Name: analysis of n-glycans from raphanus sativus cultivars using pngase h+
Uploaded: 2018-06-25T15:00:00.000+00:00
Duration: 8 min 26 s
Description: Watch this Scientific Journal Video about Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+ at JoVE.com

这项工作得到了中国自然科学基金的部分支持 (批准号31471703、A0201300537 和31671854至合资企业和李涅、赠款 31470435 G.Y.) 和100外国人才计划 (批准编号 JSB2014012 合资企业)。

1. 样品收集
<ol><li>购买不同品种的鲜萝卜 (萝卜)。</li>
</ol>2. 从萝卜中分离蛋白质
<ol><li>融汇约100克新鲜萝卜与厨房搅拌机10分钟。</li>
	<li>将浆料转移到50毫升离心管和离心机在 1.4万 x克4 摄氏度, 以消除不溶性的材料。</li>
	<li>将上清液小心地转移到新的50毫升离心管中, 加入等量的2米乙酸酸 (TCA) 溶液。 注: 添加 TCA 将沉淀可溶性 (糖) 蛋白。</li>
	<li>离心机在 1.4万 x g在4°c 为30分钟并且从颗粒 (糖) 蛋白质去除上清。</li>
	<li>用20毫升去离子水和离心机清洗颗粒, 1.4万 x克4 摄氏度, 去除可溶性寡糖和多糖。</li>
	<li>重复步骤2.5。四次。</li>
	<li>在1毫升的去离子水中重新悬浮小球, 转移到新鲜的1.5 毫升离心管。</li>
</ol>3. n-多糖的制备
<ol><li>从步骤2.7 中混合50µL 蛋白质溶液。(等于5克新鲜萝卜), 0.2 亩重组 PNGase H+ 10 毫米醋酸。</li>
	<li>孵育反应混合物在37°c 为12小时。孵化后, 离心机在 1.4万 x g 5 分钟内除去多余的蛋白质和酶。</li>
	<li>将上清液转移到新鲜的1.5 毫升离心管。</li>
</ol>4. n-多糖的纯化
<ol><li>制备固相萃取 (SPE) 柱, 以丰富n-多糖, 从反应混合物中除去盐和其他杂质, 提高荧光 2-苯酰胺 (2-AB) 标记剂对n-糖的选择性衍生。<ol>
<li>加入3毫升的去离子水洗涤柱。</li>
		<li>加入3毫升80% 乙腈溶液, 含三氟乙酸酸 (TFA, 0.1% 伏/v) 活化 SPE 柱。</li>
		<li>加入3毫升去离子水, 平衡 SPE 柱。</li>
	</ol>
</li>
	<li>将该示例从步骤3.3 转移到该列, 然后丢弃该流程。</li>
	<li>加入1.5 毫升的去离子水洗涤柱并丢弃流经。</li>
	<li>洗脱从柱上释放的n-多糖, 使用1.5 毫升水中20% 乙腈溶液, 含有 0.1% TFA (v/v) 到2毫升离心管中。</li>
	<li>室温离心蒸发去除溶剂。蒸发, 直到样品完全干燥。</li>
</ol>5. n-多糖的荧光衍生化
<ol><li>准备1毫升的 2 ab 溶液, 包括35毫米 2 AB 和0.1 米 cyanoborohydride 钠在二甲基亚砜/乙酸溶液 (7:3, v/v)。</li>
	<li>添加5µL 2 AB 溶液的干燥样品 (步骤 4.5) 来源于六不同的萝卜。漩涡每个样品直到完全地溶化。</li>
	<li>在65摄氏度孵育2小时的混合物。</li>
	<li>让样品冷却到室温5分钟, 加入5µL 去离子水和40µL 乙腈。离心管在 1.4万 x g 3 分钟。</li>
	<li>将48µL 从上清到300µL 高回收率 HPLC 瓶。将衍生物糖样品存放在摄氏-20 摄氏度, 可达一个月。</li>
</ol>6. HILIC-UPLC 多糖的分析
<ol><li>使用与在线荧光 detectorset 连接的标准 UPLC 系统, 分别在 330 nm/420 nm 的激发/发射波长上分析样品。</li>
	<li>使用疏水性相互作用液相色谱 (HILIC)-UPLC 糖柱在60°c 的柱温度为分析。</li>
	<li>通过稀释50毫升的库存溶液 (1 米甲酸铵溶液, pH 4.5), 用950毫升的液相色谱-质谱 (LCMS) 级水, 制备溶剂 A。准备库存解决方案, 如下所示:<ol>
<li>添加43毫升甲酸到700毫升的 LCMS 级 H2O。</li>
		<li>用滴状加水氨溶液 (25% 瓦特/瓦) 将 pH 值调整为4.5。</li>
		<li>将溶剂转移到测量缸内, 并填充1000毫升, LCMS 级 H2o 型存储在4-6 摄氏度, 可达3月。</li>
	</ol>
</li>
	<li>采用 LCMS 级乙腈为溶剂 B。</li>
	<li>用以下梯度洗脱分离n-多糖:<ol>
<li>从95% 的溶剂 B 开始加入溶剂 a. 将流量设置为0.5 毫升/分, 从0到44.5 分钟。</li>
		<li>从0分钟到6分钟, 逐渐减少溶剂 B 与线性梯度 to78 的比例。</li>
		<li>从6分钟到44.5 分钟, 逐渐降低溶剂 B 的比例, 线性梯度为55.9%。</li>
		<li>从44.5 分钟到46.5 分钟, 快速将溶剂 B 的比例从55.9% 降到 0%, 并在0% 处保持2分钟, 并开始洗涤柱。将流量降低到0.25 毫升/分, 从44.5 到55分钟。</li>
		<li>通过将溶剂 B 从48.5 分钟增加到50.5 分钟, 并在95% 处保持4.5 分钟, 进一步洗涤柱。</li>
		<li>将该柱重新平衡95% 溶剂 B 的起始条件为0.5 毫升/分, 从50.5 到57分钟。</li>
	</ol>
</li>
	<li>在 UPLC 系统中注入45µL 的样品。</li>
	<li>洗脱多糖由 UPLC 与保留时间在15和40分钟之间。</li>
	<li>用2毫升离心管收集每个观测到的 UPLC 峰值分数, 并通过离心蒸发干燥样品。</li>
	<li>注射约 1 pmol 2 AB 标记葡聚糖标准 (2−20葡萄糖单位), 以校准植物n-糖的分析, 并分配他们的洗脱时间为标准化的葡萄糖单位。</li>
</ol>7. MALDI 分析
<ol><li>在5µL LCMS 级水中溶解6.8 步的样品。混合1µL 的样品溶液和1µL 6-aza 2 thiothymine (的) 溶液 (0.3% 瓦特/v 在70% 伏/五水中乙腈), 并将混合物移到 MALDI-飞行样品载体上。</li>
	<li>通过按下启动按钮, 在正离子模式下使用 MALDI 质谱仪对样品进行分析。</li>
	<li>采用随附的 MALDI 分析软件对质谱进行分析。</li>
	<li>使用开放源码糖解释软件19解释频谱。设置 2 AB 标记的搜索参数, [M + Na]+, 并将精度参数设置为 2.0 Da。</li>
</ol>

