Diese Arbeit wurde teilweise durch einen Research Career Development Award des Israel Cancer Research Fund unterstützt, ein Stipendium der israelischen Nanotechnologie-Initiative und der Helmsley Charitable Trust für einen Focal Technology Area auf Nanomedicines für personalisierte Theranostics (VP-K) und National Institute der Gesundheit gewähren R01GM076360 (zu XC).

<ol>
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Eine erfolgreiche Glycan-Microarray-Fertigung erfordert eine sorgfältige Planung und beinhaltet einige wichtige Schritte im Protokoll. Dazu gehören: (1) Planung der Block- und Plattenlayouts, die alle nachfolgenden Parameter definieren ( zB Distanzen, Abstand, Probenmenge und Druck); (2) Reinigung der Stifte und Sicherstellung der Pin-Integrität, die für die Kontrolle der Spot-Homogenität entscheidend ist; (3) Aufrechterhalten hoher Feuchtigkeit während des Druckens, entscheidend für die Vermeidung von P...

Sias sind saure Zucker, die Glykinketten auf Zelloberflächen-Glykoproteinen und Glykolipiden bei Wirbeltieren abdecken. Sia-Expression wird in Krebszellen 1 modifiziert und korreliert mit Progression und / oder Metastase 2 , 3 . Zwei der großen Sia-Formen bei Säugetieren sind Neu5Ac und seine hydroxylierte Form, Neu5Gc 2 . Menschen können Neu5Gc aufgrund einer spezifischen Inaktivierung des für das CMAH-Enzym kodierenden Gens nicht synthetisieren. Diese nicht-menschliche Sia enthält metabolisch in menschliche Zellen als &quot;Selbst&quot;, die aus diätetischen Neu5Gc-reichen Nahrungsmitteln ( zB rotem Fleisch) 4 , 5 stammen . Neu5Gc ist bei niedrigen Niveaus auf den Zelloberflächen von menschlichen Epithelien und Endothelien vorhanden, aber es sammelt sich besonders bei Karzinomen an. Neu5Gc wird vom humanen humoralen Immunsystem 2 , 6 als fremd anerkannt.Die antigene Komplexität von Neu5Gc-Glykane können auf mehreren Ebenen entstehen, einschließlich Neu5Gc Modifikation, Verknüpfung, zugrunde liegenden Glykane und Gerüste, und ihre Dichte, alle reflektiert durch die Komplexität der anti-Neu5Gc Antikörperantwort beim Menschen 6. Einige dieser Antikörper dienen als Karzinom-Biomarker und potenzielle Immuntherapeutika 7 . Das Aufkommen der chemoenzymatischen Synthese verschiedener Sialoglycane 8 ebnete den Weg für eine eingehendere Analyse solcher Antikörper, erleichtert durch den Einsatz der Glycan-Microarray-Technologie 9 , 10 . So sind mit der erleichterten Vorbereitung und Manipulation von großen Bibliotheken von natürlichen und synthetischen Kohlenhydraten Glycan-Mikroarrays zu einer leistungsfähigen Hochdurchsatz-Technologie geworden, um die Wechselwirkungen von Kohlenhydraten mit einer Vielzahl von Biomolekülen 10 , 11 , </sup&gt; 12 , 13 In einem Array-Format werden minimale Mengen an Materialien verwendet, und diese multivalente Anzeige von biologisch relevanten Glykanen ermöglicht die Untersuchung von Tausenden von Bindungsinteraktionen in einem einzigen Experiment. Wichtig ist, dass diese Technologie auch auf die Biomarker-Entdeckung und die Überwachung von Immunantworten in verschiedenen Proben angewendet werden kann 7 , 12 . Die erfolgreiche Glycan-Microarray-Herstellung erfordert die Berücksichtigung von drei wichtigen Aspekten: der Roboter-Typ, die Glycan-Konjugationschemie und die Detektionsoptik. Für die Druckinstrumentenbetrachtung stehen zwei Techniken zur Verfügung: Kontakt- und berührungslose Drucker. Im