical Biochemistry.
Jun 2019
10.1016/j.ab.2019.03.004
Human semaphorin 4D (SEMA4D), a type I integral membrane glycoprotein, regulates key cellular functions (e.g. cell-cell communication, platelet activation). Its 120 kDa extracellular region can be shed from the membrane to release soluble SEMA4D (sSEMA4D). Studies on circulating sSEMA4D levels are mostly performed with poorly characterized assays and use serum and plasma as matrix. We developed and validated a sandwich ELISA utilizing two monoclonal antibodies with resolved epitopes and determined affinities. Human serum and plasma samples were analyzed, and the influence of protease activity on sSEMA4D concentration was tested by collecting samples in the presence of the protease inhibitor TAPI-1. Both antibodies recognize conformational epitopes in the sema domain. Validation for plasma (EDTA, citrate, heparin) showed valid specificity, precision, accuracy, dilution linearity, and robustness. The assay shows a calibration range from 62.5 to 2000 pmol/L with a quantification limit of 31 pmol/L. sSEMA4D was significantly higher in serum than in plasma, whereas serum and plasma levels from samples collected in the presence of TAPI-1 showed no statistical difference. This ELISA provides a reliable tool for the quantification of sSEMA4D in human plasma. Serum is not recommended as matrix due to the accumulation of shed SEMA4D during blood coagulation altering serum sSEMA4D levels.

Autoantibodies to a novel Rpp38 (Th/To) derived B-cell epitope are specific for systemic sclerosis and associate with a distinct clinical phenotype

Koenig, Martial; Bentow, Chelsea; Satoh, Minoru; Fritzler, Marvin J; Senécal, Jean-Luc; Mahler, Michael
Apr 2019
10.1093/rheumatology/kez123
Abstract Objective Detection of antinuclear antibodies and specific autoantibodies is important in the diagnosis and classification of SSc. Several proteins of the Th/To complex, including Rpp25, Rpp38 and hPop1 are the target of autoantibodies in SSc patients. However, very little is known about the epitope distribution of this autoantigen. Consequently, we screened Rpp25, Rpp38 and hPop1 for B cell epitopes and evaluated their clinical relevance. Methods Serum pools with (n = 2) and without (n = 1) anti-Th/To autoantibodies were generated and used for epitope discovery. Identified biomarker candidate sequences were then utilized to synthesize synthetic, biotinylated, soluble peptides. The peptides were tested to determine reactivity with sera from SSc cohorts (n = 202) and controls (n = 159) using a chemiluminescence immunoassay. Additionally, samples were also tested for antibodies to full-length recombinant Rpp25 antibodies by chemiluminescence immunoassay. Results Several immunodominant regions were found on the three proteins. The strongest reactivity was observed with an Rpp38 peptide (aa 229–243). Autoantibodies to the Rpp38 peptide were detected in 8/149 (5.4%) limited cutaneous SSc patients, but not in any of 159 controls (P = 0.003 by two-sided Fisher’s exact probability test). Although reactivity to the novel antigenic peptide was correlated with the binding to Rpp25 (rho = 0.44; P < 0.0001), subsets of patient sera either reacted strongly with Rpp25 or with the novel Rpp38-derived peptide. Conclusion A novel Rpp38 epitope holds promise to increase the sensitivity in the detection of anti-Th/To autoantibodies, thus enhancing the serological diagnosis of SSc.

In-depth serum proteomics reveals biomarkers of psoriasis severity and response to traditional Chinese medicine

