Name: analysis of histone antibody specificity with peptide microarrays
Uploaded: 2017-08-01T14:00:00.000+00:00
Duration: 9 min 47 s
Description: Watch this Scientific Journal Video about Analysis of Histone Antibody Specificity with Peptide Microarrays at JoVE.com

Este trabalho foi apoiado em parte pelo Van Andel Research Institute e uma bolsa de pesquisa dos Institutos Nacionais de Saúde (CA181343) para SBR

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A confiabilidade de anticorpos em aplicações de pesquisa biomédica é primordial 46 , 47 . Isto é especialmente verdadeiro na bioquímica da cromatina, dada a posição dos anticorpos como ferramentas-chave para a maioria das técnicas desenvolvidas para caracterizar a abundância e distribuição de PTM de histonas. O protocolo apresentado aqui detalha um pipeline otimizado para o projeto, fabricação e uso de microarrays de péptidos para analisar a espec...

O DNA genômico é embalado elegantemente dentro do núcleo da célula eucariótica com proteínas histonas para formar cromatina. A subunidade de repetição da cromatina é o nucleossoma, que consiste em 147 pares de bases de DNA envolvidos em torno de um núcleo octamerico de proteínas histonas - H2A, H2B, H3 e H4 1 . A cromatina é amplamente organizada em eucromatina vagamente compactada e domínios de heterocromatina bem embalados. O grau de compactação da cromatina regula a medida em que as máquinas de proteínas podem acessar o DNA subjacente para realizar processos fundamentais de DNA, tais como replicação, transcrição e reparo. Os principais reguladores da acessibilidade do genoma no contexto da cromatina são PTMs na cauda não estruturada e nos domínios principais das proteínas das histonas 2 , 3 . Os PTM Histone funcionam diretamente influenciando a estrutura da cromatina 4 e indiretamente através daH o recrutamento de proteínas leitoras e seus complexos macromoleculares associados que possuem atividades de remodelação da cromatina, enzima e andaimes 5 . Estudos sobre a função das histonas PTM nas últimas duas décadas sugerem esmagadoramente que essas marcas desempenham papéis fundamentais na regulação do destino das células, do desenvolvimento organizacional e da iniciação / progressão da doença. Alimentado pelos avanços na tecnologia proteômica baseada em espectrometria de massa, foram descobertas mais de 20 PTM de histonas únicas em mais de 80 resíduos de histonas distintas 6 . Notavelmente, essas modificações geralmente ocorrem em combinações e consistentes com a hipótese do &quot;código histônico&quot;, numerosos estudos sugerem que as proteínas leitoras são direcionadas para regiões discretas de cromatina através do reconhecimento de combinações específicas de histonas PTM 7 , 8 , 9 . Um desafio chave para avançar será atribuir funções ao grDevido a lista de PTM de histonas e para determinar como combinações específicas de PTM de histonas orquestra as funções dinâmicas associadas à cromatina. Os anticorpos são os reagentes lynchpin para a detecção de PTM de histonas. Como tal, mais de 1.000 anticorpos específicos de PTM de histonas foram comercialmente desenvolvidos para uso em pesquisa de bioquímica de cromatina. Com o rápido desenvolvimento da tecnologia de sequenciação de DNA de alto rendimento, esses reagentes estão sendo amplamente utilizados por pesquisadores individuais e iniciativas de &quot;roteiro&quot; de epigenômica em larga escala ( por exemplo , ENCODE e BLUEPRINT) em ChIP-seq (imunoprecipitação de cromatina, juntamente com sequenciação da próxima geração ) Pipelines para gerar mapas espaciais de alta resolução da distribuição de histonas PTM em todo o genoma 10 , 11 . No entanto, estudos recentes mostraram que a especificidade dos anticorpos PTM das histonas pode ser altamente variável e que esses reagentes exibem falta<html> Propriedades