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作者没有什么可透露的。

我们在这里提出的协议允许比较不同品种的萝卜的n-糖剖面。与现有协议相比, 这种方法的一个显著优点是, 多糖的酶释放与 2 AB 的衍生反应之间没有缓冲的变化。这个过程最关键的步骤是用 SPE 柱纯化n-多糖, 因为在反应混合物中除去盐或其他杂质可能会对荧光衍生效率产生负面影响。此处提出的方法仅允许对不同的n-多糖的相对丰度进行评估;绝对量化需要在步骤2.1 中添...

近年来, 植物中的n-多糖引起了越来越多的关注, 因为先前的研究突出了n-多糖作为免疫交叉反应的潜在来源, 可能引发过敏反应1,2.此前已证明, 植物糖蛋白的n-多糖会影响催化活性3,4, 热稳定性和折叠5,6或亚细胞定位和分泌物7。为了使糖结构与它们各自的功能相关联, 多糖必须首先从化学或酶的糖蛋白中释放出来。经典的化学方法释放n-和O型多糖是β消除, 其中碱性样品处理是伴随着减少与硼氢化, 以产生一个糖醇8。然而, 这一过程排除了荧光的标记, 并导致单糖单位从糖结构的减少端显著衰减。基于氢氧化铵/碳酸盐处理的化学 deglycosylation 也是常用的替代方法9。这两种化学释放方法都没有降解完整的蛋白质, 这允许在不存在同质量范围内的肽片段干扰的情况下对未标记的糖池进行质谱分析。然而, 这些方法的一个缺点是增加的降解率α1,3-fucosylated n-多糖, 一个共同的碳水化合物结构发现在植物10。另外, 使用肽的酶释放方法:n-glycanases (PNGases, EC 3.5.1.52) 也被广泛应用。重组 PNGase F (从杆菌 meningosepticum) 是最常见的选择, 允许释放所有类型的n-多糖, 除了轴承核心α1,3 岩藻糖11,12的结构。因此, PNGase (从杏仁籽中分离) 通常用于植物n-多糖13的分析。然而, 这种酶 deglycosylates 只 proteolytically 衍生糖肽, 无法 deglycosylate 本族糖蛋白14。因此, 在进一步分析之前需要进行多步骤的抽样检查, 从而导致大量的多糖损失, 尤其是低丰度的15。该方法的总体目标是以简单、稳健的方式为n-糖发布和荧光标记提供一个优化的工作流。基本的基本原理是, 最近在Terriglobus 长春发现的 PNGase H+可以 recombinantly 表达在大肠杆菌中, 可以直接从蛋白质支架中水解n-多糖酸性情况16。使用 PNGase H+替代方法的一个关键优点是荧光标记反应可以在同一反应管内进行, 而不会改变反应缓冲器17,18。低丰寡糖的制备条件简单, 回收率高, 使该方法成为多糖分析的重要工具. 该协议适用于不同植物种类的n-多糖分析。