Kontaktdruck werden 1-48 Stahlstifte in eine Multi-Well-Quellplatte mit Glycan-Lösung getaucht und auf funktionalisierten Glasrutschen durch direktes Kontaktieren der Glas-Gleitfläche entdeckt. Die Lösungsmenge deAn die Folie geleitet wird, ist eine Funktion der anhaltenden Dauer auf der Gleitfläche. Normalerweise werden die Proben zuerst auf einem Glasblock vorgespannt (um homogene Flecken zu erreichen), bevor sie auf die Gleitfläche gedruckt werden. Bei berührungslosen Druckern ( z. B. dem piezoelektrischen Drucker) werden die Glykane aus einer Glaskapillare mit kontrollierten elektrischen Signalen bedruckt. Das elektrische Signal kann fein kalibriert werden, um ein präziseres Drucken im Verhältnis zum Kontaktdruck zu erreichen. Die Größe und Morphologie der Flecken sind auch relativ homogener. Ein zusätzlicher Vorteil ist das Recycling der Probe nach dem Druck auf die Quellplatte. Trotzdem ist der Hauptnachteil von piezoelektrischen Druckern die Druckspitzenbegrenzung (4 oder 8), was zu einer sehr langen Druckdauer führt, die besondere Aufmerksamkeit auf Gleitstabilität, Temperatur, Feuchtigkeit und Probenverdampfung erfordert. Der berührungslose Tintenstrahldrucker benötigt größere Probenvolumina 14 . <p clasS = &quot;jove_content&quot;&gt; Im Gegensatz zu den begrenzten verfügbaren Optionen für Druckmethoden ist die Glycan-Konjugationschemie eine komplexere Betrachtung, mit vielen Möglichkeiten zur Auswahl. Ausgewählte Immobilisierungschemie muss sowohl die aktiven Gruppen auf den Glykanen als auch die Schieberoberflächenreaktivität berücksichtigen. Die Glycane, die auf einer bestimmten Mikroarray-Oberfläche immobilisiert werden sollen, entweder synthetisch synthetisiert oder natürlich isoliert, benötigen alle eine identische reaktive Gruppe. Darüber hinaus müssen die Glykane rein und homogen sein. Auf der anderen Seite sollte die Immobilisierungsoberfläche und die Chemie Reproduzierbarkeit und zuverlässige Befestigungsdichte bieten. Mehrere Immobilisierungsmethoden wurden mit entweder kovalenten oder nicht-kovalenten (physikalischen Absorptions-) Bindungen 10 , 11 , 12 , 13 entwickelt . Für detaillierte Informationen über gedruckte Glycan-Microarray-Technologie für die UninitiativeD Ermittler, beziehen sich auf diese ausgezeichneten Bewertungen 13 , 15 . Wichtig ist, dass die jüngsten Mindestinformationen für eine Glycomics Experiment (MIRAGE) Initiative Leitlinien für die Probenvorbereitung 16 und für die Meldung von Daten aus Glycan-Microarray-Analysen 17 zur Verbesserung der Standards in diesem wachsenden Bereich beschrieben werden. Hier beschreiben wir ein detailliertes Protokoll für die Herstellung von Sialoglycan-Mikroarrays mit einem speziellen Kontakt-Nano-Drucker im 16-Well-Format. Jeder der Glycane hat ein primäres Amin, das ihre kovalente Verbindung zu epoxyaktivierten Glasrutschen vermittelt. Wir beschreiben auch die Entwicklung und Analyse einer Folie mit verschiedenen menschlichen Serenproben, Antikörpern und Sia-bindenden Pflanzenlektinen. Sialoglycan Microarray-Assays beinhalten mehrere wichtige Schritte, die Array-Herstellung, Verarbeitung, Entwicklung und Analyse beinhalten. Array-Fertigung erfordert die Planung der arRay-Layout, Vorbereitung der Glykane und Quellplatte, Programmierung der Nano-Drucker, und das Drucken der Folien. Anschließend werden die Folien verarbeitet, entwickelt und analysiert ( Abbildung 1 ).

