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基质辅助激光解吸电离质谱分析糖类物质 总被引:1,自引:0,他引:1
基质辅助激光解吸电离质谱(MALDI-MS)是一种样品无需衍生、图谱解析简单、灵敏度高、快速便捷的分析生物样品结构的方法,已被广泛用于糖类物质的结构分析。此技术与HPLC、糖苷酶外切技术以及各种串联质谱等技术结合使用,可给出糖类物质详细的结构信息。本文介绍了基质辅助激光解吸(MALDI)离子化技术的原理、特点、与飞行时间质量分析器(TOF)联用时的相关技术和裂解方式,以及MALDI-MS在分析糖类物质时选用的基质、样品的制备、糖链碎片分析的方法和在不同糖型分析中的应用,展示了它的发展前景。随着MALDI对糖类物质分析时基质的改进、质谱分辨率的提高、质量检测范围的扩大,MALDI-MS技术必将成为糖类物质分析中强有力的工具。 相似文献
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高效液相色谱在生物医药研究中的应用第四讲高效液相色谱法在糖类研究中应用 总被引:1,自引:0,他引:1
随着科学技术的迅速发展和人们对单糖、双糖、寡糖、尤其是多糖的功能的不断深入了解,糖的研究已成为当今化学家和生物学家的研究“热点”。例如肿瘤的发生与细胞表面的糖类的变化有着密切的关系;人类血型的不同主要由于血红细胞中糖基的不同;以及最近发现的糖链在分子生物学中具有决定性的作用。所有这些发现和发明均有赖于糖类方法学的突飞猛进。高效液相色谱(HPLC)就是糖类研究方法学中的重要组 相似文献
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唾液酸酶和唾液酸糖基转移酶在生物体内许多重要的生理过程中发挥着不可或缺的作用.一般而言,唾液酸酶可以断裂糖缀合物末端与唾液酸残基相连接的糖苷键而水解唾液酸糖缀合物;唾液酸糖基转移酶以CMP-Neu5Ac为底物将唾液酸残基转移至新的糖基受体上.综述了与唾液酸相关的酶的分类、结构与催化反应机理;唾液酸酶和唾液酸糖基转移酶催化反应在有机合成中的应用;以及唾液酸酶的检测方法.对未来与唾液酸相关的酶的研究与应用进行了展望. 相似文献
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肝素是一类结构复杂的高分子糖类,以N-硫酸、6-O硫酸氨基葡萄糖和2-O硫酸艾杜糖醛酸为主要成分组成二糖。常见的肝素注射液是以未分级肝素为原料纯化灭菌制备而来,在临床上使用更为广泛的是将肝素降解得到的低分子片段,低分子肝素保留了肝素糖链基本结构,但是不同制备工艺导致其具有不同的还原及非还原端。本研究以肝素类药物为研究对象,使用肝素裂解酶玉,域及芋将肝素注射液、低分子肝素注射液(达肝素、那屈肝素、依诺肝素)、化学合成的类肝素(磺达肝素)进行酶催化降解产生二糖,结合强阴离子交换色谱( Strong anion exchange high performance liquid chromatography,SAX-HPLC)以及紫外检测器在线分析,使用市售肝素二糖标准品确定各种二糖结构。此外,使用反向离子对色谱和电喷雾质谱联用分析磺达肝素中的甲基二糖以及达肝素和那屈肝素中的脱氨二糖结构,为肝素以及类肝素药物的质量控制提供更为精确的结构信息。 相似文献
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建立了一种用非特异性酶链酶蛋白酶 E(Pronase E)从糖蛋白上释放N-糖链的方法. 以牛胰核糖核酸酶 B(Ribo B)和鸡白蛋白(Chicken Albumin)为材料, 用Pronase E代替N-糖苷酶 F(PNGase F)释放N-糖链. 当蛋白酶质量与糖蛋白质量比为1∶1时, 得到只带一个天冬氨酸(Asn)的闭环N-糖链, 称其为糖氨酸(glycan-Asn), 这样既为糖链引入了天然的-NH2活性基团, 同时还保持了糖链原有的还原端闭环结构. 以9-氯甲酸芴甲酯(Fmoc-Cl)为衍生试剂对解离后的糖氨酸进行衍生, 采用高效液相色谱-电喷雾质谱联用技术(HPLC-ESI/MS)对Fmoc-Cl糖氨酸衍生物进行分析, 建立了糖蛋白的Pronase E酶解、微量糖氨酸的Fmoc-Cl衍生以及糖氨酸衍生物的HPLC-ESI/MS分析方法, 该方法保持了N-糖链的天然结构, 便于以-NH2为功能基团进一步进行荧光标记、分离制备以及糖链与蛋白质的相互作用研究. 相似文献
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Globo-H作为一种和乳腺癌、前列腺癌相关的复杂糖类抗原,其发现为糖类疫苗开发和癌症免疫治疗带来了机遇,但如何高效、高纯地获得合成糖类抗原,以供研究和临床应用,也向寡糖合成方法学提出了挑战.综述了1995年Danishefsky首次以糖烯组装策略全合成Globo-H以来的各种新方法,如:Schmidt的三氯乙酰亚胺酯法、Boons的双向糖苷化法、Wong的基于糖基给体活性差异的一锅煮策略、Seeberger的液相线性合成和固相自动组装法、Huang的多组份反复预活化一锅煮法和最新报道的酶法.就糖合成方法学而言,硫苷法依旧可称为"明星方法",糖烯、三氯乙酰亚胺酯和氟代糖也普遍采用,磷酸酯糖基给体在固相合成中的应用正显示出其新的活力.这些方法代表了当今糖化学的水平和发展趋势. 相似文献
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Carbohydrate components in glycoproteins play a critical role in health and disease and specificity of glycoprotein biomarkers can be greatly enhanced by analysis of their sugar components containing frequently different isomers. We show that two glycan isomers, 2,3-sialyllactose and 2,6-sialyllactose (important in cancer), can be distinguished voltammetrically after their modification with osmium(VI) complexes. Using SWV at different frequencies and CV at different scan rates we found conditions for simple discrimination between these isomers at mercury and carbon electrodes. 相似文献
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A Glycan Array-Based Assay for the Identification and Characterization of Plant Glycosyltransferases
Dr. Colin Ruprecht Dr. Max P. Bartetzko Dr. Deborah Senf Dr. Anna Lakhina Dr. Peter J. Smith Dr. Maria J. Soto Hyunil Oh Dr. Jeong-Yeh Yang Dr. Digantkumar Chapla Dr. Daniel Varon Silva Prof. Dr. Mads H. Clausen Prof. Dr. Michael G. Hahn Prof. Dr. Kelley W. Moremen Prof. Dr. Breeanna R. Urbanowicz Prof. Dr. Fabian Pfrengle 《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2020,132(30):12593-12598
