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作者： Fabien Pamelard Gael Even Costin Apostol Cristian Preda Clarisse Dhaenens Vronique Fafeur Rémi Desmet Oleg Melnyk

peptide microarray technology requires bioinformatics and statistical tools to manage, store, and analyze the large amount of data produced. To address these needs, we developed a system called protein array software environment (PASE) that provides an integrated framework to manage and analyze microarray information from polypeptide chip technologies. less

关键词： Proteomics microarray peptide microarray Protein Array Nucleic Acid microarray Statistical Calculation Database Management System Data analysis peptide protein molecular interaction studies database management Web platform and statistical analyses

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HCV immunodominant peptide mapping reveals unique HLA-A*02-restricted signatures: insights for CD8⁺ T-cell-based vaccines and immunotherapies

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IMMUNOGENETICS 2025年第1期77卷 1-15页

作者： Correa-Dias, Laura Cardoso Lopes-Ribeiro, Agata Marques-Ferreira, Geovane Gomes-de-Pontes, Leticia Pereira-Santos, Thaiza Aline Reis, Erik Vinicius de Sousa Moraes, Thais de Fatima Silva Martins-Filho, Olindo Assis Barbosa-Stancioli, Edel Figueiredo da Fonseca, Flavio Guimaraes Coelho-dos-Reis, Jordana Grazziela Univ Fed Minas Gerais Lab Virol Basica & Aplicada Inst Ciencias Biol Ave Antonio Carlos6627-Pampulha BR-31270901 Belo Horizonte MG Brazil Fundacao Oswaldo Cruz Grp Integrado Pesquisas Biomarcadores Inst Rene Rachou Ave Augusto Lima 1715 BR-30190009 Belo Horizonte MG Brazil

Several barriers for the development of an HCV vaccine still exist, including the genetic diversity of the virus, and the shortage of assessable models for in vitro and in vivo assays. Therefore, in this study, HCV epitope mapping was performed for 59 polyprotein sequences from 7 HCV genotypes. Around 2,880 peptides were considered epitopes for CD8+ T cells. The peptide induction of cytokines from Th1 and/or Th2 axes of the cellular immune response was assessed, indicating a tendency for Th2 axis. In vitro evaluation was performed using peptide microarray and a recombinant HLA-A*02:01 molecule. A total of 615 peptides of high reactivity to HLA-A*02:01 were identified, with predominance of leucine and tryptophan residues, highlighting their importance for TCR-epitope binding and CD8+ T activation. Finally, HCV-derived peptide patterns restricted to HLA-A2*02:01 observed in this study provide important information for the development of a multi-epitope-based pan-genotypic vaccine against the virus.

关键词： Hepatitis C HCV Cellular immune response CD8(+ )T response HLA-A*02:01 peptide microarray

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Investigation of Immunoreactivity Profiles and Epitope Landscape in Divergent COVID-19 Trajectories and SARS-CoV-2 Variants

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JOURNAL OF PROTEOME RESEARCH 2025年第2期24卷 762-776页

作者： Bihani, Surbhi Ray, Arka Borishetty, Dhanush Tuckley, Chaitanya Salkar, Akanksha Acharjee, Arup Shrivastav, Prithviraj Shrivastav, Om Shastri, Jayanthi Agrawal, Sachee Duttagupta, Siddhartha Srivastava, Sanjeeva Indian Inst Technol Dept Biosci & Bioengn Mumbai 400076 India Indian Inst Technol Ctr Res Nanotechnol & Sci Mumbai 400076 India Univ Allahabad Fac Sci Dept Zool Mol Om Lab Prayagraj 211002 India St Ignatius Coll Lane Cove NSW 2066 Australia Kasturba Hosp Infect Dis Mumbai 400011 India Indian Inst Technol Dept Elect Engn Mumbai 400076 India