<table><tbody><tr><td>Primary-amine containing sialoglycans</td><td>Glycohub, Inc., Davis, CA, USA (http://www.glycohub.com/services)</td><td>Contact info@glycohubusa.com for compound requests</td><td>Printed glycans</td></tr><tr><td>Monosodium phosphate monohydrate</td><td>Sigma</td><td>S9638</td><td>Printing buffer component</td></tr><tr><td>Disodium phosphate heptahydrate</td><td>Sigma</td><td>S9390</td><td>Printing buffer component</td></tr><tr><td>Phosphate buffered saline</td><td>Hy-Labs</td><td>BP-507/500D</td><td>Printing buffer/ incubation/washing buffer</td></tr><tr><td>Tris-base</td><td>Sigma</td><td>T1503</td><td>Slide blocking reagent</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G-7893</td><td>Printing buffer component</td></tr><tr><td>Ethanolamine</td><td>Thermo-Fisher Scientific</td><td>0700/08</td><td>Slide blocking reagent</td></tr><tr><td>Ovalbumin (Grade V)</td><td>Sigma</td><td>A5503</td><td>Slide Blocking protein</td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P7949</td><td>Slide washing detergent</td></tr><tr><td>Alexa 555-Hydrazide</td><td>Thermo-Fisher Scientific</td><td>A20501MP</td><td>Marker on array</td></tr><tr><td>ChromPure Human IgG, whole molecule</td><td>Jackson Immunoresearch</td><td>009-000-003</td><td>Printing component</td></tr><tr><td>Biotinylated- SNA</td><td>Vector Laboratories</td><td>B-1305</td><td>Plant Lectin &amp;ndash; binding Sia-alpha2&amp;ndash;6-linked</td></tr><tr><td>Biotinylated-MALII</td><td>Vector Laboratories</td><td>B-1265</td><td>Plant Lectin &amp;ndash; binding Sia-alpha2&amp;ndash;3-linked</td></tr><tr><td>Chicken-anti Neu5Gc IgY</td><td>BioLegend</td><td>146903</td><td>Primary detection</td></tr><tr><td>Cy3-Streptavidin</td><td>Jackson Immunoresearch</td><td>016-160-0848</td><td>Biotin binding</td></tr><tr><td>Cy3-anti Human IgG</td><td>Jackson Immunoresearch</td><td>109-165-088</td><td>Secondary detection against human IgG</td></tr><tr><td>Cy3-anti Chicken IgY</td><td>Jackson Immunoresearch</td><td>703-165-155</td><td>Secondary detection against chicken IgY</td></tr><tr><td>Human sera samples</td><td>Israeli Blood Bank</td><td></td><td>Primary detection</td></tr><tr><td>Compressed Nitrogen (Grade 5)</td><td></td><td></td><td>General dusting/drying tool</td></tr><tr><td>Epoxy-coated slides</td><td>Corning</td><td>40044</td><td>Slides</td></tr><tr><td>Epoxy-coated slides</td><td>PolyAn</td><td>2D 104-00-221</td><td>Slides. In this type of slides the surface is more hydrophobic (compared to Coring slides) therefore the glycans Print Buffer would need to be supplemented with 0.005% Tween-20 to obtain 100 &amp;micro;m size spots.</td></tr><tr><td>384-well microtiter plate</td><td>Genetix</td><td>2070</td><td>Printing plate</td></tr><tr><td>VWR lab marker</td><td>VWR</td><td>52877-310</td><td>Slide labeling</td></tr><tr><td>Staining Tube</td><td>ArrayIt</td><td>MST</td><td>Slide developing tool</td></tr><tr><td>Staining bath</td><td>VWR</td><td>25608-904</td><td>Slide developing tool</td></tr><tr><td>Slides glass holders</td><td>VWR</td><td>631-9321</td><td>Slide developing tool</td></tr><tr><td>GenePix Scanner</td><td>Molecular devices</td><td>4000B</td><td>Slide scanner</td></tr><tr><td>LM-60 NanoPrinter</td><td>ArrayIt</td><td>LM-60</td><td>Array printer</td></tr><tr><td>Pins</td><td>ArrayIt</td><td>946MP3</td><td>Printing pins</td></tr><tr><td>ProPlate Module</td><td>Grace Bio-Labs</td><td>P37004</td><td>Slide developing module</td></tr><tr><td>Distilled water</td><td>Bio-Lab</td><td>2321020500</td><td>Required for arrayer and humidifier</td></tr><tr><td>Electronic Multi Pippete, 8 Channel , volume range 2-125 &amp;mu;L</td><td>Thermo-Fisher Scientific (Matrix)</td><td>MA-2131 Impact2 Equalizer 384</td><td>Multi pippete for sample dispansing into 384-well plate</td></tr></tbody></table>

