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

Diese Arbeit wurde zum Teil vom Van Andel Research Institute und einem Forschungsstipendium der National Institutes of Health (CA181343) an SBR unterstützt

Die Autoren haben nichts zu offenbaren.

Die Zuverlässigkeit der Antikörper in biomedizinischen Forschungsanwendungen liegt im Mittelpunkt 46 , 47 . Dies trifft besonders für die Chromatin-Biochemie zu, da die Position von Antikörpern als Schlüsselinstrumente für die Mehrheit der Techniken zur Charakterisierung der Häufigkeit und Verteilung von Histon-PTMs entwickelt wurde. Das hier vorgestellte Protokoll beschreibt eine optimierte Pipeline für die Konstruktion, Herstellung und Verwendung von P...

Genomische DNA ist elegant im eukaryotischen Zellkern mit Histonproteinen verpackt, um Chromatin zu bilden. Die sich wiederholende Untereinheit von Chromatin ist das Nukleosom, das aus 147 Basenpaaren von DNA besteht, die um einen oktameren Kern von Histonproteinen - H2A, H2B, H3 und H4 1 gewickelt sind. Chromatin ist weitgehend in lose gepackten Euchromatin und fest verpackten Heterochromatin-Domänen organisiert. Der Grad der Chromatin-Verdichtung regelt das Ausmaß, in dem Protein-Maschineries auf die zugrunde liegende DNA zugreifen können, um fundamentale DNA-templated Prozesse wie Replikation, Transkription und Reparatur durchzuführen. Schlüsselregulatoren der Genomzugänglichkeit im Rahmen von Chromatin sind PTMs auf dem unstrukturierten Schwanz und Kerndomänen der Histonproteine 2 , 3 . Histone-PTMs funktionieren direkt durch die Beeinflussung der Struktur von Chromatin 4 und indirekt durchH die Rekrutierung von Leserproteinen und deren assoziierten makromolekularen Komplexen, die Chromatin-Remodeling-, Enzym- und Gerüstaktivitäten haben 5 . Studien der Histon-PTM-Funktion in den vergangenen zwei Jahrzehnten überwiegend vorschlagen, dass diese Markierungen spielen Schlüsselrollen bei der Regulierung der Zelle Schicksal, organismen Entwicklung und Krankheit Einleitung / Progression. Angetrieben durch Fortschritte in der Massenspektrometrie-basierten Proteomtechnologie wurden mehr als 20 einzigartige Histon-PTMs auf mehr als 80 verschiedenen Histonresten entdeckt 6 . Bemerkenswert ist, dass diese Modifikationen häufig in Kombinationen auftreten und im Einklang mit der &quot;Histon-Code&quot; -Hypothese sind, zeigen zahlreiche Studien, dass Leserproteine ​​auf diskrete Bereiche des Chromatins durch Erkennung spezifischer Kombinationen von Histon-PTMs 7 , 8 , 9 gerichtet sind . Eine zentrale Herausforderung, die vorwärts geht, wird es sein, dem grEine Liste von Histon-PTMs und zu bestimmen, wie spezifische Kombinationen von Histon-PTMs die mit Chromatin verbundenen dynamischen Funktionen orchestrieren. Antikörper sind die Lynchpin-Reagenzien für den Nachweis von Histon-PTMs. Als solche wurden mehr als 1.000 Histon-PTM-spezifische Antikörper kommerziell für den Einsatz in der Chromatin-Biochemie-Forschung entwickelt. Mit der rasanten Entwicklung der DNA-Sequenzierungstechnologie mit hohem Durchsatz werden diese Reagenzien ausgiebig von einzelnen Forschern und großflächigen epigenomischen &quot;Roadmap&quot; -Initiativen ( zB ENCODE und BLUEPRINT) in ChIP-seq (Chromatin-Immunpräzipitation gepaart mit Sequenzierung der nächsten Generation) eingesetzt ) Pipelines zur Erzeugung hochauflösender räumlicher Karten der Histon-PTM-Verteilung genomweit 10 , 11 . Allerdings haben jüngste Studien gezeigt, dass die Spezifität von Histon-PTM-Antikörpern sehr variabel sein kann und dass diese Reagenzien unf<html> Austretende Eigenschaften wie z. B. Off-Target-Epitop-Erkennung, starker positiver und negativer Einfluss durch benachbarte PTMs und Schwierigkeiten, die Modifikationsreihenfolge an einem