这项工作部分得到以色列癌症研究基金研究职业发展奖的支持，以色列国家纳米技术计划和赫尔姆斯利慈善信托基金会为个人化支持纳米医学（VP-K）和国家卫生署授予R01GM076360（至XC）。

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作者没有什么可以披露的。

成功的聚糖微阵列制造需要仔细的规划，并包括协议中的几个重要步骤。这些包括:（1）规划定义所有后续参数（ 例如距离，间距，样品量和打印）的块和板布局; （2）清洁引脚并确保引脚的完整性，这对于控制点均匀性至关重要; （3）在印刷过程中保持高湿度，在长时间打印运行期间避免样品蒸发至关重要，这可能损害光斑均匀性; （4）选择正确的对齐和分析参数，这可以影响结果（ <em...

Sias是覆盖细胞表面糖蛋白上的聚糖链和脊椎动物中糖脂的酸性糖。 SIA表达在癌细胞1改性，并与进展和/或转移2,3相关。哺乳动物中主要的Sia形式的两种是Neu5Ac及其羟基化形式Neu5Gc 2 。由于编码CMAH酶的基因的特异性失活，人类不能合成Neu5Gc。这种非人的新航代谢合并到人体细胞的"自我"，从富含膳食中Neu5Gc的食物（ 如红肉）始发4,5。 Neu5Gc在人上皮细胞和内皮细胞表面的低水平存在，但特别积累在癌中。 Neu5Gc被人体液免疫系统2认为是外来的， 6 。Neu5Gc-聚糖的抗原性复杂性可能出现在多个水平，包括Neu5Gc修饰，连锁，底层聚糖和支架及其密度，都反映在人类6抗体Neu5Gc抗体的复杂性6 。一些抗体作为癌生物标志物和潜在的免疫治疗7。不同sialoglycans 8的酶法合成的到来铺平了道路这样的抗体的更深入的分析，通过使用聚糖微阵列技术9,10容易。因此，具有天然和合成碳水化合物的大文库的制备容易和操纵，聚糖微阵列已经成为一个强大的高通量技术用于研究与生物分子10，11的无数的碳水化合物的相互作用， </sup&gt; 12，13。以阵列形式，使用最少量的材料，并且生物相关聚糖的这种多重显示允许在单个实验中调查数千个结合相互作用。重要的是，这种技术也可以应用于生物标志物发现以及各种样品7中，12监测免疫应答。 成功的聚糖微阵列制造需要考虑三个重要方面:打印机器人类型，聚糖共轭化学和检测光学。关于印刷仪器的考虑，有两种技术可供选择:联系和非接触式打印机。在接触印刷中，将1-48个钢针浸入含有聚糖溶液的多孔源板中，并通过直接接触玻璃载片表面而在功能化玻璃载片上点样。溶液量活动到幻灯片是滑动表面上持续时间的函数。通常，样品首先在玻璃块上预先点样（达到同质点），然后再将其印刷在载玻片上。在非接触式打印机（ 例如，压电式打印机）中，使用受控电信号从玻璃毛细管印刷聚糖。可以对电信号进行精细校准，以实现相对于接触印刷的更精确的印刷。斑点的大小和形态也相对更均匀。另外的优点是在打印后将样品回收到源极板。然而，压电式打印机的主要缺点是打印头限制（4或8），导致打印时间非常长，需要特别注意滑动稳定性，温度，湿度和样品蒸发。非接触喷墨打印机需要更大的样品体积14 。 <p class ="jove_content"&gt;与印刷方法的有限可用选项相反，聚糖缀合化学是一个更复杂的考虑因素，有许多选择可供选择。选择的固定化学必须考虑聚糖上的活性基团和载玻片表面的反应性。要固定在特定微阵列表面上的合成或天然分离的聚糖都需要相同的反应性基团。此外，聚糖需要是纯的和均一的。另一方面，固定化表面和化学应提供重复性和可靠的附着密度。多种固定化方法已经开发了共价或非共价的（物理吸收）附件10，11，12，13。关于印刷聚糖微阵列技术的非常详细的信息ð调查，把这些优秀回顾13，15。重要的是，最近提供的一个糖尿病实验最低限度信息（MIRAGE）计划描述了样品制备的准则16，并报告了聚糖微阵列分析17的数据，以提高这一增长领域的标准。 在这里，我们描述了使用16孔格式的特定接触式纳米打印机制造sialoglycan微阵列的详细协议。每个聚糖具有介导它们与环氧基活化玻璃载片的共价键的伯胺。我们还描述了使用各种人血清样品，抗体和Sia结合植物凝集素的一个载玻片的开发和分析。 Sialoglycan微阵列测定涉及几个主要步骤，包括阵列制造，加工，开发和分析。阵列制造需要规划射线布局，制备聚糖和源极板，编程纳米打印机和打印幻灯片。随后，对载玻片进行处理，显影和分析（ 图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