profiling anti-neu5gc igg in human sera with a sialoglycan microarray assay

Die Zellen werden mit einem Mantel von Kohlenhydrat-Ketten (Glykane) bedeckt, der gewöhnlich bei Krebs verändert wird und der Variationen in der Sialinsäure (Sia) -Expression beinhaltet. Dies sind saure Zucker, die ein 9-Kohlenstoff-Grundgerüst haben und diese Kappe Wirbeltiere Glykane auf Zelloberflächen. Zwei der wichtigsten Sia-Formen bei Säugetieren sind N- Acetylneuraminsäure (Neu5Ac) und ihre hydroxylierte Form, N- Glykolylneuraminsäure (Neu5Gc). Menschen können keine endogenen Neu5Gc aufgrund der Inaktivierung des Gens, das für Cytidin 5&#39;monophosphat-Neu5Ac (CMP-Neu5Ac) Hydroxylase (CMAH) kodiert, produzieren. Ausländische Neu5Gc wird von menschlichen Zellen durch den diätetischen Verzehr von rotem Fleisch und Molkerei erworben und erscheint anschließend auf diversen Glykanen auf der Zelloberfläche, die sich hauptsächlich auf Karzinomen ansammeln. Folglich haben die Menschen zirkulierende Anti-Neu5Gc-Antikörper, die vielfältige Rollen bei Krebs und anderen chronischen Entzündungs-vermittelten Erkrankungen spielen und potenzielle diagnostische und therapeutische Medikamente werdenRgets Hier beschreiben wir einen Hochdurchsatz-Sialoglycan-Microarray-Assay, um solche Anti-Neu5Gc-Antikörper in den menschlichen Seren zu beurteilen. Neu5Gc-haltige Glykane und deren abgestimmte Kontrollpaare (Neu5Ac-haltige Glykane) mit jeweils einem primären Amin-Amin sind kovalent an epoxidbeschichtete Glasscheiben gebunden. Wir veranschaulichen das Drucken von 56 Folien im 16-Well-Format mit einem speziellen Nano-Drucker, der bis zu 896 Arrays pro Druck erzeugen kann. Jede Folie kann verwendet werden, um 16 verschiedene menschliche Serenproben für die Bewertung von Anti-Neu5Gc-Antikörperspezifität, Intensität und Diversität zu screenen. Das Protokoll beschreibt die Komplexität dieses robusten Tools und bietet eine grundlegende Leitlinie für diejenigen, die die Reaktion auf Neu5Gc diätetische Kohlenhydrat-Antigen in verschiedenen klinischen Proben in einem Array-Format zu untersuchen.

Array Drucken, Entwicklung und Analyse: Das Drucken eines Sialoglycan-Mikroarrays mit mehreren Glycan-Proben und menschlichen IgG-STD-Kurven in 16 verschiedenen Blöcken erfordert eine sorgfältige Kalibrierung, um sicherzustellen, dass alle Proben in allen 16 Blöcken pro Folie und allen Folien im gleichen Drucklauf gleichmäßig wie möglich gedruckt werden. Daher sind mehrere Kalibrierungsexperimente erforderlich, bevor die sp...

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Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay

Profiling Anti-Neu5Gc IgG in menschlichen Seren mit einem Sialoglycan-Microarray-Assay

Ein Sialoglycan-Mikroarray-Assay kann verwendet werden, um Anti-Neu5Gc-Antikörper in menschlichen Seren zu bewerten, was es zu einem potentiellen Hochdurchsatz-Diagnosetest für Krebs und andere chronisch entzündungsvermittelte menschliche Krankheiten macht.

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) ...

profiling-anti-neu5gc-igg-human-sera-with-sialoglycan-microarray

Research

JoVE Journal

Behavior

9.4K Aufrufe.  Tel Aviv University.  Ein Sialoglycan-Mikroarray-Assay kann verwendet werden, um Anti-Neu5Gc-Antikörper in menschlichen Seren zu bewerten, was es zu einem potentiellen Hochdurchsatz-Diagnosetest für Krebs und andere chronisch entzündungsvermittelte menschliche Krankheiten macht. 

