The authors thank the Thin Film Technology Division, Instrument Technology Research Center (ITRC) and National Applied Research Laboratories, Hsinchu Science Park, Taiwan. This work was supported by the National Science Council (grant no. MOST 105-0210-01-13-01) and Academia Sinica.

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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycan+Array+on+Aluminum+Oxide-Coated+Glass+Slides+Through+Phosphonate+Chemistry.">Glycan Array on Aluminum Oxide-Coated Glass Slides Through Phosphonate Chemistry.</a> J. Am. Chem. Soc. 132, 13371-13380 (2010).</li><li>Tseng, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Aluminum+Oxide-Coated+Glass+Slides+for+Glycan+Microarrays.">Preparation of Aluminum Oxide-Coated Glass Slides for Glycan Microarrays.</a> ACS Omega. 1, 773-783 (2016).</li><li>Shivatare, S. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Hiv-1+gp120+V1/V2+Domain+with+Broadly+Neutralizing+Antibody+PG9.">Structure of Hiv-1 gp120 V1/V2 Domain with Broadly Neutralizing Antibody PG9.</a> Nature. 480, 336-343 (2011).</li><li>Julien, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+Recognition+of+the+HIV-1+Trimer+by+Broadly+Neutralizing+Antibody+PG9.">Asymmetric Recognition of the HIV-1 Trimer by Broadly Neutralizing Antibody PG9.</a> Proc Natl Acad Sci U S A. 110, 4351-4356 (2013).</li><li>Willis, J. 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The authors have nothing to disclose.

A class of HIV-1 bNAbs including PG9, PG16, and PGTs 128, 141 - 145 were reported to be highly potent in neutralizing 70 - 80% of circulating HIV-1 isolates. The epitopes of these bNAbs are highly conserved among the variants of the entire HIV-1 group M, therefore they may guide the effective immunogen design for an HIV vaccine to elicit neutralizing antibodies23,24,25. As a part of our ongoing efforts to identify the glycan epi...

N-glycans on glycoproteins are covalently linked to the asparagine (Asn) residue of the consensus Asn-Xxx-Ser/Thr sequon, which affect several biological processes such as protein conformation, antigenicity, solubility, and lectin recognition1,2. The chemical synthesis of N-linked oligosaccharides represents a significant synthetic challenge because of their huge structural micro heterogeneity and highly branched architecture. Careful selection of protecting groups to tune reactivity of building blocks, achieving selectivity at anomeric centers, and proper use of promoter/activator(s) are key elements in synthesis of complex oligosaccharides. To solve this problem of complexity, a great amount of work to advance N-glycan synthesis was reported recently3,4. In spite of these robust approaches, finding an effective method for the preparation of a wide range of N-glycans (~20,000) remains a major challenge.
The rapid mutation rate of HIV-1 to achieve the extensive genetic diversity and its ability to escape from neutralizing antibody response, is among the greatest challenges to develop a safe and prophylactic vaccine against HIV-15,6,7. One effective tactic that HIV uses to avoid the host immune response is the post-translational glycosylation of envelope glycoprotein gp120 with a diverse N-linked glycans derived from the host glycosylation machinery8,9. A recent report regarding the precise analysis of recombinant monomeric HIV-1 gp120 glycosylation from human embryonic kidney (HEK) 293T cells suggests the occurrence of structural microheterogeneity with a characteristic cell-specific pattern10,11,12. Therefore, understanding the glycan specificities of HIV-1 bNAbs requires well characterized gp120 related N-glycan structures in a quantity sufficient for analysis.
The discovery of glycan microarray technology provided high throughput-based exploration of specificities of a diverse range of carbohydrate-binding proteins, viruses/bacterial adhesins, toxins, antibodies, and lectines13,14. The systematic glycans arrangement in an arrayed chip-based format could determine problematic low affinity protein-glycan interactions through multivalent presentation15,16,17,18. This chip-based glycan arrangement conveniently appears to effectively mimic cell-cell interfaces. To enrich the technology and overcome the uneven issue associated with conventional array formats, our group recently developed a glycan array on an aluminum oxide-coated glass (ACG) slide using phosphonic acid-ended glycans to enhance the signal intensity, homogeneity, and sensitivity19,20.
To improve the current understanding about glycan epitopes of newly isolated HIV-1 broadly neutralizing antibodies (bNAbs), we have developed a highly efficient modular strategy for the preparation of a broad array of N-linked glycans21,22 to be printed on an ACG slide (see Figure 1). Specificity profiling studies of HIV-1 bNAbs on an ACG array offered the unusual detection of hetero-glycan binding behavior of highly potent bNAb PG9 that was isolated from HIV infected individuals23,24,25.