avoráveis, como o reconhecimento do epitopo fora do alvo, forte influência positiva e negativa dos PTM vizinhos e dificuldade em discriminar a ordem de modificação sobre um resíduo particular ( por exemplo , mono-, di- ou trimetilisina) 12 , 13 , 14 , 15 , 16, 17, 18. Portanto, o controle de qualidade rigoroso dos reagentes de anticorpos específicos de PTM de histonas é necessário para interpretar com precisão os dados gerados com esses reagentes valiosos. A tecnologia Microarray permite a interrogação simultânea de milhares de interações macromoleculares em um formato de alto rendimento, reprodutível e miniaturizado. Por esta razão, uma variedade de plataformas de microarray foram criadas para analisar proteína-DNA 19 ,&quot;, 20, proteína-proteína 21 e interações proteína-péptido 22. Na verdade, as microarrays de péptidos de histonas emergiram como uma plataforma de descoberta informativa para pesquisa de bioquímica de cromatina, permitindo o perfil de alto rendimento dos escritores, borrachas e leitores de histonas PTMs 15 , 23 , 24 e também para a análise da especificidade do anticorpo das histonas 17 , 25. Além da sua aplicação na pesquisa de cromatina e epigenética, os arrays de péptidos de histona possuem utilidade potencial como teste diagnóstico / prognóstico para lúpus eritematoso sistêmico e outras doenças autoimunes em que os anticorpos anti- Os autoanticorpos da cromatina são gerados 26 , 27 . Aqui, descrevemos um pipeline integrado que desenvolvemos para projetar, fabricar e queGerando microarrays de péptidos de histona para gerar perfis de especificidade para anticorpos que reconhecem histonas e seus PTMs. O pipeline é facilitado pela ArrayNinja, uma aplicação de software de código aberto e interativa que desenvolvemos recentemente, que integra os estágios de design e análise das experiências de microarrays 28 . ArrayNinja funciona melhor no Google Chrome. Resumidamente, uma impressora de microarray de contato robotizado é usada para depositar uma biblioteca de péptidos de histona conjugados com biotina em posições definidas em lâminas de microscópio de vidro revestidas com estreptavidina. As matrizes podem então ser usadas em um formato de ensaio competitivo e paralelo para interrogar interações anticorpo-epítopo ( Figura 1 ). A biblioteca de péptidos consiste em centenas de péptidos sintéticos únicos que abrigam PTMs (acetilação de lisina, metilação de lisina / arginina e fosforilação de serina / treonina) e em combinações relevantes em grande parte derivadas de conjuntos de dados proteômicos. Métodos para síntese e validação de péptidos<html> São detalhados em outro lugar 23 . Os dados gerados a partir dos nossos atuais esforços de rastreamento de anticorpos de PTM de histonas utilizando esta plataforma de matriz são arquivados em um recurso público da Web, a Histone Antibody Specificity Database (www.histoneantibodies.com). Notavelmente, as microarrays de péptidos de histonas fabricadas com variações deste protocolo também foram utilizadas amplamente para caracterizar a atividade dos domínios de leitor PTM de histona 8 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 e, mais recentemente, para histona de perfil Atividades de gravador e borracha PTM 24 . /files/ftp_upload/55912/55912fig1.jpg &quot;/&gt; Figura 1: Desenho dos desenhos animados do procedimento Stepwise para triagem de anticorpos em um microarray de péptido de histona. Os péptidos de histona biotinilados que abrigam modificações pós-translacionais definidas (círculos vermelho e azul) são co-impressos com biotina-fluoresceína em vidro revestido com estreptavidina. As interações positivas são visualizadas como fluorescência vermelha. <a href="http://ecsource.jove.com/files/ftp_upload/55912/55912fig1large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>

