这项工作是由美国能源生物科学研究所授予OO0G01。

OLIMP方法是体现在双子叶植物，xyloglucan的细胞壁中存在的主要半纤维素，使用的是内切葡聚糖酶的glycosylhydrolase。该方法是证明整个拟南芥植物组织源。酶和细胞外基质材料上使用相同的步骤取决于所需的分析，可替代。 1。细胞墙隔离<ol><li>收获五个为期5天的老整个拟南芥幼苗或您所需材料的同等数额，并转移到1.5ml的反应管。确保该材料置于试管底部。 </li><li>反应管在液氮中的样品和管理单元冻结顶部添加两个3毫米金属球。 </li><li>研磨冷冻样品使用一个球磨（下午2时30分至2分钟，25HZ）。 </li><li>加入1ml 70％的含水乙醇样品。删除使用磁铁的金属球。涡彻底。 </li><li>离心14000 RPM为10分钟，以颗粒不溶于醇残留物含有细胞壁物质的样品。 </li><li>小心取出上清液吸。确保不打扰颗粒。 </li><li>氯仿/甲醇（1:1 V / V）溶液，加入1毫升。涡彻底悬浮颗粒。 </li><li>在10分钟14000转离心样品，并小心取出上清液吸。确保不打扰颗粒。 </li><li>使用浓缩干燥样品。 </li></ol> 2。溶低聚糖<ol><li>对于增溶在其低聚糖的形式，尤其是多糖悬浮在一个适当的缓冲液25μl的干燥颗粒。内切葡聚糖酶50MM酸铵，PH4.5，是使用。新增的内切葡聚糖酶0.2U。涡的悬挂和自旋液体下来。 </li><li>孵育16h后的样品在37 ° C的孵化器，在300RPM晃动。 </li><li>取出的精华（水）使用一个集中的溶剂。 </li></ol> 3。 MALDI - TOF分析发布低聚糖<ol><li> BioRex MSZ 501阳离子交换树脂用于去除盐类和酶消化的样品。条件放置在一个空列等分和大量的水冲洗珠小珠。 </li><li> 5-10 BioRex珠消化和干燥样品。然后添加加入10μl水，可用于数额较小的水材料5μL。溶液中浸泡简要纺纱管珠。在室温下孵育至少10分钟。 </li><li>现货2μL矩阵（2,5 - 二羟基苯甲酸（DHB），在水中10mg/ml的）到MALDI靶盘。下导致同质矩阵化学晶体的真空溶剂蒸发矩阵。 </li><li>脱盐，目标板上的矩阵晶体样品溶液点2μL。 如果你想现场的大量样品，只保留了3分钟的最大的斑点。这一时限保证，不会干，还首次发现样品。等待一个额外的2分钟，以确保重新溶解在去年发现样品基质和样品寡糖分子充分混合。再用干的真空状态下的目标板。 样本/矩阵结构应在不到1分钟，使同质结晶结晶。 </li><li>放入一个的MALDI - TOF质谱仪的目标板。机应积极reflectron模式，加快20000V的电压和延迟为350 nm;选定的质量范围应达500-3000（可能会改变依赖于预期的低聚物）。开始发射激光，并收集每个样品100-200光谱，这是编译生成一个具有代表性的平均光谱（图2A）。代表特定寡糖的离子可以由他们过充质量（M / Z）的比例确定。所有感兴趣的离子的离子强度（峰高）应增加高达100％，在每个样品中的低聚糖的相对丰度（图2B）。 </li></ol> 原位 OLIMP分析4。 OLIMP也可用于直接省略任何墙壁准备步骤的组织。作为一个例子黄化拟南芥胚轴用于植物组织源。同样，一个内切葡聚糖酶是用来确定的半纤维素xyloglucan的结构。酶和组织材料可以代替所需的分析，根据使用相同的步骤。 <ol><li>收获一个黄化苗和它放到一个空的MALDI靶盘，让苗干。 </li><li>添加在所需的位置上干苗木0.5μL内切葡聚糖酶（0.4U/μl，50MM酸铵溶解，PH4.5）。确保酶下降触及部分目标板。 </li><li>放置在密闭容器中，饱和湿度的MALDI靶盘，以避免酶的下降变干。在会议厅内孵育1板6H在37 ° C。 </li><li>干MALDI靶盘，在真空条件下的植物组织和酶下降。 </li><li>加入0.5μL矩阵（道亨银行;为10mg / L）的每个干酶点之上。静置2分钟。 </li><li>在真空条件下干燥MALDI靶盘。 </li><li>分析样品的MALDI - TOF。地方组织和酶/矩阵（S）现货成的MALDI - TOF质谱仪的目标板。机应积极reflectron模式，加快20000V的电压和延迟为350 nm;选定的质量范围应达500-3000（可能会改变依赖于预期的低聚物）。 </li><li>启动激光发射到基质/样品的晶体组织旁边。确保你不打组织本身，在这种情况下，分子的飞行时间将关闭。收集20-50％左右谱斑，这是编译生成一个代表性的平均光谱（图3）。代表特定寡糖的离子可以由他们过充质量（M / Z）的比例确定。离子强度（离子身高），可报告和所有感兴趣的离子应增加高达100％，在每个样品中的低聚糖的相对丰度。 </li></ol> 5。代表性的成果在拟南芥中的半纤维素xyloglucan OLIMP分析的一个例子是如图2所示。由于离子质量差异和特点已知酶（内切葡聚糖酶）的特异性的离子可以被分配到具体的寡糖结构（图2A）。显然无法区分，结构异构体。各种低聚糖的相对丰度测定的基本假设（图2B）是他们的质谱响应因子这些低聚糖是非常类似的。如下所示，OLIMP量化高度重复性。然而，请注意，该方法的鲁棒性是高度依赖的信号信噪比的各种低聚糖离子。例如低聚糖的盐或较低的数额污染可以减少这个比例。 该组织本身（原位分析）的OLIMP分析，可以非常小和定义地区的研究涉及很少的样品制备。这里介绍的例子（图3）没有质的（相同的离子），但数量差异（离子强度的变化）之间的拍摄和拟南芥幼苗根组织的观察。 OLIMP方法的排列，从而导致组织的“成像”。 <img src="/files/ftp_upload/2046/2046fig1.jpg" alt="图1" /> 图1。OLIMP使用作为一种植物源的整个拟南芥幼苗的过程流程图。首先，该组织是浸渍和细胞壁物质准备（图片修改野等10）。然后释放一个特定的壁多糖寡糖使用特定的水解酶。最后，可溶性低聚糖的相对丰度使用MALDI - TOF质谱测定。 <img src="/files/ftp_upload/2046/2046fig2.jpg" alt="图2" /> 图2。OLIMP确定在拟南芥中的黄化苗xyloglucan低聚糖的相对丰度。一）代表xyloglucan寡糖的质谱，每个离子代表一个特定的寡糖结构，结构异构体无法加以区分。 B）为低聚糖的相对丰度测定相应条形图。 <img src="/files/ftp_upload/2046/2046fig3.jpg" alt="图3" /> 图3。 原位 OLIMP使用为例，分析拟南芥黄化苗。每种酶/消化液滴（彩色圆圈）可以独立进行分析，和一个可以得到相应的质谱和分析。 <img src="/files/ftp_upload/2046/2046fig4.jpg" alt="图4" /> 图4。OLIMP，消化，用木聚糖酶（Megazyme）芒草叶材料获得的半纤维素木聚糖谱。