This study aimed to elucidate the complexity of the humoral immune response in COVID-19 patients with varying disease trajectories using a SARS-CoV-2 whole proteome peptide microarray chip. The microarray, containing 5347 peptides spanning the entire SARS-CoV-2 proteome and key variants of concern, was used to analyze IgG responses in 10 severe-to-recovered, 9 nonsevere-to-severe cases, and 10 control case (5 pre-pandemic and 5 SARS-CoV-2-negative) plasma samples. We identified 1151 IgG-reactive peptides corresponding to 647 epitopes, with 207 peptides being cross-reactive across 124 epitopes. Nonstructural protein 3 (nsp3) exhibited the highest number of total and unique epitopes, followed by the spike protein. nsp12 had the most number of cross-reactive epitopes. peptides from the spike protein and nsps 2, 3, 5, and 13 were notably associated with recovery. Additionally, specific mutations in SARS-CoV-2 variants were found to alter peptide immunoreactivity, with some mutations (e.g., G142D, L452R, and N501Y) enhancing and others (e.g., R190S and E484 K) reducing immune recognition. These findings have critical implications for the development of diagnostics, vaccines, and therapeutics. Understanding the distribution of epitopes and the impact of viral mutations on antigenicity provides insights into immune evasion mechanisms, informing strategies for controlling COVID-19 and future coronavirus outbreaks.

关键词： SARS-CoV-2 COVID-19 peptide microarray z-score Mann-Whitney U test Kruskal-Wallis test epitope IgG immuneresponse mutation cross-reactivity

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peptide microarrays with site-specifically immobilized synthetic peptides for antibody diagnostics

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SENSORS AND ACTUATORS B-CHEMICAL 2006年第2期113卷 655-663页

作者： Andresen, H Grötzinger, C Zarse, K Birringer, M Hessenius, C Kreuzer, OJ Ehrentreich-Förster, E Bier, FF Fraunhofer Inst Biomed Engn Dept Mol Bioanalyt & Bioelect D-14558 Potsdam Germany Peptides & Elephants GmbH D-14558 Potsdam Germany Charite Univ Med Berlin Dept Gastroenterol & Hepatol D-13353 Berlin Germany Univ Potsdam Inst Biochem & Biol Potsdam Germany

peptide microarrays bear the potential to discover molecular recognition events on protein level, particularly in the field of molecular immunology, in a manner and with an efficiency comparable to the performance of DNA microarrays. We developed a novel peptide microarray platform for the detection of antibodies in liquid samples. The system comprises site-specific solution phase coupling of biotinylated peptides to NeutrAvidin, localized microdispensing of peptide-NeutrAvidin conjugates onto activated glass slides and a fluorescence immuno sandwich assay format for antibody capture and detection. Our work includes synthetic peptides deduced from amino acid sequences of immunodominant linear epitopes, such as the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein and three domains of the Human coronavirus 229E polymerase polyprotein. We demonstrate that our method produces peptide arrays with excellent spot morphology which are capable of specific and sensitive detection of monoclonal antibodies from fluid samples. (c) 2005 Elsevier B.V. All rights reserved.

关键词： antibody diagnostics fluorescence immuno assay linear epitope peptide microarray site-specific immobilization

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peptide microarrays for detailed, high-throughput substrate identification, kinetic characterization, and inhibition studies on protein kinase A

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ANALYTICAL BIOCHEMISTRY 2009年第2期387卷 150-161页

作者： Hilhorst, Riet Houkes, Liesbeth van den Berg, Adrienne Ruijtenbeek, Rob PamGene Int BV NL-5200 BJ Shertogenbosch Netherlands

A microarray-based mix-and-measure, nonradioactive multiplex method with real-time detection was used for substrate identification, assay development, assay optimisation, and kinetic characterization of protein kinase A (PKA). The peptide arrays included either up to 140 serine/threonine-containing peptides or a concentration series of a smaller number of peptides. In comparison with existing singleplex assays, data quality was high, variation in assay conditions and reagent consumption were reduced considerably, and assay development could be accelerated because phosphorylation kinetics were monitored simultaneously on 4,12, or 96 arrays. PKA was shown to phosphorylate many peptides containing known PKA phosphorylation sites as well as some new substrates. The kinetic behavior of the enzyme and the mechanism of inhibition by AMP-PNP, staurosporin, and PKA inhibitor peptide on the peptide microarray correlated well with data from homogeneous assays. Using this multiplex setup, we showed that the kinetic parameters of PKA and the potency of PKA inhibitors can be affected by the sequence of the peptide substrate. The technology enables kinetic monitoring of kinase activity in a multiplex setting such as a cell or tissue lysate. Finally, this high-throughput method allows fast identification of peptide substrates for serine/threonine kinases that are still uncharacterized. (C) 2009 Elsevier Inc. All rights reserved.