<table><tbody><tr><td>Acetic acid</td><td>Sigma Aldrich</td><td>64197</td><td></td></tr><tr><td>Acetonitrile</td><td>Sigma Aldrich</td><td>75058</td><td></td></tr><tr><td>Acetic anhydride</td><td>Sigma Aldrich</td><td>108247</td><td></td></tr><tr><td>Anhydrous magnesium sulfate</td><td>Sigma Aldrich</td><td>7487889</td><td></td></tr><tr><td>Boron trifluoride ethyl etherate</td><td>Sigma Aldrich</td><td>109637</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma Aldrich</td><td>9048468</td><td></td></tr><tr><td>Bio-Gel P2 polyacrylamide</td><td>Bio-Rad</td><td>1504118</td><td></td></tr><tr><td>Bis(cyclopentadienyl)hafnium(IV) dichloride</td><td>Sigma Aldrich</td><td>12116664</td><td></td></tr><tr><td>&amp;beta;-1, 4 Galactosyl transferases from bovine milk</td><td>Sigma Aldrich</td><td>48279</td><td></td></tr><tr><td>BioDot Cartesion technology with robotic pin SMP3 (Stealth Micro Spotting Pins)</td><td>Arrayit</td><td></td><td></td></tr><tr><td>Cerium ammonium molybdate</td><td>TCI</td><td>C1794</td><td></td></tr><tr><td>Cerium ammonium nitrate</td><td>Sigma Aldrich</td><td>16774213</td><td></td></tr><tr><td>Clean glass slide&amp;nbsp;</td><td>Schott&amp;nbsp;</td><td></td><td></td></tr><tr><td>Cytidine-5&amp;prime;-monophospho-N-acetylneuraminic acid</td><td>Sigma Aldrich</td><td>3063716</td><td></td></tr><tr><td>Deuterated chloroform</td><td>Sigma Aldrich</td><td>865496</td><td></td></tr><tr><td>Donkey Anti-Human IgG (Alexa Fluor647 conjugated</td><td>Jackson Immuno Research, USA</td><td>709605098</td><td></td></tr><tr><td>Dichloromethane</td><td>Sigma Aldrich</td><td>75092</td><td></td></tr><tr><td>Diethylaminosulfur trifluoride</td><td>Sigma Aldrich</td><td>38078090</td><td></td></tr><tr><td>Dimethylformamide</td><td>Sigma Aldrich</td><td>68122</td><td></td></tr><tr><td>Ethyl acetate</td><td>Sigma Aldrich</td><td>141786</td><td></td></tr><tr><td>Ethylene glycol</td><td>Acros Organic</td><td>107211</td><td></td></tr><tr><td>FAST frame slide incubation chambers</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Guanosine 5&#039;-diphospho-b-L-fucose disodium salt&amp;nbsp;</td><td>Sigma Aldrich</td><td>15839700</td><td></td></tr><tr><td>Lab tracer 2.0 software&amp;nbsp;</td><td></td><td></td><td>Section 4 of the Protocol</td></tr><tr><td>GenePix Pro 4300A reader (microarray image analysis)</td><td>moleculardevices</td><td></td><td>www.moleculardevices.com</td></tr><tr><td>GraphPad Prism Software (Image processing )</td><td>GraphPad Software, Inc</td><td></td><td>http://www.graphpad.com/guides/prism/6/user-guide/</td></tr><tr><td>Lithium hydroxide</td><td>Sigma Aldrich</td><td>1310652</td><td></td></tr><tr><td>Manganese chloride</td><td>Sigma Aldrich</td><td>7773015</td><td></td></tr><tr><td>Methanol</td><td>Sigma Aldrich</td><td>67561</td><td></td></tr><tr><td>N-butanol</td><td>Sigma Aldrich</td><td>71363</td><td></td></tr><tr><td>Oxalic acid</td><td>Acros Organic</td><td>144627</td><td></td></tr><tr><td>Palladium hydroxide</td><td>Sigma Aldrich</td><td>12135227</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>10010023</td><td></td></tr><tr><td>Pyridine</td><td>Sigma Aldrich</td><td>110861</td><td></td></tr><tr><td>P-Toluene sulfonic acid monohydrate</td><td>Sigma Aldrich</td><td>773476</td><td></td></tr><tr><td>Silver triflate</td><td>Sigma Aldrich</td><td>2923286</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma Aldrich</td><td>144558</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma Aldrich</td><td>7647145</td><td></td></tr><tr><td>Sodium hydrogen carbonate</td><td>Sigma Aldrich</td><td>144558</td><td></td></tr><tr><td>Sodium methoxide&amp;nbsp;</td><td>Sigma Aldrich</td><td>124414</td><td></td></tr><tr><td>Sodium sulfate</td><td>Sigma Aldrich</td><td>7757826</td><td></td></tr><tr><td>Toluene&amp;nbsp;</td><td>Sigma Aldrich</td><td>108883</td><td></td></tr><tr><td>Tris buffer&amp;nbsp;</td><td>Amresco</td><td>N/A</td><td>Ultra-pure grade</td></tr><tr><td>Tween-20</td><td>Amresco</td><td>9005645</td><td></td></tr><tr><td>Uridine diphosphate galactose (UDP-galactose)</td><td>Sigma Aldrich</td><td>137868521</td><td></td></tr></tbody></table>

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.