<ol>
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OLIMP这里介绍的方法，使一个非常敏感和快速的分析，在细胞外基质聚合物。 OLIMP结合酶的释放与随后的MALDI - TOF分析的低聚物。一个的MALDI - TOF谱的生成需要不到一分钟，因此，OLIMP是适合的应用范围广泛，包括高通量的研究，如突变屏幕。 OLIMP不仅限于植物多糖，但有可能被应用于广泛的聚合物，只有通过具体的水解酶限制。然而，OLIMP限制，不能得到的聚合物的绝对丰度。 正如前面提到OLIMP可用于研究多种，在细胞外基质的物种多样性多糖的结构。作为一个例子，如图4代表主要草种的半纤维素，聚糖OLIMP频谱。在这里，来自温带草芒草的细胞壁物质消化使用木聚糖酶。

<table><tbody><tr><td>2,5-dihydroxybenzoic acid</td><td>Sigma-Aldrich</td><td>37550</td><td>10mg/mL in water</td></tr><tr><td>BioRex MSZ 501(D) Resin</td><td>Bio-Rad</td><td>142-7425</td><td></td></tr><tr><td>Endoglucanase</td><td>Megazyme</td><td>E-CELTR</td><td></td></tr><tr><td>Xylanase M6</td><td>Megazyme</td><td>E-XYRU6</td><td></td></tr><tr><td>3mm metal balls</td><td>Retsch</td><td>22.455.0011</td><td></td></tr><tr><td>Beat mill</td><td>Retsch</td><td>Mixer Mill MM400</td><td></td></tr><tr><td>MALDI-TOF</td><td>Shimadzu Corporation</td><td>Axima Performance</td><td></td></tr><tr><td>MALDI target plate</td><td>Kratos Analytical</td><td>DE4555TA</td><td></td></tr><tr><td>SpeedVac</td><td>Eppendorf</td><td>Vacufuge 5301</td><td></td></tr><tr><td>Vacuum manifold</td><td>EMD Millipore</td><td>MSVMHTS00</td><td></td></tr><tr><td>Vacuum pump</td><td>Welch Allyn</td><td>DryFast Ultra 2032</td><td></td></tr></tbody></table>