Serie: Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

Massenspektrometrische Analyse Glycosphingolipid Antigene

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

Forschung

Immunologie und Infektion

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise &#945;-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial &#946;-mannoside linkage. We further attached the N-glycans to the surface of an aluminum oxide-coated glass (ACG) slide to create a covalent mixed array for the analysis of hetero-ligand interaction with an HIV antibody. In particular, the binding behavior of a newly isolated HIV-1 broadly neutralizing antibody (bNAb), PG9, to the mixture of closely spaced Man5GlcNAc2 (Man5) and 2,6-di-sialylated bi-antennary complex type N-glycan (SCT) on an ACG array, opens a new avenue to guide the effective immunogen design for HIV vaccine development. In addition, our ACG array embodies a powerful tool to study other HIV antibodies for hetero-ligand binding behavior.

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated.

Chemo-enzymatische Synthese von N- Glykanen für Array-Entwicklung und HIV-Antikörper-Profiling

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are ...

Chemie

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and determine glycan alterations in diseases. Most commercially available biopharmaceutical proteins are glycoproteins. The efficacy of these drugs is affected by glycosylation patterns. Therefore, an in-depth characterization method for the N-glycans is necessary. Here, we present a comprehensive approach for qualitative and quantitative analysis of N-glycans using hydrophilic interaction liquid chromatography equipped with fluorescence detection and tandem mass spectrometry (HILIC-FLD-MS/MS). N-glycans were released from glycoproteins with a facile method and labeled by a procainamide fluorophore tag in the strategy. Subsequently, the procainamide labeled N-glycans were analyzed by a HILIC-FLD-MS/MS technique. In this approach, N-glycan structures were confirmed by the tandem mass spectrometric analysis, whereas fluorescence detection was used for the quantitative analysis. An application for data analysis of the detected N-glycan peaks is described in the study. This protocol can be applied to any glycoprotein extracted from various species.

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

N-glycan profiling of glycoproteins is essential for discovering novel biomarkers and understanding glycan functions in cellular events. Additionally, N-glycan analysis of protein biopharmaceuticals is very important for human use. In this current article, a high-throughput strategy for identifying and quantifying N-glycan structures was presented using the HILIC-FLD-MS/MS technique.

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and ...

Biochemie

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Ein miniaturisiertes Glycan Microarray-Test zur Beurteilung der Avidität und Spezifität von Influenza-A-Virus Hämagglutininarten

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing termini of oligo- and polysaccharides, as well as its unique chemical characteristics, sialic acid is involved in a multitude of different receptor-ligand interactions. By modifying the expression of sialic acid on the cell surface, sialic acid-dependent interactions will consequently be influenced. This can be helpful to investigate sialic acid-dependent interactions and has the potential to influence certain diseases in a beneficial way. Via metabolic glycoengineering (MGE), the expression of sialic acid on the cell surface can be modulated. Herein, cells, tissues, or even entire animals are treated with C2-modified derivatives of N-acetylmannosamine (ManNAc). These amino sugars act as sialic acid precursor molecules and therefore are metabolized to the corresponding sialic acid species and expressed on glycoconjugates. Applying this method produces intriguing effects on various biological processes. For example, it can drastically reduce&#160;the expression of polysialic acid (polySia) in treated neuronal cells and thus affects neuronal growth and differentiation. Here, we show the chemical synthesis of two of the most common C2-modified N-acylmannosamine derivatives, N-propionylmannosamine (ManNProp) as well as N-butanoylmannosamine (ManNBut), and further show how these non-natural amino sugars can be applied in cell culture experiments. The expression of modified sialic acid species is quantified by high performance liquid chromatography (HPLC) and further analyzed via mass spectrometry. The effects on polysialic acid expression are elucidated via Western blot using a commercially available polysialic acid antibody.

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid is a typical monosaccharide-unit found in glycoconjugates. It is involved in a plethora of molecular and cellular interactions. Here we present a method to modify cell surface sialic acid expression using metabolic glycoengineering with N-acetylmannosamine derivatives.

Metabolische Glykan der Sialinsäure Säure mit N-Acyl-Mannosamines geändert

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing ...