细胞覆盖着通常在癌症中被改变并包括唾液酸（Sia）表达变化的碳水化合物链（聚糖）的斗篷。这些是具有9碳骨架并且在细胞表面上覆盖脊椎动物聚糖的酸性糖。哺乳动物中主要的Sia形式中的两种是N-乙酰神经氨酸（Neu5Ac）及其羟基化形式， N-乙酰神经氨酸（Neu5Gc）。由于编码胞苷5&#39;-单磷酸-Neu5Ac（CMP-Neu5Ac）羟化酶（CMAH）的基因失活，人类不能产生内源性Neu5Gc。外源Neu5Gc由人类细胞通过红肉和乳制品的饮食消耗而获得，随后出现在细胞表面的多种聚糖上，主要集中在癌上。因此，人类具有循环的抗Neu5Gc抗体，其在癌症和其他慢性炎症介导的疾病中起着不同的作用，并且正在成为潜在的诊断和治疗rgets。在这里，我们描述了一种高通量sialoglycan微阵列测定法来评估人血清中的这种抗Neu5Gc抗体。含有Neu5Gc的聚糖及其配对的对照（含Neu5Ac的聚糖），每个具有核心伯胺，共价连接到环氧树脂涂布的玻璃片上。我们举例说明了使用特定的纳米打印机，可以以16孔格式打印56张幻灯片，每台打印机最多可生成896个阵列。每张幻灯片可用于筛选16种不同的人血清样品，以评估抗Neu5Gc抗体的特异性，强度和多样性。该协议描述了这种强大的工具的复杂性，并提供了一个基本准则，旨在探讨在阵列格式的不同临床样品中对Neu5Gc膳食碳水化合物抗原的反应。

阵列打印，开发和分析: 打印具有多个聚糖样品的sialoglycan微阵列和16个不同块中的人IgG STD曲线需要彻底校准，以确保所有样品在每个幻灯片所有16个块中以及在相同打印运行中的所有幻灯片尽可能均匀地打印。因此，在确定特定印刷参数之前需要进行多次校准实验，包括每种样品的缓冲液组合物，载玻片类型和制造，384孔板型，...

Watch this Scientific Journal Video about Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay at JoVE.com

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

使用唾液酸多糖微阵列测定在人血清中分析抗Neu5Gc IgG

可以使用sialoglycan微阵列测定来评估人血清中的抗Neu5Gc抗体，使其成为癌症和其他慢性炎症介导的人类疾病的潜在的高通量诊断测定。

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Behavior

9.4K 次观看.  Tel Aviv University. 可以使用sialoglycan微阵列测定来评估人血清中的抗Neu5Gc抗体，使其成为癌症和其他慢性炎症介导的人类疾病的潜在的高通量诊断测定。 

视频: 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.

鞘糖脂抗原的质谱分析

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

科研

免疫与感染

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.

用于阵列发育和 HIV 抗体分析的糖的化学酶法合成

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 ...

化学

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 ...

生物化学

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.

小型化聚糖微阵列测定评定A型流感病毒血凝素的亲和力和特异性

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.

利用N酰基修饰 Mannosamines 对唾液酸的代谢 Glycoengineering

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 ...

生物工程

<meta charset="utf8"></head><body>使用单吸头中的微珠阵列 （BIST） 进行基于凝集素的聚糖自动化分析

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>作者聚焦:使用全自动系统推进蛋白质糖基化研究

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.

使用全自动系统对重组蛋白进行快速糖质定性评估

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.

印制糖类阵列是分析小动物循环抗碳水化合物抗体汇辑的一种敏感技术

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.

化学阻断抗体芯片复用高通量的复杂样品中特定蛋白糖基化的剖析

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

使用唾液酸多糖微阵列测定在人血清中分析抗Neu5Gc IgG

摘要

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此视频中的章节

鞘糖脂抗原的质谱分析

用于阵列发育和 HIV 抗体分析的糖的化学酶法合成

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

小型化聚糖微阵列测定评定A型流感病毒血凝素的亲和力和特异性

利用N酰基修饰 Mannosamines 对唾液酸的代谢 Glycoengineering

使用全自动系统对重组蛋白进行快速糖质定性评估

使用全自动系统对重组蛋白进行快速糖质定性评估

使用全自动系统对重组蛋白进行快速糖质定性评估

印制糖类阵列是分析小动物循环抗碳水化合物抗体汇辑的一种敏感技术

化学阻断抗体芯片复用高通量的复杂样品中特定蛋白糖基化的剖析