<table><tbody><tr><td>Printing Buffer</td><td>ArrayIt</td><td>PPB</td><td></td></tr><tr><td>BSA</td><td>Omnipure</td><td>2390</td><td></td></tr><tr><td>Streptavidin-coated glass microscope slides</td><td>Greiner Bio-one</td><td>439003-25</td><td></td></tr><tr><td>polypropylene 384 well plate</td><td>Greiner Bio-one</td><td>784201</td><td></td></tr><tr><td>Biotin-fluorescein</td><td>Sigma</td><td>53608</td><td></td></tr><tr><td>contact microarray printer</td><td>Aushon</td><td>2470</td><td>Aushon 2470 Microarray Printer</td></tr><tr><td>contact microarray printer</td><td>Gene Machines</td><td>OmniGrid 100</td><td>OmniGrid Microarray Printer</td></tr><tr><td>PBS</td><td>Invitrogen</td><td>14190</td><td></td></tr><tr><td>Blocking Buffer</td><td>ArrayIt</td><td>SBB</td><td></td></tr><tr><td>Hydrophobic wax pen</td><td>Vector Labs</td><td>H-4000</td><td>ImmEdge Hydrophobic Barrier PAP Pen</td></tr><tr><td>Silicon Gasket</td><td>Grace Bio-labs</td><td>622511</td><td></td></tr><tr><td>Hybridization Vessel</td><td>Thermo Scientific</td><td>267061</td><td>or similar vessel</td></tr><tr><td>Fluorescent-dye conjugated secondary antibody</td><td>Life Technologies</td><td>A-21244</td><td>Alexa Fluor 647 (anti-rabbit)</td></tr><tr><td>Fluorescent-dye conjugated secondary antibody</td><td>Life Technologies</td><td>A-21235</td><td>Alexa Fluor 647 (anti-mouse)</td></tr><tr><td>Wax Imprinter</td><td>ArrayIt</td><td>MSI48</td><td></td></tr><tr><td>Tween-20</td><td>Omnipure</td><td>9490</td><td></td></tr><tr><td>Microarray Scanner</td><td>Innopsys</td><td>InnoScan 1100AL</td><td>or equivalent microarray scanner</td></tr><tr><td>EipTitan Histone Peptide Microarray</td><td>Epicypher</td><td>112001</td><td></td></tr><tr><td>AbSurance Pro Histone Peptide Microarray</td><td>Millipore</td><td>16668</td><td></td></tr><tr><td>MODified Histone Peptide Array</td><td>Active Motif</td><td>13001</td><td></td></tr><tr><td>Histone Code Peptide Microarrays</td><td>JPT</td><td>His_MA_01</td><td></td></tr><tr><td>Wax</td><td>Royal Oak</td><td>GulfWax</td><td>for wax imprinter</td></tr><tr><td>Humidified Microarray Slide Hybridization Chamber</td><td>VWR</td><td>97000-284</td><td></td></tr><tr><td>High throughput microscope slide washing chamber</td><td>ArrayIt</td><td>HTW</td><td></td></tr><tr><td>Microscope slide centrifuge</td><td>VWR</td><td>93000-204</td><td></td></tr><tr><td>Antibody 1</td><td>Abcam</td><td>8898</td><td></td></tr><tr><td>Antibody 2</td><td>Millipore</td><td>07-473</td><td></td></tr><tr><td>Biotinylated histone peptide</td><td>EpiCypher</td><td>12-0001</td><td>Example peptide. Similar peptides with various modifications are available from several commercial sources.</td></tr><tr><td>ImageMagick</td><td></td><td></td><td>https://www.imagemagick.org/script/index.php</td></tr><tr><td>ArrayNinja</td><td></td><td></td><td>https://rothbartlab.vai.org/tools/</td></tr></tbody></table>