<table><tbody><tr><td>&lt;strong&gt;Chemicals:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Trichloroacetic acid&amp;nbsp;</td><td>SCR, Shanghai</td><td>80132618</td><td></td></tr><tr><td>Acetic acid glacial</td><td>Huada, Guangzhou</td><td>64-19-7</td><td></td></tr><tr><td>Acetonitrile</td><td>General-reagent</td><td>G80988C</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Energy chemical</td><td>W810031</td><td></td></tr><tr><td>2-aminobenzamide</td><td>Heowns, Tianjin</td><td>A41900</td><td></td></tr><tr><td>Sodium cyanoborohydride</td><td>J&amp;amp;K Scientific Ltd</td><td>314162</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Huada, Guangzhou</td><td>67-68-5</td><td></td></tr><tr><td>2AB-labeled dextran ladder, 200 pmol</td><td>Agilent Technologies</td><td>AT-5190-6998</td><td></td></tr><tr><td>6-Aza-2-thiothymine&amp;nbsp;</td><td>Sigma</td><td>275514</td><td></td></tr><tr><td>&lt;strong&gt;Tools/Materials:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Kitchen blender</td><td>Bear, Guangzhou</td><td>LLJ-A10T1</td><td></td></tr><tr><td>Centrifuge</td><td>Techcomp</td><td>CT15RT</td><td></td></tr><tr><td>Centrifugal Evaporator</td><td>Hualida, Taicang</td><td>LNG-T120</td><td></td></tr><tr><td>SPE column</td><td>Supelco</td><td>Supelclean ENVI Carb SPE column</td><td></td></tr><tr><td>MALDI-TOF mass spectrometer</td><td>Bruker</td><td>Autoflex</td><td></td></tr><tr><td>&lt;strong&gt;HPLC Analysis:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>High-recovery HPLC vial</td><td>Agilent Technologies</td><td>&amp;nbsp;# 5188-2788</td><td></td></tr><tr><td>HPLC System</td><td>Shimadzu</td><td>Nexera</td><td></td></tr><tr><td>Fluorescence Detector for HPLC</td><td>Shimadzu</td><td>RF-20Axs&amp;nbsp;</td><td></td></tr><tr><td>Column oven</td><td>Hengxin</td><td>CO-2000</td><td></td></tr><tr><td>HPLC Column</td><td>Waters</td><td>Acquity UPLC BEH glycan column</td><td>2.1 &amp;times; 150 mm, 1.7 &amp;mu;m particle size</td></tr><tr><td>LCMS-grade Water</td><td>Merck Millipore</td><td>#WX00011</td><td></td></tr><tr><td>LCMS-grade Acetonitrile</td><td>Merck Millipore</td><td># 100029</td><td></td></tr><tr><td>Formic acid</td><td>Aladdin</td><td>F112034</td><td></td></tr><tr><td>Ammonia solution</td><td>Aladdin</td><td>A112080</td><td></td></tr></tbody></table>

analysis of n-glycans from raphanus sativus cultivars using pngase h+

近年来, 植物的碳水化合物基团受到了相当大的关注, 因为它们是交叉反应, 引起过敏性免疫反应的潜在来源。此外, 碳水化合物结构在植物代谢中也起着至关重要的作用。在这里, 我们提出了一个简单和快速的方法, 以制备和分析不同品种的萝卜 (萝卜) 的n-多糖, 利用n-glycanase 的具体释放植物衍生碳水化合物结构。为了达到这一目的, 萝卜组织匀浆的粗乙酸酸沉淀处理 PNGase H+, 并标记使用 2-苯酰胺作为荧光标记。随后用超效液相色谱 (UPLC) 分离法和基质辅助激光解吸电离-飞行时间 (MALDI) 质谱法对糖样品进行了分析, 详细结构对萝卜衍生n-糖结构的相对 abundancies 进行评价和量化。该协议也可用于对不同植物种类的n-多糖进行分析, 对多糖对人体健康的作用和作用的进一步研究有一定的参考价值。

图 1显示了描述的协议的示意图, 包括从萝卜中分离 (糖) 蛋白、 n-多糖的制备、UPLC 分析以及这些成分的 MALDI--MS 分析。图 2显示了萝卜品种衍生物多糖的代表性 UPLC 图谱。图 3显示了2衍生物n-糖结构用 MALDI-飞行质谱法获得的结果。图 4显示了每个萝卜品种的n-</e...