关键词： peptide microarray PKA cAMP-dependent protein kinase A Substrate identification Enzymology Michaelis constant Kinetics Mechanism of action IC50 Staurosporin PKA inhibitor peptide AMP-PNP

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peptide microarrays for the characterization of antigenic regions of human chromogranin A

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PROTEOMICS 2005年第14期5卷 3600-3603页

作者： Chiari, M Cretich, M Corti, A Damin, F Pirri, G Longhi, R CNR ICRM I-20131 Milan Italy Ist Sci San Raffaele Dept Biol & Technol Res Immunobiotechnol Unit Milan Italy

microarraying peptides is a powerful proteomics technique for studying molecular recognition events. Since peptides have small molecular mass, they are not easily accessible when adsorbed onto solid supports. Moreover, peptides can lack a well-defined three-dimensional structure, and therefore a correct orientation is essential to promote the interaction with their target. In this work, we investigated the suitability as a peptide array substrate of a glass slide coated with a copolymer of N,N-dimethylacrylamide, N,N-acryloyloxysuccinimide, and [3-(methacryloyl-oxy)propyl]trimethoxysilyl. This polymeric surface was used as substrate for peptides in the characterization of linear antigenic sites of human chromogranin A, a useful tissue and serum marker for neuroendocrine tumors and a precursor of many biologically active peptides. The microarray support provided sufficient accessibility of the ligand, with no need for a spacer, as the polymer chains prevent interaction of immobilized peptides with substrate. In addition, the polymeric surface constitutes an aqueous micro-environment in which linear epitopes are freely exposed despite peptide random orientation. The results reported in this article are in accordance with those obtained in conventional ELISA assays using biotinylated and non-biotinylated peptides.

关键词： human chromogranin A peptide microarray polymer coating

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Chemoselective Strategies to peptide and Protein Bioprobes Immobilization on microarray Surfaces

Chemoselective Strategies to Peptide and Protein Bioprobes I...

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作者： Alessandro Gori Renato Longhi

Ordered and reproducible bioprobe immobilization onto sensor surfaces is a critical step in the development of reliable analytical devices. A growing awareness of the impact of the immobilization scheme on the consistency of the generated data is driving the demand for chemoselective approaches to immobilize biofunctional ligands, such as peptides, in a predetermined and uniform fashion. Herein, the most intriguing strategies to selective and oriented peptide immobilization are described and discussed. The aim of the current work is to provide the reader a general picture on recent advances made in this field, highlighting the potential associated with each chemoselective strategy. Case studies are described to provide illustrative examples, and cross-references to more topic-focused and exhaustive reviews are proposed throughout the text. less

关键词： peptide microarray Click Chemistry microarray Oriented immobilization Chemoselective reactions

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Evaluation of protein kinase activities of cell lysates using peptide microarrays based on surface plasmon resonance imaging

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ANALYTICAL BIOCHEMISTRY 2008年第2期375卷 223-231页

作者： Mori, Takeshi Inamori, Kazuki Inoue, Yusuke Han, Xiaoming Yamanouchi, Go Niidome, Takuro Katayama, Yoshiki Kyushu Univ Fac Engn Dept Appl Chem Fukuoka 8190395 Japan Kyushu Univ Ctr Future Chem Fukuoka 8190395 Japan Toyobo Co Ltd Biotechnol Frontier Project Fukui 9140047 Japan Kyushu Univ Grad Sch Syst Life Sci Fukuoka 8190395 Japan CREST Japan Sci & Technol Agcy Kawaguchi Saitama 3320012 Japan

We developed a peptide microarray based on surface plasmon resonance (SPR) imaging for monitoring protein kinase activities in cell lysates. The substrate peptides of kinases were tethered to the microarray surface modified with a self-assembled monolayer of an alkanethiol with triethylene glycol terminus to create a low nonspecific binding surface. The phosphorylation of the substrate peptides immobilized on the surface was detected with the following phosphate specific binders by amplifying SPR signals: anti-phosphotyrosine antibody for tyrosine kinases and Phos-tag biotin (a phosphate-specific ligand with biotin tag) for serine/threonine kinases. Using the microarray, 9 kinds of protein kinases were evaluated as a pattern of phosphorylation of 26 kinds of substrate peptides. The pattern was unique for each protein kinase. The microarray could be used to evaluate the inhibitory activities of kinase inhibitors. The microarray was applied successfully for kinase activity monitoring of cell lysates. The chemical stimuli responsive activity changes of protein kinases in cell lysates could also be monitored by the peptide microarray. Thus, the peptide microarray based on SPR imaging would be applicable to cell-based drug discovery, diagnosis using tissue lysates, and biochemical studies to reveal signal transduction pathways. (c) 2007 Elsevier Inc. All rights reserved.