Xu, Meng; Deng, Jingwen; Xu, Kaikun; Zhu, Tiansheng; Han, Ling; Yan, Yuhong; Yao, Danni; Deng, Hao; Wang, Dan; Sun, Yaoting; Chang, Cheng; Zhang, Xiaomei; Dai, Jiayu; Yue, Liang; Zhang, Qiushi; Cai, Xue; Zhu, Yi; Duan, Hu; Liu, Yuan; Li, Dong; Zhu, Yunping; Radstake, Timothy R. D. J.; Balak, Deepak M.W.; Xu, Danke; Guo, Tiannan; Lu, Chuanjian; Yu, Xiaobo
Theranostics.
Apr 2019
10.7150/thno.31144
Serum and plasma contain abundant biological information that reflect the body’s physiological and pathological conditions and are therefore a valuable sample type for disease biomarkers. However, comprehensive profiling of the serological proteome is challenging due to the wide range of protein concentrations in serum. Methods: To address this challenge, we developed a novel in-depth serum proteomics platform capable of analyzing the serum proteome across ~10 orders or magnitude by combining data obtained from Data Independent Acquisition Mass Spectrometry (DIA-MS) and customizable antibody microarrays. Results: Using psoriasis as a proof-of-concept disease model, we screened 50 serum proteomes from healthy controls and psoriasis patients before and after treatment with traditional Chinese medicine (YinXieLing) on our in-depth serum proteomics platform. We identified 106 differentially-expressed proteins in psoriasis patients involved in psoriasis-relevant biological processes, such as blood coagulation, inflammation, apoptosis and angiogenesis signaling pathways. In addition, unbiased clustering and principle component analysis revealed 58 proteins discriminating healthy volunteers from psoriasis patients and 12 proteins distinguishing responders from non-responders to YinXieLing. To further demonstrate the clinical utility of our platform, we performed correlation analyses between serum proteomes and psoriasis activity and found a positive association between the psoriasis area and severity index (PASI) score with three serum proteins (PI3, CCL22, IL-12B). Conclusion: Taken together, these results demonstrate the clinical utility of our in-depth serum proteomics platform to identify specific diagnostic and predictive biomarkers of psoriasis and other immune-mediated diseases.

Automated laser-assisted synthesis of microarrays for infectious disease research

Paris, Grigori; Heidepriem, Jasmin; Tsouka, Alexandra; Mende, Marco; Eickelmann, Stephan; Loeffler, Felix F.
Mar 2019
10.1117/12.2516781
We developed a next-generation method for chemical in–situ combinatorial biomolecule array synthesis. This allows for an unprecedented combinatorial freedom in the automated chemical synthesis of molecule arrays with very high spot densities. Key feature of this new method is an automated positioning and laser transfer process: Small solid material spots are rapidly transferred from a donor film to an acceptor surface, requiring only minute amounts of materials. The transfer is performed with different and easy-to-produce donor slides. Each donor slide bears a thin polymer film, embedding one type of monomer. The coupling reaction occurs in a separate heating step, where the matrix becomes viscous and building blocks can diffuse within the material and couple to the acceptor surface. Since these transferred material spots are only several nanometers thin, this method allows for a consecutive multi-layer material deposition of e.g. activation reagents and amino acids. Subsequent heat-induced mixing facilitates an in–situ activation and coupling of the monomers. Positioning several of such resin spots, containing different chemical reagents, on top of each other, will enable for the first time in such small dimensions unique chemical synthesis strategies for each spot. Amount and concentration of the deposited materials can be adjusted with the laser parameters. Employing similar arrays, we can analyze the human immune response towards the proteome of different pathogens. We screened several peptide array replicas with different patient sera. The screenings resulted in significant hits in several proteins with interesting implications for future diagnostics and vaccine development.

Methods of Selecting Binding Reagents

Mallick, Parag; Egertson, Jarrett
Feb 2019
Methods and systems are provided herein for selecting an affinity reagent which binds a desired peptide epitope in a plurality of sequence contexts. The method relies on obtaining a peptide library, each peptide having the sequence αΧβ, wherein X is the desired peptide epitope, wherein each of a and β comprise an amino acid, using the peptide library to select an affinity reagent.

Bayesian Analysis of MicroScale Thermophoresis Data to Quantify Affinity of Protein:Protein Interactions with Human Survivin

Garcia-Bonete, Maria-Jose; Jensen, Maja; Recktenwald, Christian V.; Rocha, Sandra; Stadler, Volker; Bokarewa, Maria; Katona, Gergely
Sci Rep.
Dec 2017
10.1038/s41598-017-17071-0
A biomolecular ensemble exhibits different responses to a temperature gradient depending on its diffusion properties. MicroScale Thermophoresis technique exploits this effect and is becoming a popular technique for analyzing interactions of biomolecules in solution. When comparing affinities of related compounds, the reliability of the determined thermodynamic parameters often comes into question. The thermophoresis binding curves can be assessed by Bayesian inference, which provides a probability distribution for the dissociation constant of the interacting partners. By applying Bayesian machine learning principles, binding curves can be autonomously analyzed without manual intervention and without introducing subjective bias by outlier rejection. We demonstrate the Bayesian inference protocol on the known survivin:borealin interaction and on the putative protein-protein interactions between human survivin and two members of the human Shugoshin-like family (hSgol1 and hSgol2). These interactions were identified in a protein microarray binding assay against survivin and confirmed by MicroScale Thermophoresis.