Watch this Scientific Journal Video about Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+ at JoVE.com

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

萝卜品种多糖的 PNGase 研究

本文介绍了一种简便、快速的方法, 对不同品种萝卜 (萝卜) 的n-多糖进行了制备和分析。

In recent years, the carbohydrate moieties of plants have received considerable attention, as they are a potential source of cross-reactive, ...

analysis-of-n-glycans-from-raphanus-sativus-cultivars-using-pngase-h

Research

JoVE Journal

Biochemistry

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

6.5K 次观看.  Nanjing Agricultural University. 本文介绍了一种简便、快速的方法, 对不同品种萝卜 (萝卜) 的n-多糖进行了制备和分析。

视频: Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts of purified material. Here, we describe a simple method for gaining detailed polysaccharide structural information, including resolution of structural isomers. For polysaccharide analysis by gel electrophoresis (PACE), plant cell wall material is hydrolyzed with glycosyl hydrolases specific to the polysaccharide of interest (e.g., mannanases for mannan). Large format polyacrylamide gels are then used to separate the released oligosaccharides, which have been fluorescently labeled. Gels can be visualized with a modified gel imaging system (see Table of Materials). The resulting oligosaccharide fingerprint can either be compared qualitatively or, with replication, quantitatively. Linkage and branching information can be established using additional glycosyl hydrolases (e.g., mannosidases and galactosidases). Whilst this protocol describes a method for analyzing glucomannan structure, it can be applied to any polysaccharide for which characterized glycosyl hydrolases exist. Alternatively, it can be used to characterize novel glycosyl hydrolases using defined polysaccharide substrates.

A protocol for the structural analysis of polysaccharides by gel electrophoresis (PACE), using mannan as an example, is described.

植物甘露聚糖细胞壁多糖的结构表征

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts ...

科研

生物化学

A Quantitative Glycomics and Proteomics Combined Purification Strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

定量糖组学和蛋白质组学组合式净化策略

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

化学

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 ...

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 ...

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

N -连接糖链的糖蛋白在哺乳动物细胞中的脉冲大通分析

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

生物学

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the specific roles of glycans, the relationship between their structures and their impact on the functions of proteins. A common method for glycan analysis is to employ exoglycosidase cleavage to release N-linked glycans from glycoproteins or glycopeptides using Peptide-N-Glycosidase F (PNGase F). However, the glycan-protein linkages in bacteria are different and there is no enzyme available to release glycans from bacterial glycoproteins. In addition, free glycans have also been described in mammalian cells, bacteria, yeast, plants, and fish. In this article, we present a method that can characterize the N-linked glycosylation system in Campylobacter jejuni by detecting asparagine (Asn)-linked and free glycans that are not attached to their target proteins. In this method, total proteins from C. jejuni were digested by Pronase E with a higher enzyme to protein ratio (2:1&#8722;3:1) and a longer incubation time (48&#8722;72 h). The resulted Asn-glycans and free glycans were then purified using porous graphitic carbon cartridges, permethylated, and analyzed by mass spectrometry.

This study presents a method for glycomics analysis of glycoproteins by a combination of pronase E digestion, permethylation, and mass spectrometry analysis. This method is capable of analyzing all types of N-linked glycans, including bacterial N-glycans.

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the ...

<meta charset="utf8"></head><body>使用糖组学指导的糖蛋白质组学分析结直肠癌组织

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.

<meta charset="utf8"></head><body>糖组学引导的糖蛋白质组学有助于在复杂的肿瘤微环境中全面分析糖蛋白质组

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.

糖组学引导的糖蛋白质组学有助于在复杂的肿瘤微环境中全面分析糖蛋白质组 

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

癌症研究

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

使用具体远藤和exoglycosidases糖基化蛋白质的鉴定和表征

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

萝卜品种多糖的 PNGase 研究

摘要

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此视频中的章节

植物甘露聚糖细胞壁多糖的结构表征

定量糖组学和蛋白质组学组合式净化策略

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

N -连接糖链的糖蛋白在哺乳动物细胞中的脉冲大通分析

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

糖组学引导的糖蛋白质组学有助于在复杂的肿瘤微环境中全面分析糖蛋白质组

糖组学引导的糖蛋白质组学有助于在复杂的肿瘤微环境中全面分析糖蛋白质组

糖组学引导的糖蛋白质组学有助于在复杂的肿瘤微环境中全面分析糖蛋白质组

使用具体远藤和exoglycosidases糖基化蛋白质的鉴定和表征