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1.
糖组学是继基因组学、蛋白质组学之后,又一门新兴的学科,其主要是研究糖分子的结构与功能.糖是一类比核酸、蛋白质更加独特的生物分子,它们不仅是生物体储存能量和释放能量的主要物质,更是生物体内的信息传递分子,并且在生理和病理过程中扮演着重要的角色,如细胞间的识别作用、炎症以及自身免疫疾病等.在结构上,糖类物质更为复杂,具有宏观不均一性(蛋白质上有多个糖基化位点)和微观不均一性(同一结合位点上可以连接不同的多糖),所以糖链的结构解析一直是糖组学研究的难题.相较于传统的分析方法,质谱法具有高灵敏度、高精度、高通量等优势,被认为是在糖链结构解析过程中重要的分析方法.本文综述了质谱、多级质谱、液相色谱-质谱、毛细管电泳-质谱等方法在糖组学中糖链结构解析的研究进展.  相似文献   

2.
糖组学是继基因组学、蛋白质组学之后,又一门新兴的学科,其主要是研究糖分子的结构与功能.糖是一类比核酸、蛋白质更加独特的生物分子,它们不仅是生物体储存能量和释放能量的主要物质,更是生物体内的信息传递分子,并且在生理和病理过程中扮演着重要的角色,如细胞间的识别作用、炎症以及自身免疫疾病等.在结构上,糖类物质更为复杂,具有宏观不均一性(蛋白质上有多个糖基化位点)和微观不均一性(同一结合位点上可以连接不同的多糖),所以糖链的结构解析一直是糖组学研究的难题.相较于传统的分析方法,质谱法具有高灵敏度、高精度、高通量等优势,被认为是在糖链结构解析过程中重要的分析方法.本文综述了质谱、多级质谱、液相色谱-质谱、毛细管电泳-质谱等方法在糖组学中糖链结构解析的研究进展.  相似文献   

3.
基于超滤膜辅助的糖蛋白全N-连接糖链的富集和质谱解析   总被引:1,自引:1,他引:0  
糖基化作为一种常见的蛋白质翻译后修饰,对蛋白质的空间结构、生物功能等具有重要的影响.解析糖蛋白糖链结构有助于更清楚地认识糖蛋白及其功能.本研究建立了一种基于超滤膜富集血清中糖蛋白全N-连接糖链,并利用质谱技术对糖链结构进行分析的方法.根据糖蛋白及其糖链结构之间的分子质量差异,利用Millipore公司的10 ku超滤膜富集血清糖蛋白上酶解(PNGase F)释放的全N-连接糖链,并使用MALDI-TOF/TOF-MS解析糖链结构.通过该技术可以从血清中富集并鉴定到23种独特的N-连接的糖链结构,并且利用二级质谱进行了结构确认.该方法可以被用于从大量生物样本中富集糖蛋白全N-连接糖链,可以达到快速、高通量地解析糖蛋白N-连接糖链的目的.  相似文献   

4.
糖类抗原125(CA125)被认为是卵巢癌诊断的“金标准”,但在临床应用中普遍存在着特异性不高的问题.肿瘤形成和发展过程中常伴有糖基化修饰异常和糖链结构的改变,不同的肿瘤具有特异的异常糖链结构.近年来,借助凝集素芯片、多重质谱分析等糖蛋白组学和糖组学研究技术,发现不同来源CA125的O-糖链和N-糖链结构存在着明显的微观不均一性,以这些特征性糖链结构为标志物,可以显著提高CA125对卵巢癌的诊断特异性.在过去的10年,研究者们除对CA125糖链结构和糖基化模式做了深入的研究外,还利用糖组的研究方法,直接对来自卵巢癌患者血液、体液(腹水、囊泡液等)中糖蛋白的糖链做了精细的结构解析,结果显示,可有效鉴别卵巢癌患者和健康志愿者的特异性N-糖链结构,有可能成为灵敏度高和特异性好的卵巢癌生物标志物.卵巢癌生物标志物研究发展的总趋势是从传统的对蛋白质的定性和定量研究,逐步转向于对标志物糖基化修饰和特异性糖链结构的鉴定以及定量分析.本文从糖组学的视角,对卵巢癌标志物糖组学的研究现状及发展趋势进行了综述和展望.  相似文献   