bestimmten Rest ( z. B. Mono-, Di- oder Tri-Methyllysin) 12 , 13 , 14 , 15 zu unterscheiden , 16 , 17 , 18 Daher ist eine rigorose Qualitätskontrolle von Histon-PTM-spezifischen Antikörperreagenzien notwendig, um die mit diesen wertvollen Reagenzien erzeugten Daten genau zu interpretieren. Die Microarray-Technologie ermöglicht die gleichzeitige Abfrage von Tausenden von makromolekularen Wechselwirkungen in einem hochdurchsatz-, reproduzierbaren und miniaturisierten Format. Aus diesem Grund wurde eine Vielzahl von Microarray-Plattformen geschaffen, um die Protein-DNA 19 zu analysieren ,„&gt; 20, Protein-Protein - 21 und Protein-Peptid - Wechselwirkungen 22. In der Tat, Histon - Peptidchips haben als informative Plattform zur Entdeckung für Chromatin - Biochemie Forschung entstanden, Hochdurchsatz - Profilierung der Autoren, Radiergummis ermöglicht, und die Leser von Histon PTM 15 , 23 , 24 und auch für die Analyse der Histon-Antikörper-Spezifität 17 , 25. Über ihre Anwendung in der Chromatin- und Epigenetikforschung hinaus haben Histon-Peptid-Arrays potentielle Nutzen als diagnostischer / prognostischer Test für systemischen Lupus erythematodes und andere Autoimmunkrankheiten, bei denen Anti- Chromatin-Autoantikörper werden erzeugt 26 , 27 . Hier beschreiben wir eine integrierte Pipeline, die wir für die Konstruktion, Fertigung und Ausführung entwickelt habenRindende Histon-Peptid-Mikroarrays zur Erzeugung von Spezifitätsprofilen für Antikörper, die Histone und ihre PTMs erkennen. Die Pipeline wird von ArrayNinja erleichtert, ein Open-Source, interaktive Software - Anwendung , die wir vor kurzem entwickelt, die die Design- und Analysestufen Microarray - Experimente 28 integriert. ArrayNinja funktioniert am besten in Google Chrome. Kurz gesagt wird ein Roboter-Kontakt-Mikroarray-Drucker verwendet, um eine Bibliothek von Biotin-konjugierten Histon-Peptiden an definierten Positionen auf Streptavidin-beschichteten Glasmikroskop-Objektträgern abzuscheiden. Arrays können dann in einem kompetitiven und parallelen Assayformat verwendet werden, um Antikörper-Epitop-Wechselwirkungen abzufragen ( Abbildung 1 ). Die Peptidbibliothek besteht aus Hunderten von einzigartigen synthetischen Peptiden, die PTMs (Lysinacetylierung, Lysin / Arginin-Methylierung und Serin / Threonin-Phosphorylierung) allein und in relevanten Kombinationen, die weitgehend von Proteomik-Datensätzen abgeleitet sind, beherbergen. Methoden zur Peptidsynthese und Validierung<html> Sind an anderer Stelle bekannt 23 . Daten, die aus unseren laufenden Histon-PTM-Antikörper-Screening-Bemühungen unter Verwendung dieser Array-Plattform generiert werden, werden auf einer öffentlichen Webressource, der Histone Antibody Specificity Database (www.histoneantibodies.com) archiviert. Bemerkenswerterweise wurden auch Histon-Peptid-Mikroarrays, die mit Variationen dieses Protokolls hergestellt wurden, ausgiebig verwendet, um die Aktivität der Histon-PTM-Leserdomänen 8 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 zu charakterisieren und in jüngerer Zeit Histon zu profilieren PTM-Schriftsteller und Radiergummi-Aktivitäten 24 . /files/ftp_upload/55912/55912fig1.jpg &quot;/&gt; Abbildung 1: Karikatur Darstellung der schrittweisen Vorgehensweise zur Antikörper-Screening auf einem Histon-Peptid-Mikroarray. Biotinylierte Histon-Peptide, die definierte posttranslationale Modifikationen (rote und blaue Kreise) beherbergen, werden mit Biotin-Fluorescein auf Streptavidin-beschichtetem Glas co-gedruckt. Positive Wechselwirkungen werden als rote Fluoreszenz visualisiert. <a href="http://ecsource.jove.com/files/ftp_upload/55912/55912fig1large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</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