A recombinant BBSome core complex and how it interacts with ciliary cargo

Klink, Björn Udo; Zent, Eldar; Juneja, Puneet; Kuhlee, Anne; Raunser, Stefan; Wittinghofer, Alfred
Nov 2017
10.7554/eLife.27434
Cilia are small, antenna-like structures on the surface of eukaryotic cells that harbor a unique set of sensory proteins, including GPCRs and other membrane proteins. The transport of these proteins involves the BBSome, an eight-membered protein complex that is recruited to ciliary membranes by the G-protein Arl6. BBSome malfunction leads to Bardet-Biedl syndrome, a ciliopathy with severe consequences. Short ciliary targeting sequences (CTS) have been identified that trigger the transport of ciliary proteins. However, mechanistic studies that relate ciliary targeting to BBSome binding are missing. Here we used heterologously expressed BBSome subcomplexes to analyze the complex architecture and to investigate the binding of GPCRs and other receptors to the BBSome. A stable heterohexameric complex was identified that binds to GPCRs with interactions that only partially overlap with previously described CTS, indicating a more complex recognition than anticipated. Arl6•GTP does not affect these interactions, suggesting no direct involvement in cargo loading/unloading.

Antibody fingerprints in lyme disease deciphered with high density peptide arrays

Weber, Laura K.; Isse, Awale; Rentschler, Simone; Kneusel, Richard E.; Palermo, Andrea; Hubbuch, Jürgen; Nesterov-Mueller, Alexander; Breitling, Frank; Loeffler, Felix F.
Eng. Life Sci..
Oct 2017
10.1002/elsc.201700062
Lyme disease is the most common tick-borne infectious disease in Europe and North America. Previous studies discovered the immunogenic role of a surface-exposed lipoprotein (VlsE) of Borreliella burgdorferi. We employed high density peptide arrays to investigate the antibody response to the VlsE protein in VlsE-positive patients by mapping the protein as overlapping peptides and subsequent in-depth epitope substitution analyses. These investigations led to the identification of antibody fingerprints represented by a number of key residues that are indispensable for the binding of the respective antibody. This approach allows us to compare the antibody specificities of different patients to the resolution of single amino acids. Our study revealed that the sera of VlsE-positive patients recognize different epitopes on the protein. Remarkably, in those cases where the same epitope is targeted, the antibody fingerprint is almost identical. Furthermore, we could correlate two fingerprints with human autoantigens and an Epstein-Barr virus epitope; yet, the link to autoimmune disorders seems unlikely and must be investigated in further studies. The other three fingerprints are much more specific for B. burgdorferi. Since antibody fingerprints of longer sequences have proven to be highly disease specific, our findings suggest that the fingerprints could function as diagnostic markers that can reduce false positive test results.

Identification of Streptococcus cristatus peptides that repress expression of virulence genes in Porphyromonas gingivalis

Ho, Meng-Hsuan; Lamont, Richard J.; Xie, Hua
Sci Rep.
May 2017
10.1038/s41598-017-01551-4
Dental plaque is a complex multispecies biofilm, and is a direct precursor of periodontal disease. The virulence of periodontal pathogens, such as Porphyromonas gingivalis, is expressed in the context of this polymicrobial community. Previously, we reported an antagonistic relationship between Streptococcus cristatus and P. gingivalis, and identified arginine deiminase (ArcA) of S. cristatus as the signaling molecule to which P. gingivalis responds by repressing the expression and production of FimA protein. Here we demonstrate that direct interaction between P. gingivalis and S. cristatus is necessary for the cell-cell communication. Two surface proteins of P. gingivalis, PGN_0294 and PGN_0806, were found to interact with S. cristatus ArcA. Using a peptide array analysis, we identified several P. gingivalis-binding sites of ArcA, which led to the discovery of an 11-mer peptide with the native sequence of ArcA that repressed expression of fimbriae and of gingipains. These data indicate that a functional motif of ArcA is sufficient to selectively alter virulence gene expression in P. gingivalis, and PGN_0294 and PGN_0806 may serve as receptors for ArcA. Our findings provide a molecular basis for future rational design of agents that interfere with the initiation and formation of a P. gingivalis-induced pathogenic community.

Peptide array functionalization via the Ugi four-component reaction

Ridder, B.; Mattes, D. S.; Nesterov-Mueller, A.; Breitling, F.; Meier, M. A. R.
Chem. Commun..
May 2017
10.1039/C7CC01945A
The Ugi four-component reaction was investigated as a tool for the functionalization of peptide arrays via post-synthetic side-chain modification, mimicking post-translational processes. Additionally, as a proof of concept for the synthesis of peptidomimetics on arrays, the integration of an Ugi unit into a growing peptide chain was demonstrated.

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