5.
糖类抗原125(CA125)被认为是卵巢癌诊断的"金标准",但在临床应用中普遍存在着特异性不高的问题.肿瘤形成和发展过程中常伴有糖基化修饰异常和糖链结构的改变,不同的肿瘤具有特异的异常糖链结构.近年来,借助凝集素芯片、多重质谱分析等糖蛋白组学和糖组学研究技术,发现不同来源CA125的O-糖链和N-糖链结构存在着明显的微观不均一性,以这些特征性糖链结构为标志物,可以显著提高CA125对卵巢癌的诊断特异性.在过去的10年,研究者们除对CA125糖链结构和糖基化模式做了深入的研究外,还利用糖组的研究方法,直接对来自卵巢癌患者血液、体液(腹水、囊泡液等)中糖蛋白的糖链做了精细的结构解析,结果显示,可有效鉴别卵巢癌患者和健康志愿者的特异性N-糖链结构,有可能成为灵敏度高和特异性好的卵巢癌生物标志物.卵巢癌生物标志物研究发展的总趋势是从传统的对蛋白质的定性和定量研究,逐步转向于对标志物糖基化修饰和特异性糖链结构的鉴定以及定量分析.本文从糖组学的视角,对卵巢癌标志物糖组学的研究现状及发展趋势进行了综述和展望.  相似文献   

6.
目前,小分子药物研究已经进入平台期,而大分子生物药物迅速发展起来。糖药物作为生物药中尤为重要的一类,被广泛应用于肿瘤、心脑血管疾病等的预防和治疗。然而无论骨架糖链还是修饰性糖链,都具有结构复杂且微不均一性的特点,这对现代分析技术提出了较高的要求。随着软电离技术、多级质谱技术的发展以及分辨率和灵敏度的提高,质谱技术已被广泛应用于糖类药物的分析和表征。对糖药物、质谱技术进行了简要的介绍,并以多糖药物和糖缀合物类药物为例阐述了质谱在糖药物结构表征方面的技术进展,此外还就质谱在糖药物新药及仿制药物研发和注册方面的应用进行了讨论。  相似文献   

7.
蛋白质糖基化作为最普遍、最重要的蛋白质修饰,一直是组学研究的焦点之一.近十几年来,N-连接糖蛋白质组学研究普遍采用的方法是将糖链与所修饰的多肽分开进行分析.该策略虽降低了分析难度,却也丢失了糖链与蛋白质糖基化位点间重要的对应关系信息.近年来,完整糖肽的质谱分析策略和方法逐步建立起来.总体而言,要实现对完整糖肽的直接质谱分析,首先需要从复杂样品中富集完整糖肽以消除非糖基化多肽对完整糖肽分析的影响,然后在质谱分析中还需要根据糖肽特性调整相应质谱分析参数,最后在后续数据分析中还需要开发相应的分析软件以完成完整糖肽中多肽序列和糖链组成或结构的鉴定.本文即从以上三个主要方面系统阐述目前N-完整糖肽分析中常用的质谱和数据分析策略和方法,并进一步在糖肽谱图识别、母离子单同位素分子质量校正、数据库选择以及假阳性率评估和控制等方面都进行了逐一探讨.完整糖肽的直接质谱分析有助于获取糖链和糖基化位点间的对应关系信息,可为生物标志物发现和疾病致病机理等研究提供更有力的糖蛋白质组学研究工具.  相似文献   

8.
蛋白质糖基化作为最普遍、最重要的蛋白质修饰,一直是组学研究的焦点之一.近十几年来,N-连接糖蛋白质组学研究普遍采用的方法是将糖链与所修饰的多肽分开进行分析.该策略虽降低了分析难度,却也丢失了糖链与蛋白质糖基化位点间重要的对应关系信息.近年来,完整糖肽的质谱分析策略和方法逐步建立起来.总体而言,要实现对完整糖肽的直接质谱分析,首先需要从复杂样品中富集完整糖肽以消除非糖基化多肽对完整糖肽分析的影响,然后在质谱分析中还需要根据糖肽特性调整相应质谱分析参数,最后在后续数据分析中还需要开发相应的分析软件以完成完整糖肽中多肽序列和糖链组成或结构的鉴定.本文即从以上三个主要方面系统阐述目前N-完整糖肽分析中常用的质谱和数据分析策略和方法,并进一步在糖肽谱图识别、母离子单同位素分子质量校正、数据库选择以及假阳性率评估和控制等方面都进行了逐一探讨.完整糖肽的直接质谱分析有助于获取糖链和糖基化位点间的对应关系信息,可为生物标志物发现和疾病致病机理等研究提供更有力的糖蛋白质组学研究工具.  相似文献   