关键词： peptide microarray kinase SPR cell lysate inhibitor drug stimulation

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Functional peptide microarrays for specific and sensitive antibody diagnostics

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PROTEOMICS 2006年第5期6卷 1376-1384页

作者： Andresen, H Grötzinger, C Zarse, K Kreuzer, OJ Ehrentreich-Förster, E Bier, FF Fraunhofer Inst Biomed Engn Dept Mol Bioanalyt & Bioelect D-14558 Potsdam Nuthetal Germany Peptides&elephants GmbH Potsdam Nuthetal Germany Charite Univ Med Berlin Dept Gastroenterol & Hepatol Berlin Germany Univ Potsdam Inst Biochem & Biol Potsdam Germany

peptide microarrays displaying biologically active small synthetic peptides in a high-density format provide an attractive technology to probe complex samples for the presence and/or function of protein analytes. We present a new approach for manufacturing functional peptide microarrays for molecular immune diagnostics. Our method relies on the efficiency of site-specific solution-phase coupling of biotinylated synthetic peptides to NeutrAvidin (NA) and localized microdispensing of peptide-NA-complexes onto activated glass surfaces. Antibodies are captured in a sandwich manner between surface immobilized peptide probes and fluorescence-labeled secondary antibodies. Our work includes a total of 54 peptides derived from immunodominant linear epitopes of the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein, and three domains of the Human coronavirus polymerase polyprotein and their cognate mAbs. By using spacer molecules of different type and length for NA-mediated peptide presentation, we show that the incorporation of a minimum spacer length is imperative for antibody binding, whereas the peptide immobilization direction has only secondary importance for antibody affinity and binding. We further demonstrate that the peptide array is capable of detecting low-picomolar concentrations of mAbs in buffered solutions and diluted human serum with high specificity.

关键词： antibody microspot immunoassay on-chip epitope mapping peptide microarray site-specific immobilization

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Development of peptide microarrays for epitope mapping of antibodies against the human TSH receptor

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JOURNAL OF IMMUNOLOGICAL METHODS 2006年第1-2期315卷 11-18页

作者： Andresen, Heiko Zarse, Kim Groetzinger, Carsten Hollidt, Joerg-M. Ehrentreich-Foerster, Eva Bier, Frank F. Kreuzer, Oliver J. Fraunhofer Inst Biomed Engn Dept Mol Bioanalyt & Bioelect D-14558 Potsdam Germany Charite Univ Med Berlin Dept Gastroenterol & Hepatol D-13353 Berlin Germany Invent Diagnost GMBH D-16761 Hennigsdorf Germany Univ Potsdam Inst Biochem & Biol Potsdam Germany Peptides & Elephants GMBH D-14558 Potsdam Germany

Accurate characterization of the antigen binding region of antibodies is of great value in many fields of research, assay development and clinical diagnostics. Up to now, there is an unmet clinical need to use antibodies as diagnostic markers for the prediction of both prognosis and therapeutic response. To this end, comprehensive but differentiated immunoassays need to be generated. We have developed a peptide microarray for the diagnosis and epitope mapping of anti-thyrotropin receptor antibodies. The primary sequence of the human thyrotropin receptor (hTSHR) was represented by a library of 251 synthetic peptides. The peptides were site-specifically immobilized in a two-step procedure first by coupling of biotinylated peptides to hydrazide-modified streptavidin and then utilizing a subsequent chemoselective reaction between the hydrazide linkers of the streptavidin and an aldehyde coated glass surface. The technology was used to map the epitopes of seven commercially available murine monoclonal antibodies specific for the human TSH receptor (mTSHRAb). A previously unknown epitope recognized by mTSHRAb 4Cl was identified at amino acids (AA) 379 through 384 and the epitope recognized by mTSHRAb A9 was also localized (AA 214-222). Previously identified epitopes recognized by mTSHRAbs 2C11 (AA 349-360), 28 (AA 34-39), 49 (AA 289-297), A7 (AA 406411) and A 10 (AA 34-39) were confirmed. The peptide microarray exhibited excellent performance in single and multiplex antibody analysis and high specificity. This technology may have potential as a multi-determinate in vitro diagnostic assay for the differential analysis of a heterogeneity of antibodies involved in the pathogenesis of autoimmune diseases. (c) 2006 Elsevier B.V. All rights reserved.

关键词： peptide microarray epitope mapping TSH receptor site-specific immobilization autoimmune disease muliplex immunoassay

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