Growing plants with modified cell wall compositions is a promising strategy to improve resistance to pathogens, increase biomass digestibility, and tune other important properties. In order to alter biomass architecture, a detailed knowledge of cell wall structure and biosynthesis is a prerequisite. We report here a glycan array-based assay for the high-throughput identification and characterization of plant cell wall biosynthetic glycosyltransferases (GTs). We demonstrate that different heterologously expressed galactosyl-, fucosyl-, and xylosyltransferases can transfer azido-functionalized sugar nucleotide donors to selected synthetic plant cell wall oligosaccharides on the array and that the transferred monosaccharides can be visualized “on chip” by a 1,3-dipolar cycloaddition reaction with an alkynyl-modified dye. The opportunity to simultaneously screen thousands of combinations of putative GTs, nucleotide sugar donors, and oligosaccharide acceptors will dramatically accelerate plant cell wall biosynthesis research. 相似文献
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A Glycan Array‐Based Assay for the Identification and Characterization of Plant Glycosyltransferases
Colin Ruprecht Max P. Bartetzko Deborah Senf Anna Lakhina Peter J. Smith Maria J. Soto Hyunil Oh Jeong‐Yeh Yang Digantkumar Chapla Daniel Varon Silva Mads H. Clausen Michael G. Hahn Kelley W. Moremen Breeanna R. Urbanowicz Fabian Pfrengle 《Angewandte Chemie (International ed. in English)》2020,59(30):12493-12498
Growing plants with modified cell wall compositions is a promising strategy to improve resistance to pathogens, increase biomass digestibility, and tune other important properties. In order to alter biomass architecture, a detailed knowledge of cell wall structure and biosynthesis is a prerequisite. We report here a glycan array‐based assay for the high‐throughput identification and characterization of plant cell wall biosynthetic glycosyltransferases (GTs). We demonstrate that different heterologously expressed galactosyl‐, fucosyl‐, and xylosyltransferases can transfer azido‐functionalized sugar nucleotide donors to selected synthetic plant cell wall oligosaccharides on the array and that the transferred monosaccharides can be visualized “on chip” by a 1,3‐dipolar cycloaddition reaction with an alkynyl‐modified dye. The opportunity to simultaneously screen thousands of combinations of putative GTs, nucleotide sugar donors, and oligosaccharide acceptors will dramatically accelerate plant cell wall biosynthesis research. 相似文献
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Sampling of Glycan‐Bound Conformers by the Anti‐HIV Lectin Oscillatoria agardhii agglutinin in the Absence of Sugar 下载免费PDF全文
Marta G. Carneiro Leonardus M. I. Koharudin David Ban T. Michael Sabo Pablo Trigo‐Mourino Adam Mazur Christian Griesinger Angela M. Gronenborn Donghan Lee 《Angewandte Chemie (International ed. in English)》2015,54(22):6462-6465