9.
不同转移潜能膀胱癌细胞糖组相对定量分析   总被引:1,自引:1,他引:0  
膀胱癌是发生在膀胱黏膜组织上的一种恶性肿瘤,是泌尿系统中最常见的恶性肿瘤,早期(非肌层浸润型膀胱癌)阶段的诊断和治疗是降低膀胱癌死亡率的最有效方式.肿瘤的发生过程与糖链表达的改变有着密切的关系,而定量分析膀胱癌发生过程中糖链的表达变化尚未有研究.本研究以2株人膀胱正常上皮细胞系(HCV29、HUCV1),1株非肌层浸润性膀胱癌细胞系(KK47),和3株浸润性膀胱癌细胞系(YTS1、J82、T24)为研究材料,应用本室建立的利用乙酰肼修饰糖链唾液酸,以及[12C6]-和[13C6]-苯胺同位素修饰糖链还原性末端技术,然后利用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS),进行膀胱上皮细胞不同病理状态的糖组相对定量分析.从6株细胞中共鉴定出52种N-连接糖链结构,并定量分析了不同类型的糖链在不同细胞中的分布差异,发现唾液酸化、岩藻糖化的N-连接糖链在膀胱癌肿瘤细胞恶化过程中呈现显著升高的趋势,同时平分型糖链和高甘露糖型N-连接糖链也呈表达升高趋势,说明这些糖链结构的表达变化与膀胱癌发生关系密切,从而有助于进一步阐明膀胱癌发生过程中糖链相关的分子机理.  相似文献   

10.
蛋白质糖基化修饰的鉴定是蛋白质翻译后修饰分析中最具挑战性的任务之一,近几年尤其受到关注.快速发展的质谱技术为规模化的蛋白质糖基化修饰研究提供了有效的手段.与其他基于质谱技术的翻译后修饰鉴定相比,糖基化鉴定的难点在于糖链是大分子而且存在微观不均一性,另外糖链本身可以在串联质谱中碎裂且与肽段的碎裂规律不同,导致蛋白质组学的质谱解析方法和软件难以完整地鉴定肽段序列和糖链结构.完整N-糖肽的鉴定是糖基化分析的热点内容之一,针对N-糖肽的鉴定,近年来,人们开发了多种多样的质谱解析方法,其中包括用N-糖酰胺酶切除糖链后鉴定N-糖基化位点的方法、基于电子转运裂解的糖肽肽段鉴定、基于高能碰撞裂解与电子转运裂解联用或碰撞诱导裂解与三级谱联用的完整N-糖肽鉴定等等.本文对这些质谱解析方法进行了整理和综述,简要指出了目前完整糖肽鉴定软件存在的一些不足,展望了未来的发展方向.  相似文献   

11.
Negative ion electrospray (ESI) fragmentation spectra derived from anion-adducted glycans were evaluated for structural determination of N-linked glycans and found to be among the most useful mass spectrometric techniques yet developed for this purpose. In contrast to the more commonly used positive ion spectra that contain isobaric ions formed by losses from different regions of the molecules and often lead to ambiguous deductions, the negative ion spectra contain ions that directly reflect structural features such as the branching pattern, location of fucose, and the presence of bisecting GlcNAc. These structural features are sometimes difficult to determine by traditional methods. Furthermore, the spectra give structural information from mixtures of isomers and from single compounds. The method was evaluated with well-characterized glycans from IgG and used to explore structures of N-linked glycans released from serum glycoproteins with the aim of identifying biomarkers for cancer. Quantities of glycans were measured by ESI and by matrix-assisted laser desorption ionization mass spectrometry; each technique produced virtually identical results for the neutral desialylated glycans.  相似文献   