A modular chemo-enzymatic strategy for the synthesis of a wide array of N-glycans is presented in Figure 1. The strategy is based on the fact that diversity can be created at beginning by chemo-enzymatic synthesis of the three important modules, followed by the &#945;-specific mannosylation at the 3-O and/or 6-O position of the mannose residue of the common core trisaccharide of N-glycans. Considering the structural diversi...

Watch this Scientific Journal Video about Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling at JoVE.com

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-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 ...

chemo-enzymatic-synthesis-n-glycans-for-array-development-hiv

Research

JoVE Journal

Chemistry

8.7K Views.  Academia Sinica. Een modulaire benadering van de synthese van N- glycanen voor bevestiging aan een aluminium oxide-gecoate glazen schuif (ACG dia) als een microarray glycan heeft ontwikkeld en het gebruik ervan voor de profilering van een HIV in grote lijnen neutraliserende antilichamen aangetoond.

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

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

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) expression. These are acidic sugars that have a 9-carbon backbone and that cap vertebrate glycans on cell surfaces. Two of the major Sia forms in mammals are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated form, N-glycolylneuraminic acid (Neu5Gc). Humans cannot produce endogenous Neu5Gc due to the inactivation of the gene encoding cytidine 5&#39;monophosphate-Neu5Ac (CMP-Neu5Ac) hydroxylase (CMAH). Foreign Neu5Gc is acquired by human cells through the dietary consumption of red meat and dairy and subsequently appears on diverse glycans on the cell surface, accumulating mostly on carcinomas. Consequently, humans have circulating anti-Neu5Gc antibodies that play diverse roles in cancer and other chronic inflammation-mediated diseases and that are becoming potential diagnostic and therapeutic targets. Here, we describe a high-throughput sialoglycan microarray assay to assess such anti-Neu5Gc antibodies in the human sera. Neu5Gc-containing glycans and their matched pairs of controls (Neu5Ac-containing glycans), each with a core primary amine, are covalently linked to epoxy-coated glass slides. We exemplify the printing of 56 slides in a 16-well format using a specific nano-printer capable of generating up to 896 arrays per print. Each slide can be used to screen 16 different human sera samples for the evaluation of anti-Neu5Gc antibody specificity, intensity, and diversity. The protocol describes the complexity of this robust tool and provides a basic guideline for those aiming to investigate the response to Neu5Gc dietary carbohydrate antigen in diverse clinical samples in an array format.

A sialoglycan microarray assay can be used to evaluate anti-Neu5Gc antibodies in human sera, making it a potential high-throughput diagnostic assay for cancer and other chronic inflammation-mediated human diseases.

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

Behavior

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.

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

Immunology and Infection

Profiling of Colorectal Cancer Tissues Using Glycomics-Guided Glycoproteomics

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven glycomics and glycoproteomics have emerged as powerful approaches to analyze liberated glycans and intact glycopeptides, respectively, offering a deeper understanding of the heterogeneous glycoproteome in the tumor microenvironment (TME). This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology, which firstly employs porous graphitized carbon-LC-MS/MS-based glycomics to elucidate the glycan structures and their quantitative distribution in the glycome of tumor tissues, cell populations, or bodily fluids being investigated. This allows for a comparative glycomics analysis to identify altered glycosylation across patient groups, disease stages, or conditions, and, importantly, serves to enhance the downstream glycoproteomics analysis of the same sample(s) by creating a library of known glycan structures, thus reducing the data search time and the glycoprotein misidentification rate. Focusing on N-glycoproteome profiling, the sample preparation steps for the glycomics-guided glycoproteomics method are detailed in this protocol, and key aspects of the data collection and analysis are discussed. The glycomics-guided glycoproteomics method provides quantitative information on the glycoproteins present in the TME and their glycosylation sites, site occupancy, and site-specific glycan structures. Representative results are presented from formalin-fixed paraffin-embedded tumor tissues from colorectal cancer patients, demonstrating that the method is sensitive and compatible with tissue sections commonly found in biobanks. Glycomics-guided glycoproteomics, therefore, offers a comprehensive view into the heterogeneity and dynamics of the glycoproteome in complex TMEs, generating robust biochemical data required to better understand the glycobiology of cancers.