Lectins from different sources have been shown to interfere with HIV infection by binding to the sugars of viral‐envelope glycoproteins. Three‐dimensional atomic structures of a number of HIV‐inactivating lectins have been determined, both as free proteins and in glycan‐bound forms. However, details on the mechanism of recognition and binding to sugars are elusive. Herein we focus on the anti‐HIV lectin OAA from Oscillatoria agardhii: We show that in the absence of sugars in solution, both the sugar‐free and sugar‐bound protein conformations that were observed in the X‐ray crystal structures exist as conformational substates. Our results suggest that glycan recognition occurs by conformational selection within the ground state; this model differs from the popular “excited‐state” model. Our findings provide further insight into molecular recognition of the major receptor on the HIV virus by OAA. These details can potentially be used for the optimization and/or development of preventive anti‐HIV therapeutics. 相似文献
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西妥昔单抗具有较复杂的糖基化修饰,在抗原结合片段(Fab)和可结晶片段(Fc)的重链上都含有2个N-糖基化位点,其中Fab段的糖基化最为复杂,要研究清楚该位点的糖基化修饰,开发专一性切糖技术和稳定的聚糖比例分析方法是当前迫切需要解决的难题。以中国仓鼠卵巢(CHO)细胞表达的西妥昔单抗为研究对象,使用β-N-乙酰氨基葡萄糖苷酶(Endo F2)开发了一种快速Fab段聚糖释放的方法,利用超高效液相色谱-高分辨质谱(UPLC-HRMS)进行了定性和聚糖比例分析。第一步对抗体原液进行非变性酶切,抗体原液经超纯水稀释后,加入糖苷酶Endo F2进行酶切,通过质谱对质量数的解析,结果表明Endo F2酶切时间5 min, Fab段的聚糖就能完全切除,而Fc段的聚糖不受影响,实现了快速酶切,而且切糖具有很好的专一性。第二步对Fab段聚糖进行比例分析,将释放的聚糖经对氨基苯甲酰胺(2-AB)荧光标记后使用超高效液相色谱联用荧光检测器(FLR)进行检测,在亲水作用色谱(HILIC)柱上得到良好的分离并可以进行稳定地聚糖比例分析。3次独立试验结果表明,酶切后的质谱图基本一致,且聚糖的比例结果也基本一致,表明Endo F2酶切方法和聚糖比例分析方法都具有较好的稳定性和可靠性。此外,通过测定来自两个不同工艺生产的样品,数据显示两者的糖谱上具有非常明显的差异,表明利用开发的方法可以实现对抗体生产工艺进行监测研究,对抗体生产工艺的评估具有非常重要的意义。 相似文献
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Dai Z Kawde AN Xiang Y La Belle JT Gerlach J Bhavanandan VP Joshi L Wang J 《Journal of the American Chemical Society》2006,128(31):10018-10019
Here we present the first report on nanoparticle-based biosensing of glycan markers of diseases. The protocol relies on the competition between a nanocrystal (CdS)-tagged sugar and the target sugar for the binding sites of surface-confined lectin and monitoring the extent of competition through highly sensitive electrochemical detection of the captured nanocrystal. This development is expected to allow decentralized detection of carbohydrate moieties and lectin-carbohydrate interactions to be performed more rapidly, sensitively, inexpensively, and reliably. 相似文献
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Janardan Singh Yadav 《International journal of quantum chemistry》1983,23(4):1441-1450
The glycan moiety of the bacterial peptidoglycan consists of alternatingly β(1 → 4) linked disaccharides of N-acetylglucosamine (NAG) and its 3-O-D lactic derivative, N-acetyl β-D-muramic acid (NAM). PCILO conformational energy calculations have been carried out for NAG–NAM and NAM–NAG disaccharides to see whether or not the glycan strands possess a chitinlike structure as suggested by earlier workers. In agrement with recent experimental findings, the present results also suggest that the chitinlike structure is energetically disallowed. Furthermore, the bulky N-acetyl substituents at C2 positions of the two sugar molecules are found to be relatively less important in stabilizing mutual orientations of the two pyranosyl rings. 相似文献