oligo mass profiling (olimp) of extracellular polysaccharides

介导的细胞对环境的直接接触是在许多生物细胞外基质。细胞外基质的一个共同点是，它们包含在复杂形式的糖蛋白糖基，蛋白多糖，和/或多糖。例子包括人类和动物细胞组成的纤维状蛋白和蛋白多糖或多糖的植物和真菌的细胞壁主要的细胞外基质，蛋白多糖/糖脂细菌的细胞壁。所有这些glycostructures都在细胞与细胞和细胞环境的通信和信号中扮演重要的角色。 一个非凡的细胞外基质的复杂的例子是在目前较高的植物细胞墙。他们的墙几乎完全是由糖，干重的75％，这个星球上最丰富的生物聚合物组成。因此，研究如何最好的利用这些材料作为一个碳中性的可再生资源，以取代化石燃料衍生石化进行。燃料转换的主要挑战仍然是酶解或化学降解，由于在目前这种独特的生物复合材料的独特glycostructures墙顽抗。 在这里，我们目前的植物细胞壁glycostructures的快速，灵敏的分析方法。这种方法OLIGO质谱分析（OLIMP）是基于从墙体材料促进特定glycosylhydrolases和后续分析的可溶性寡糖混合物使用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF/MS的低聚糖的酶释放1）（图1）。 OLIMP要求墙壁只有5000细胞为一个完整的分析，可以对组织本身2的执行，并适合高通量分析 3 。虽然由OLIMP溶解低聚糖的绝对数额不能确定的各种寡糖离子的相对丰度，可以划定从大众给予在墙上的原生多糖的替代模式的见解光谱。 OLIMP可用于分析的墙聚合物的种类繁多，仅由 4个特定酶的可用性的限制。例如，在植物细胞壁的酶聚合物的分析可用来分析的半纤维素xyloglucan 使用xyloglucanase 5，11，12，13，木聚糖使用远藤-β-（1-4），木聚糖酶6,7 ，或使用相结合的一个多聚半乳糖醛酸酶和methylesterase 8果胶多糖。此外，使用OLIMP glycosylhydrolase，甚至糖基转移酶活动相同的原则，可以监测和确定9。

Watch this Scientific Journal Video about OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides at JoVE.com

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

描述一个快速的方法是争取到在细胞外基质多糖结构的见解。该方法采用glycosylhydrolases特异性，灵敏度和质量允许微量的材料加以分析法的优势。这种技术直接用于组织本身的适应性。

胞外多糖的OLIGO质谱分析（OLIMP）

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

oligo-mass-profiling-olimp-of-extracellular-polysaccharides

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Biology

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

13.6K 次观看.  University of California, Berkeley. 描述一个快速的方法是争取到在细胞外基质多糖结构的见解。该方法采用glycosylhydrolases特异性，灵敏度和质量允许微量的材料加以分析法的优势。这种技术直接用于组织本身的适应性。

视频: OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

改善凝胶还原β-消除综合O连接和磺基糖组学质谱

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

科研

化学

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

细胞外基质的Glycoproteomics:使用质谱联用完整糖肽分析的方法

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

生物化学

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 ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

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 ...

免疫与感染

Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The identification and characterization of these biomolecules can improve knowledge about their secretion pathways and help to manipulate them. Furthermore, some of these biomolecules are also interesting in terms of biotechnological applications. Described here are two protocols for easy and rapid isolation of cyanobacterial released carbohydrate polymers and proteins. The method for isolation of released carbohydrate polymers is based on conventional precipitation techniques of polysaccharides in aqueous solutions using organic solvents. This method preserves the characteristics of the polymer and simultaneously avoids the presence of contaminants from cell debris and culture medium. At the end of the process, the lyophilized polymer is ready to be used or characterized or can be subjected to further rounds of purification, depending on the final intended use. Regarding the isolation of the cyanobacterial exoproteome, the technique is based on the concentration of the cell-free medium after removal of the major contaminants by centrifugation and filtration. This strategy allows for reliable isolation of proteins that reach the extracellular milieu via membrane transporters or outer membrane vesicles. These proteins can be subsequently identified using standard mass spectrometry techniques. The protocols presented here can be applied not only to a wide range of cyanobacteria, but also to other bacterial strains. Furthermore, these procedures can be easily tailored according to the final use of the products, purity degree required, and bacterial strain.

Here, protocols for the isolation of cyanobacterial released carbohydrate polymers and isolation of their exoproteomes are described. Both procedures embody key steps to obtain polymers or proteins with high purity degrees that can be used for further analysis or applications. They can also be easily adapted according to specific user needs.

向外观察:分离氰化物释放的碳水化合物聚合物和蛋白质

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The ...

Polysome Profiling without Gradient Makers or Fractionation Systems

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories that have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here, a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided. Strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol. However, this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.

This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

无需梯度测定器或分馏系统的多聚体分析

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...

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 ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

糖基谱研究植物细胞壁聚合物，用微阵列

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

生物学

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

胞外多糖的OLIGO质谱分析（OLIMP）

摘要

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此视频中的章节

改善凝胶还原β-消除综合O连接和磺基糖组学质谱

细胞外基质的Glycoproteomics:使用质谱联用完整糖肽分析的方法

植物甘露聚糖细胞壁多糖的结构表征

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

鞘糖脂抗原的质谱分析

向外观察:分离氰化物释放的碳水化合物聚合物和蛋白质

无需梯度测定器或分馏系统的多聚体分析

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

糖基谱研究植物细胞壁聚合物，用微阵列

再加上高灵敏度指纹碳水化合物自动分析模块化高通量筛选胞外多糖平台