This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology that offers comprehensive insights into the heterogeneous glycoproteome in complex tumor microenvironments required to better understand the glycobiology of cancers.

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven ...

Cancer Research

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Bioengineering

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.

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.

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

Biology

Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and the use of four-arm, fluorescent PEG-antigen conjugates to selectively identify antigen-specific B cells through avid engagement with cognate B cell receptors. Using modular click chemistry and commercially available fluorophore kit chemistries, we demonstrated the versatility of preparing customized fluorescent PEG-conjugates by creating distinct arrays for proteolipid protein (PLP139-151) and insulin, which are important autoantigens in murine models of multiple sclerosis and type 1 diabetes, respectively. Assays were developed for each fluorescent conjugate in its respective disease model using flow cytometry. Antigen arrays were compared to monovalent autoantigen to quantify the benefit of multimerization onto PEG backbones. Finally, we illustrated the utility of this platform by isolating and assessing anti-insulin B cell responses after antigen stimulation ex vivo. Labeling insulin-specific B cells enabled the amplified detection of changes to co-stimulation (CD86) that were otherwise dampened in aggregate B cell analysis. Together, this report enables the production and use of fluorescent antigen arrays as a robust tool for probing B cell populations.

This protocol details the customizable production and use of fluorescent probes for labeling antigen-specific B cells.

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and ...

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies. Glycoengineering typically requires the generation of a stable knockout or knockin cell line using methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9. Monoclonal antibodies produced by engineered cells are then characterized using mass spectrometric methods to determine if the desired glycoprofile has been obtained. This strategy is time-consuming, technically challenging, and requires specialists. Therefore, an alternative strategy that utilizes streamlined protocols for genetic glycoengineering and glycan detection may assist endeavors toward optimal antibodies. In this proof-of-concept study, an IgG-producing Chinese hamster ovary cell served as an ideal host to optimize glycoengineering. Short interfering RNA targeting the Fut8 gene was delivered to Chinese hamster ovary cells, and the resulting changes in FUT8 protein expression were quantified. The results indicate that knockdown by this method was efficient, leading to a ~60% reduction in FUT8. Complementary analysis of the antibody glycoprofile was performed using a rapid yet highly sensitive technique: capillary gel electrophoresis and laser-induced fluorescence detection. All knockdown experiments showed an increase in afucosylated glycans; however, the greatest shift achieved in this study was ~20%. This protocol simplifies glycoengineering efforts by harnessing in silico design tools, commercially synthesized gene targeting reagents, and rapid quantification assays that do not require extensive prior experience. As such, the time efficiencies offered by this protocol may assist investigations into new gene targets.

The glycosylation pattern of an antibody determines its clinical performance, thus industrial and academic efforts to control glycosylation persist. Since typical glycoengineering campaigns are time- and labor-intensive, the generation of a rapid protocol to characterize the impact of glycosylation genes using transient silencing would prove useful.

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. ...

Elucidating Glycan Structure and Occurrence Using Microarray Polymer Profiling

Microarray Polymer Profiling (MAPP) for High-Throughput Glycan Analysis

Microarray polymer profiling (MAPP) is a robust and reproducible approach to systematically determine the composition and relative abundance of glycans and glycoconjugates within a variety of biological samples, including plant and algal tissues, food materials, and human, animal, and microbial samples. Microarray technology underpins the efficacy of this method by providing a miniaturized, high-throughput screening platform, allowing thousands of interactions between glycans and highly specific glycan-directed molecular probes to be characterized concomitantly, using only small amounts of analytes. Constituent glycans are chemically and enzymatically fractionated, before being sequentially extracted from the sample and directly immobilized onto nitrocellulose membranes. The glycan composition is determined by the attachment of specific glycan-recognizing molecular probes to the extorted and printed molecules. MAPP is complementary to conventional glycan analysis techniques, such as monosaccharide and linkage analysis and mass spectrometry. However, glycan-recognizing molecular probes provide insight into the structural configurations of glycans, which can aid in elucidating biological interactions and functional roles.

Author Spotlight: MAPP Protocol &#8211; Advancing Glycan Analysis 

Microarray polymer profiling (MAPP) is a high-throughput technique for compositional analysis of glycans in biological samples.

Microarray polymer profiling (MAPP) is a robust and reproducible approach to systematically determine the composition and relative abundance of glycans ...

Detecting Circulating Anti-Glycan Antibodies with a Printed Glycan Array

Source: Olivera-Ardid, S. et al., <a href="https://app.jove.com/v/57662">Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals.</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates printed glycan array technology for analyzing circulating anti-glycan antibodies in mouse serum. The antibodies in the serum interact with specific glycans on the chip, which is confirmed by immunostaining followed by fluorescence scanning.

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

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