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Glycans are chains of carbohydrates attached to proteins (glycoproteins and proteoglycans) or lipids (glycolipids). Glycosylation is a post-translational modification and glycans have a wide range of functions in the human body including involvement in oncological diseases. Change in a glycan structure can not only indicate the presence of a pathological process but, more importantly, in some cases also its stage. Thus, a glycan analysis has the potential to be an effective and reliable tool in cancer diagnostics. Lectins are proteins responsible for natural biorecognition of glycans; even carbohydrate moieties still attached to proteins or whole cells can be recognised by lectins, which makes them an ideal candidate for designing label-free biosensors for glycan analysis. This review seeks to summarise evidence that the glycoprofiling of biomarkers by lectin-based biosensors can be of significant help in detecting prostate cancer. 相似文献
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Prof. Cristina De Castro Dr. Immacolata Speciale Prof. Garry Duncan Dr. David D. Dunigan Dr. Irina Agarkova Prof. Rosa Lanzetta Dr. Luisa Sturiale Dr. Angelo Palmigiano Prof. Domenico Garozzo Prof. Antonio Molinaro Prof. Michela Tonetti Prof. James L. Van Etten 《Angewandte Chemie (International ed. in English)》2016,55(2):654-658
N‐glycosylation is a fundamental modification of proteins and exists in the three domains of life and in some viruses, including the chloroviruses, for which a new type of core N‐glycan is herein described. This N‐glycan core structure, common to all chloroviruses, is a pentasaccharide with a β‐glucose linked to an asparagine residue which is not located in the typical sequon N‐X‐T/S. The glucose is linked to a terminal xylose unit and a hyperbranched fucose, which is in turn substituted with a terminal galactose and a second xylose residue. The third position of the fucose unit is always linked to a rhamnose, which is a semiconserved element because its absolute configuration is virus‐dependent. Additional decorations occur on this core N‐glycan and represent a molecular signature for each chlorovirus. 相似文献
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The current project describes the chemoenzymatic modification of bovine ribonuclease B (RNase B) to contain a single glycosylation site with a known glycan. A reactive disaccharide oxazoline derivative was synthesized and stereospecifically added to deglycosylated RNase B through endo-β-N-acetylglucosaminidase M catalyzed chemoenzymatic transglycosylation. Oxazoline formation conditions were optimized using mass spectrometry, and the product verified based on its collision-induced dissociation (CID) mass spectrum. Enzymatic removal of native glycans as well as formation of the desired homogeneous product was also monitored using mass spectrometry. LC-MS(n) using four sequential rounds of CID was used to verify that the original glycosylation site had been reorganized to contain the new glycan. The techniques described herein are not limited to this analyte or glycan and should be amenable to the synthesis of numerous homogeneous glycoconjugates with judicious choice of enzyme/substrate combinations. The combined use of chemoenzymatic synthesis and mass spectrometry-based characterization shows promise for the development of homogeneous glycoprotein reference materials. A well-defined glycoprotein standard containing a single glycan of known composition, linkage and stereochemistry would be of great value for the comparison and evaluation of glycoprotein analysis techniques. 相似文献