analysis of histone antibody specificity with peptide microarrays

As modificações pós-translacionais (PTMs) nas proteínas das histonas são amplamente estudadas quanto aos seus papéis na regulação da estrutura da cromatina e da expressão gênica. A produção em massa e a distribuição de anticorpos específicos para as PTM das histonas facilitaram grandemente a pesquisa nessas marcas. Como os anticorpos PTM das histonas são reagentes-chave para muitas aplicações de bioquímica de cromatina, a análise rigorosa da especificidade do anticorpo é necessária para a interpretação precisa dos dados e o progresso contínuo no campo. Este protocolo descreve uma tubagem integrada para o projeto, fabricação e uso de microarrays de péptidos para perfilar a especificidade de anticorpos de histonas. Os aspectos de design e análise deste procedimento são facilitados pelo ArrayNinja, um pacote de software de código aberto e interativo desenvolvido recentemente para simplificar a personalização de formatos de impressão em microarray. Este pipeline foi usado para exibir um grande número de antibióticos de histona PTM, amplamente utilizados e comercializados.S, e os dados gerados a partir dessas experiências estão disponíveis gratuitamente através de um banco de dados de especificações de anticorpos de histona online e em expansão. Além das histonas, a metodologia geral aqui descrita pode ser aplicada amplamente à análise de anticorpos específicos de PTM.

Este protocolo tem sido usado para projetar e fabricar uma plataforma de microarrays de péptidos para a análise da especificidade de anticorpos de PTM das histonas. A matriz consulta uma biblioteca de mais de 300 características peptídicas únicas (20 a 40 resíduos de comprimento) representando muitas das combinações conhecidas de PTMs encontradas nas proteínas histonas núcleo e variante 38 . Este pipeline tem sido um campo de trabalho para a triagem de m...

Watch this Scientific Journal Video about Analysis of Histone Antibody Specificity with Peptide Microarrays at JoVE.com

Analysis of Histone Antibody Specificity with Peptide Microarrays

Análise da especificidade de anticorpo com histona com microarrays de péptidos

Este manuscrito descreve métodos para a aplicação de tecnologia de microarrays de péptidos para o perfil específico de anticorpos que reconhecem histonas e suas modificações pós-tradução.

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

analysis-of-histone-antibody-specificity-with-peptide-microarrays

Research

JoVE Journal

Biochemistry

39.9K visualizações.  Van Andel Research Institute.  Este manuscrito descreve métodos para a aplicação de tecnologia de microarrays de péptidos para o perfil específico de anticorpos que reconhecem histonas e suas modificações pós-tradução. 

Vídeo: Analysis of Histone Antibody Specificity with Peptide Microarrays

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera. However, in humans there exist a far greater variety of HLA types, with no two individuals other than identical twins who can share the same combination of HLA sequences. While advanced technologies for HLA typing and direct sequencing can precisely capture any mismatches in DNA sequence between a donor's and recipient's HLA, the SAB assay, due to its limited variety in sequence representation, is unable to precisely detect alloantibodies specifically against the donor HLA mismatches. We sought to develop a complementary method using a different technology to detect and characterize anti-donor HLA antibodies on a personalized basis. The screening tool is a custom peptide array of donor HLA-derived sequences for probing post-transplant sera of the organ recipient to assess the risk for antibody-mediated rejection. On a single array for one donor-recipient pair, up to 600 unique peptides are made based on the donor's HLA protein sequences, each peptide carrying at least one mismatched residue in a 15-amino acid sequence. In our pilot experiments to compare antigen patterns for pre- and post-transplant sera on these arrays, we were able to detect anti-HLA signals with the resolution that also allowed us to pinpoint the immune epitopes involved. These personalized antigen arrays allow high-resolution detection of donor-specific HLA epitopes in organ transplantation.

Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients.

Personalizado do Peptide matrizes para a deteção de aloanticorpos HLA no transplante de órgãos

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

Bioquímica

Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science of investigating the composition of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS) technology platforms. Among all the steps in an MS-based immunopeptidomics workflow, sample preparation is critically important for capturing high-quality data of therapeutic relevance. Here, step-by-step instructions are described to isolate MHC class I and II-associated peptides by immunoaffinity purification from quality control samples, from mouse (EL4 and A20), and human (JY) cell lines more specifically. The various reagents and specific antibodies are thoroughly described to isolate MHC-associated peptides from these cell lines, including the steps to verify the beads-binding efficiency of the antibody and the elution efficiency of the MHC-peptide complexes from the beads. The protocol can be used to establish and standardize an immunopeptidomics workflow, as well as to benchmark new protocols. Moreover, the protocol represents a great starting point for any non-experts in addition to foster the intra- and inter-laboratory reproducibility of the sample preparation procedure in immunopeptidomics.