12.
Binding of cell surface glycans by influenza hemagglutinin controls viral attachment and infection of host cells. This binding is a three-way interaction between viral proteins, host glycans, and viral glycans; many structural details of this interaction have been difficult to resolve. Here, we use a series of 100-ns molecular dynamics simulations to further analyze available crystallographic data on hemagglutinin-ligand interactions. Based on our simulations, we predict that the viral glycans contact the host glycans within 1-2 residues of the ligand-binding site. We also predict that the glycan-glycan interactions contain both stabilizing and destabilizing components. These predictions suggest a structural means to explain why changes to viral glycosylation alter the efficiency and selectivity of ligand binding. We also predict that the proximity of these interactions to the ligand-binding pocket will impact the binding affinity of small glycomimetic ligands analogous to the influenza neuraminidase inhibitors currently in clinical use.  相似文献   

13.
Carbohydrate moieties of salivary mucins play various roles in life processes, especially as a microbial trapping agent. While structural details of the salivary O-glycans from several mammalian sources are well studied, very little information is currently available for the corresponding N-glycans. The existence of N-glycans alongside O-glycans on mucin isolated from rat sublingual gland has previously been implicated by total glycosyl compositional analysis but the respective structural data are both lacking. The advent of facile glycomic mapping and sequencing methods by mass spectrometry (MS) has enabled a structural reinvestigation into many previously unsolved issues. For the first time, high energy collision induced dissociation (CID) MALDI-MS/MS as implemented on a TOF/TOF instrument was applied to permethyl derivatives of mucin type O-glycans and N-glycans, from which the linkage specific fragmentation pattern could be established. The predominant O-glycans carried on the rat sublingual mucin were defined as sialylated core 3 and 4 types whereas the N-glycans were determined to be non-bisected hybrid types similarly carrying a sialylated type II chain. The masking effect of terminal sialylation on the tight binding of rat sublingual mucin to Galβ1→4GlcNAc specific lectins and three oligomannose specific lectins were clearly demonstrated in this study.  相似文献   

14.
High-throughput and automated techniques (mass spectrometry in particular) allow increasingly rapid structural analysis of complex glycans. Information concerning the primary structure (composition, sequence and linkages), three-dimensional structure (including dynamics) and interactions of glycans is now available in sufficient quantity to justify the maintenance of databases and search facilities. Several such resources (both commercial and open access) are now available as web tools. To derive the full value of glycan databases, it will be necessary to develop a universally accepted machine-readable structural representation of glycans.  相似文献   

15.
Over a half of all proteins are glycosylated, and their proper glycosylation is essential for normal function. Unfortunately, because of structural complexity of nonlinear branched glycans and the absence of genetic template for their synthesis, the knowledge about glycans is lagging significantly behind the knowledge about proteins or DNA. Using a recently developed quantitative high throughput glycan analysis method we quantified components of the plasma N-glycome in 99 children with attention-deficit hyperactivity disorder (ADHD), 81 child and 5 adults with autism spectrum disorder, and a total of 340 matching healthy controls. No changes in plasma glycome were found to associate with autism spectrum disorder, but several highly significant associations were observed with ADHD. Further structural analysis of plasma glycans revealed that ADHD is associated with increased antennary fucosylation of biantennary glycans and decreased levels of some complex glycans with three or four antennas. The design of this study prevented any functional conclusions about the observed associations, but specific differences in glycosylation appears to be strongly associated with ADHD and warrants further studies in this direction.  相似文献   

16.
Differences in the fertilization behavior of Xenopus borealis from X. laevis and X. tropicalis suggest differences in the glycosylation of the egg jellies. To test this assumption, O-linked glycans were chemically released from the egg jelly coat glycoproteins of X. borealis. Over 50 major neutral glycans were observed, and no anionic glycans were detected from the released O-glycan pool. Preliminary structures of ~30 neutral oligosaccharides were determined using matrix-assisted laser desorption/ionization (MALDI) infrared multiphoton dissociation tandem mass spectrometry (MS). The mass fingerprint of a group of peaks for the core-2 structure of O-glycans was conserved in the tandem mass spectra and was instrumental in rapid and efficient structure determination. Among the 29 O-glycans, 22 glycans contain the typical core-2 structure, 3 glycans have the core-1 structure and 2 glycans contained a previously unobserved core structure with hexose at the reducing end. There were seven pairs of structural isomers observed in the major O-linked oligosaccharides. To further elucidate the structures of a dozen O-linked glycans, specific and targeted exoglycosidase digestions were carried out and the products were monitored with MALDI-MS. Reported here are the elucidated structures of O-linked oligosaccharides from glycoproteins of X. borealis egg jelly coats. The structural differences in O-glycans from jelly coats of X. borealis and its close relatives may provide a better understanding of the structure-function relationships and the role of glycans in the fertilization process within Xenopodinae.  相似文献   