Posttranslationale Modifikationen (PTMs) auf Histonproteinen sind weitgehend untersucht für ihre Rollen bei der Regulierung der Chromatinstruktur und der Genexpression. Die Massenproduktion und -verteilung von Antikörpern, die spezifisch für Histon-PTMs sind, hat die Forschung auf diesen Markierungen stark erleichtert. Als Histon-PTM-Antikörper sind Schlüsselreagenzien für viele Chromatin-Biochemie-Anwendungen, eine rigorose Analyse der Antikörperspezifität ist notwendig für eine genaue Dateninterpretation und fortgesetzte Fortschritte auf dem Gebiet. Dieses Protokoll beschreibt eine integrierte Pipeline für die Konstruktion, Herstellung und Verwendung von Peptid-Mikroarrays zur Profilierung der Spezifität von Histon-Antikörpern. Die Design- und Analyseaspekte dieses Verfahrens werden durch ArrayNinja, ein Open-Source- und interaktives Softwarepaket, das wir vor kurzem entwickelt haben, um die Anpassung von Microarray-Druckformaten zu optimieren, erleichtert. Diese Pipeline wurde verwendet, um eine große Anzahl von kommerziell verfügbaren und weit verbreiteten Histon-PTM-Antibiotika zu screenenS und Daten aus diesen Experimenten sind frei verfügbar durch eine Online-und erweiterte Histone Antibody Specificity Database. Jenseits von Histonen kann die hier beschriebene allgemeine Methodik weitgehend auf die Analyse von PTM-spezifischen Antikörpern angewendet werden.

Dieses Protokoll wurde verwendet, um eine Peptid-Mikroarray-Plattform für die Analyse der Histon-PTM-Antikörperspezifität zu entwerfen und herzustellen. Das Array fragt eine Bibliothek von mehr als 300 einzigartigen Peptidmerkmalen (20 - 40 Reste in der Länge) ab, die viele der bekannten Kombinationen von PTMs darstellen, die auf Kern- und Variantenhistonproteinen gefunden wurden 38 . Diese Pipeline war ein Arbeitspferd für das Screening von vielen weit verbr...

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

Analyse der Histon-Antikörper-Spezifität mit Peptid-Microarrays

Dieses Manuskript beschreibt Methoden zur Anwendung der Peptid-Microarray-Technologie auf die Spezifitätsprofilierung von Antikörpern, die Histone und ihre posttranslationalen Modifikationen erkennen.

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 Aufrufe.  Van Andel Research Institute.  Dieses Manuskript beschreibt Methoden zur Anwendung der Peptid-Microarray-Technologie auf die Spezifitätsprofilierung von Antikörpern, die Histone und ihre posttranslationalen Modifikationen erkennen. 

Serie: 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.

Personalisierte Peptidarrays für die Erkennung von HLA Alloantibodies in Organtransplantation

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

Forschung

Biochemie

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.

Immunpeptidomik: Isolierung von Maus- und humanen MHC-Klasse-I- und II-assoziierten Peptiden für die Massenspektrometrie-Analyse

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.

Peptid-Scanning-gestützte Identifizierung eines monoklonalen Antikörpers anerkannte Linear B-Zell-Epitop

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

Immunologie und Infektion

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-Virus spezifische diagnostische Epitop Entdeckung

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.

Chemisch-Antikörper blockiert Microarray für Multiplex-High-Throughput-Profiling von spezifischen Protein-Glykosylierung bei 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 ...

Biologie

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.

Analyse von HBV-spezifischen CD4-T-Zell-Reaktionen und Identifizierung von HLA-DR-beschränkten CD4-T-Zell-Epitopen auf Basis einer Peptidmatrix

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.

Ein hoher Durchsatz MHC II-Bindungsassay für die quantitative Analyse von Peptid-Epitope

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.

Ermittlung von Protein-Protein-Interaktionsstellen unter Verwendung von Peptid-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.

Spezifitätsanalyse von Protein Lysin Methyltransferasen Mit SPOT Peptidarrays

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.

Ein Peptid-Array-basierter Nachweis von Anti-Donor-HLA-Alloantikörpern bei Organempfängern

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