Biotechnik

<meta charset="utf8"></head><body>Automatisierte Lektin-basierte Glykanprofilierung mit Bead Array in a Single Tip (BIST)

Rapid Glyco-Qualitative Assessment of Recombinant Proteins Using a Fully Automated System

Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, including biopharmaceuticals. Glycan structures exhibit significant heterogeneity, varying with production cell types, culture conditions, and purification methods. Consequently, monitoring and evaluating the glycan structures of recombinant proteins is vital, particularly in biopharmaceutical production. The lectin microarray, a technique complementary to mass spectrometry, boasts high sensitivity and ease of use. However, it typically requires more than a day to yield results. To adapt it to non-glycoscience research or drug product process development, an automated, high-throughput alternative is needed. Therefore, the world's first fully automated lectin-based glycan profiling system was developed, utilizing the "bead array in a single tip (BIST)" technology concept. This system allows for the preparation and storage of lectin-immobilized beads in units of 1,000, with customizable parallel insertion orders for various purposes. This article presents a practical protocol for research involving "glyco-qualified" recombinant proteins. After testing their reactivity against 12 polyacrylamide-glycan conjugates, 15 lectins were selected to increase the system's versatility. In addition, the sample labeling process was optimized by switching from Cy3 to biotin, reducing the overall processing time by 30 min. For immediate data qualification, lectin-binding signals are displayed as a dotcode on the top monitor. The system's reliability was confirmed through day-to-day reproducibility tests, repeatability tests, and long-term storage tests, with a coefficient of variation of &lt;10%. This user-friendly and rapid glyco-analyzer has potential applications in the quality monitoring of endogenous glycoproteins for biomarker evaluation and validation. This method facilitates analysis for those new to glycoscience, thereby broadening its practical utility.

<meta charset="utf8"></head><body>Autor im Rampenlicht: Weiterentwicklung der Proteinglykosylierungsforschung mit einem vollautomatischen System

A system for the automated and rapid analysis of glycans on proteins was previously developed. This article presents the protocol for "glyco-qualitative analysis," optimized for a broad spectrum of users, such as those engaged in analyzing glycan structures in biopharmaceuticals and other glycoconjugate materials.

Schnelle glykoqualitative Bewertung rekombinanter Proteine mit einem vollautomatischen System

Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, ...

Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the individual immune condition determines the success in combating internal and external potential threat signals, but also the existence of a particular pattern of circulating anti-glycan antibodies (and their serological level variation) could be a significant marker of the onset and progression of certain pathological conditions. Here, we describe a Printed Glycan Array (PGA)-based methodology that offers the opportunity to measure hundreds of glycan targets with very high sensitivity; using a minimal amount of sample, which is a common restriction present when small animals (rats, mice, hamster, etc.) are used as models to address aspects of human diseases. As a representative example of this approach, we show the results obtained from the analysis of the repertoire of natural anti-glycan antibodies in BALB/c mice. We demonstrate that each BALB/c mouse involved in the study, despite being genetically identical and maintained under the same conditions, develops a particular pattern of natural anti-carbohydrate antibodies. This work claims to expand the use of PGA technology to investigate repertoire (specificities) and the levels of circulating anti-carbohydrates antibodies, both in health and during any pathological condition.

This work shows the potential of printed glycan array (PGA) technology for the analysis of circulating anti-carbohydrate antibodies in small animals.

Gedruckte Glycan Array: Eine sensible Technik für die Analyse des Repertoires der zirkulierenden Antikörper Anti-Kohlenhydrat in Kleintiere

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the ...

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 ...

Profiling Anti-Neu5Gc IgG in menschlichen Seren mit einem Sialoglycan-Microarray-Assay

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Massenspektrometrische Analyse Glycosphingolipid Antigene

Chemo-enzymatische Synthese von N- Glykanen für Array-Entwicklung und HIV-Antikörper-Profiling

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Ein miniaturisiertes Glycan Microarray-Test zur Beurteilung der Avidität und Spezifität von Influenza-A-Virus Hämagglutininarten

Metabolische Glykan der Sialinsäure Säure mit N-Acyl-Mannosamines geändert

Schnelle glykoqualitative Bewertung rekombinanter Proteine mit einem vollautomatischen System

Schnelle glykoqualitative Bewertung rekombinanter Proteine mit einem vollautomatischen System

Schnelle glykoqualitative Bewertung rekombinanter Proteine mit einem vollautomatischen System

Gedruckte Glycan Array: Eine sensible Technik für die Analyse des Repertoires der zirkulierenden Antikörper Anti-Kohlenhydrat in Kleintiere

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