17.
The structural diversity of glycoprotein N-linked oligosaccharides is determined by the expression and regulation of glycosyltransferase activities and by the availability of the appropriate acceptor/donor substrates. Cells in different tissues and in different developmental stages utilize these control points to manifest unique glycan expression patterns in response to their surroundings. The activity of a Toll-like receptor, called Tollo/Toll-8, induces a pattern of incompletely defined, but neural specific, glycan expression in the Drosophila embryo. Understanding the full extent of the changes in glycan expression that result from altered Tollo/Toll-8 signaling requires characterization of the complete N-linked glycan profile of both wild-type and mutant embryos. N-Linked glycans harvested from wild-type or mutant embryos were subjected to direct structural analysis by analytic and preparative high pressure liquid chromatography, by multidimensional mass spectrometry, and by exoglycosidase digestion, revealing a predominance of high mannose and paucimannose glycans. Di-, mono-, and nonfucosylated forms of hybrid, complex biantennary, and triantennary glycans account for 12% of the total wild-type glycan profile. Two sialylated glycans bearing N-acetylneuraminic acid were detected, the first direct demonstration of this modification in Drosophila. Glycan profiles change during normal development consistent with increasing alpha-mannosidase II and core fucosyl-transferase enzyme activities, and with decreasing activity of the Fused lobes processing hexosaminidase. In tollo/toll-8 mutants, a dramatic, expected loss of difucosylated glycans is accompanied by unexpected decreases in monofucosylated and nonfucosylated hybrid glycans and increases in some nonfucosylated paucimannose and biantennary glycans. Therefore, tollo/toll-8 signaling influences flux through several processing steps that affect the maturation of N-linked glycans.  相似文献   

18.
The natural heterogeneity of glycoprotein glycans requires that chromatography be an essential part of structural elucidation. The isomeric nature of oligosaccharide structure requires chromatography which is selective for not only composition, size and anomerity, but also ring substitutions and branching configurations. HPAEC is sensitive to these structural features and thus, has become an important new method for understanding the elusive function of glycoprotein glycans.  相似文献   

19.
Sulfated polysaccharides, like the glycosaminoglycan (GAG) heparin, are known to exhibit anticoagulant properties when certain structural features are present. The structural requirement for this action is well-established for heparin, in which a pentasaccharide motif plays a key role for keeping the high-affinity interaction to antithrombin. Over the last years of this glycomic era, several novel anticoagulant sulfated glycans have been described. Those from marine sources have been awakening special attention mainly because of their impressive anticoagulant effects together with structural uniqueness. The commonest of these glycans are the sulfated fucans (SFs), the sulfated galactans (SGs), and the marine invertebrate GAGs like the fucosylated chondroitin sulfate and ascidian dermatan sulfate. Since these marine sulfated glycans do not bear within their polymeric chains the specific pentasaccharide motif of heparin, other structural features must be necessary to trigger the anticoagulant effect. The objective of this report is to present the anticoagulant motifs of the marine SFs, SGs and GAGs.  相似文献   

20.
It has been shown that free oligosaccharides derived from N-linked glycans accumulate in the cytosol of animal cells. Most of the glycans have only a single GlcNAc at their reducing termini (Gn1 glycans), whereas the original N-glycans retain N,N′-diacetylchitobiose at their reducing termini (Gn2 glycans). Under the conditions of high-performance liquid chromatography (HPLC) mapping established for pyridylamine (PA)-labeled Gn2 N-glycans, Gn1 glycans are not well retained on reversed-phase HPLC, making simultaneous analysis of Gn1 and Gn2 glycans problematic. We introduced a dual gradient (i.e., pH and butanol gradient) for the separation of Gn1 and Gn2 glycans in a single reversed-phase HPLC. Determination of elution time for various standard Gn2 high-mannose-type glycans, as well as Gn1 glycans found in the cytosol of animal cells, showed that elution of Gn1 and Gn2 glycans could be separated. Sufficient separation for most of the structural isomers could be achieved for Gn1 and Gn2 glycans. This HPLC, therefore, is a powerful method for identification of the structures of PA-labeled glycans, especially Gn1-type glycans, isolated from the cytosol of animal cells.  相似文献   

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