Here, we present a protocol for the purification of MHC class I and class II peptide complexes from mouse and human cell lines providing high-quality immunopeptidomics data. The protocol focuses on sample preparation using commercially available antibodies.

Imunopeptidomics: Isolamento de Peptídeos MHS de classe I e II associados para análise de espectrometria de massa

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science ...

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

Identificação de um epítopo de célula B linear Monoclonal Antibody-péptido reconhecido Digitalização-assistida

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

Imunologia e Infecção

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

Zika vírus específico diagnóstico Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

Microarray de anticorpos quimicamente bloqueada para Multiplexed perfil de alta capacidade de glicosilação proteína específica em amostras complexas

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Biologia

Analysis of HBV-Specific CD4 T-cell Responses and Identification of HLA-DR-Restricted CD4 T-Cell Epitopes Based on a Peptide Matrix

CD4 T cells play important roles in the pathogenesis of chronic hepatitis B. As a versatile cell population, CD4 T cells have been classified as distinct functional subsets based on the cytokines they secreted: for example, IFN-&#947; for CD4 T helper 1 cells, IL-4 and IL-13 for CD4 T helper 2 cells, IL-21 for CD4 T follicular helper cells, and IL-17 for CD4 T helper 17 cells. Analysis of hepatitis B virus (HBV)-specific CD4 T cells based on cytokine secretion after HBV-derived peptides stimulation could provide information not only about the magnitude of HBV-specific CD4 T-cell response but also about the functional subsets of HBV-specific CD4 T cells. Novel approaches, such as transcriptomics and metabolomics analysis, could provide more detailed functional information about HBV-specific CD4 T cells. These approaches usually require isolation of viable HBV-specific CD4 T cells based on peptide-major histocompatibility complex-II multimers, while currently the information about HBV-specific CD4 T-cell epitopes is limited. Based on an HBV-derived peptide matrix, a method has been developed to evaluate HBV-specific CD4 T-cell responses and identify HBV-specific CD4 T-cell epitopes simultaneously using peripheral blood mononuclear cells samples from chronic HBV infection patients.

Based on a hepatitis B virus (HBV)-derived peptide matrix, HBV-specific CD4 T-cell responses could be evaluated in parallel with identification of HBV-specific CD4 T-cell epitopes.

Análise das respostas e identificação de epítopos de células T T específicas do HBV com base em uma matriz de peptídeos

CD4 T cells play important roles in the pathogenesis of chronic hepatitis B. As a versatile cell population, CD4 T cells have been classified as distinct ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Um Ensaio de Ligação de Alto Rendimento II do MHC para análise quantitativa de Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

A identificação da proteína-proteína Locais de interacção Usando Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

Especificidade Análise de Proteína Lisina Metiltransferases Usando SPOT Peptide Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays ...

A Peptide Array-Based Detection of Anti-donor HLA Alloantibodies in Organ Recipients

Source: Liu, P., et al. <a href="https://app.jove.com/v/56278">Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation.</a> J. Vis. Exp. (2017).
This video demonstrates an assay to detect anti-donor HLA alloantibodies in organ recipients. A peptide array is synthesized to represent potential epitopes for antibody-mediated organ rejection. The array is incubated with the recipient&#39;s serum, containing alloantibodies that bind to their target epitopes. Antibody binding is detected using chemiluminescence to identify donor-specific HLA epitopes that incite alloantibody reactivity.

Uma detecção baseada em matriz de peptídeos de aloanticorpos HLA anti-doadores em receptores de órgãos

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Design of Custom Array Layout and ...