糖组学研究中糖蛋白糖链结构分析技术

遗传信息由DNA传递至蛋白质,再经蛋白质翻译后糖基化修饰形成糖蛋白.与DNA、蛋白质相比,糖蛋白糖链结构更加多样,功能更加复杂,在一些重大的生理、病理事件中发挥着重要调节作用;而糖链如此复杂的功能是由其多样的结构决定的,糖链结构是糖组学研究的重要内容.本文就近年来糖组学研究中糖蛋白样品的提取分离、糖链释放及结构分析的基本方法及相关技术进展作了简要介绍.     

基于质谱技术研究黑色素瘤血清特异性O-糖链

对接种和未接种B16黑色素瘤细胞的C57小鼠进行血清O-糖链比较糖组学研究,寻找黑色素瘤血清特异性O-糖链.小鼠血清10μL,β-消除反应释放O-糖链.反应混合物经石墨化炭黑固相萃取小柱(GCC SPE)分离纯化后,用于MALDI-Qit-TOF-MS分析.通过Launchpad软件采集并输出质谱数据,MATLAB进行数据解析,找到了10个稳定出现的差异糖链质谱峰.利用串联质谱分析了其中5个主要差异糖链的结构.     

Pronase E酶解释放糖蛋白N-糖链的方法及荧光标记衍生物的LC-MS分析

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.     

花生致敏糖蛋白Ara h1糖链决定簇的质谱分析

以花生种子总蛋白及其主要致敏糖蛋白Ara h1为研究对象,采用"一釜法"对蛋白上的糖链进行释放并同时进行衍生化标记,通过C18固相萃取柱纯化,以电喷雾质谱(ESI-MS)、多级串联质谱(MSn)和亲水性液相色谱-质谱联用(HILIC-MS)进行结构解析和定量分析.结果表明,蛋白Ara h1共有10条N-糖链,其中7条为高甘露糖型,2条为木糖修饰,另外1条为与过敏原相关的核心α1,3-Fuc修饰N-糖链,其含量约占总糖链的12.45%.     

基于固相萃取的微量糖蛋白糖基化修饰表征技术的建立

结合自制亲水固相萃取富集柱和生物质谱鉴定技术,实现了糖基化蛋白质核糖核酸酶B的糖含量测定、糖基化位点确认、聚糖富集及结构表征,以及不同糖型相对丰度分析。结果表明:其糖含量8.47%,糖基化位点为34位的Asn,糖链主要为5种高甘露糖型结构(Man5-9GlcNAc2)。所建立的HILIC富集技术,有利于针对微量生物样本,如生物工程药物糖蛋白及重要功能糖蛋白,开展位点特异性糖链结构解析,为糖蛋白质的药效或功能研究提供线索。     

银杏种子糖蛋白的SDS-PAGE分离及N-糖链的质谱分析

利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分离了银杏种子中的糖蛋白组分, 进一步用氨水催化释放N-糖链, 并采用电喷雾离子质谱(ESI-MS)及在线液相色谱-质谱联用(LC-UV-MS/MS²)等技术对胶条上释放的N-糖链进行了定性定量分析. 结果表明, 从银杏种子中分离得到11种糖蛋白, 共检测到11条N-糖链, 包括高甘露糖型(4.88%)和复杂型(95.12%) 2种类型, 其中分子量为21000, 36000和50000的糖蛋白所释放的核心α-1,3-岩藻糖和β-1,2-木糖修饰的N-糖链所占比率较高, 分别为68.23%, 64.37%和75.09%. 本研究对进一步研究银杏糖蛋白功能具有重要意义.     

亲水相互作用色谱结合串联质谱多重碎裂模式在完整糖肽研究中的应用

蛋白质的糖基化修饰在生理及病理过程中发挥着重要作用。由于技术条件的限制,传统的糖蛋白分析方法中,通常分别鉴定蛋白质和糖链两者的结构,而忽略了蛋白质与糖链的连接关系。本研究旨在以完整糖肽作为检测对象,对糖肽中肽段序列和糖链结构的同步解析。采用亲水相互作用色谱对完整糖肽进行分离纯化,联合生物质谱中碰撞诱导解离(CID)、高能诱导解离(HCD)、电子转移解离(ETD)等裂解模式,对完整糖肽的糖基化位点、糖链结构、肽段序列等进行全方面的解析。结果表明,亲水相互作用色谱中样品与填料比1∶50可有效富集糖肽,采用30%CID能量,主要产生糖苷键断裂的碎片,为糖链的结构组成分析提供了线索;采用25%HCD能量,在低分子量区域提供了糖链的特征离子信息,并产生明确的糖肽Y1特征离子;ETD保留了完整的修饰基团而产生肽段骨架的断裂,可以提供有效的肽段序列信息。本研究结合亲水相互作用色谱与质谱仪中的多种碎裂方式,为完整糖肽的结构解析提供了一种快速、有效的研究方案。     

小角X光散射在蛋白质及其复合物领域的研究进展

小角X射线散射(SAXS)是能够表征多种形态的样品,解析从原子到几百纳米尺度上微结构的一种重要技术手段.蛋白质复合物正是在这一空间尺度范围表现出了丰富的生理活性.近年来基于分子结构药物设计的飞速发展,极大地促进了SAXS在研究蛋白质复合物中的应用.本文简要介绍了SAXS解析结构的原理、仪器设备和数据采集分析方法.围绕蛋白质复合物的两方面研究热点,溶液中的蛋白构象以及蛋白质复合物中的结构和相互作用解析,对SAXS在该领域的研究进展和一般分析方法进行了综述.     

N-糖链唾液酸连接异构体的质谱分析方法研究进展

蛋白质在翻译过程中、翻译过程后会发生糖基化. 糖基化会以直接或间接的方式影响蛋白质的功能及其相互作用, 并与多种人类疾病有关, 其中, 唾液酸化N-糖链在一些重要的生理和病理过程中发挥关键作用. 已知的唾液酸与相邻单糖之间的连接方式包括α-2,3-、α-2,6-、α-2,8-、α-2,9-连接, 连接方式不同的唾液酸化N-糖链在细胞活动、生命体的生理和病理过程中的功能往往不同. 质谱技术是分析N-糖链的重要工具, 它能够快速和灵敏地检测N-糖链, 通过将色谱技术以及衍生化方法等与质谱联用可以实现对唾液酸化N-糖链及其连接异构体的分离和检测. 本文主要围绕α-2,3-和α-2,6-连接的唾液酸化N-糖链进行综述, 介绍它们的结构和在细胞活动及疾病中不同的功能, 并综述近年来基于质谱的唾液酸化N-糖链的连接异构体分析方法以及这些方法在生物医学领域的应用, 并对未来的生物医学研究提供新的思路和途径.     

基于固定化Pronase E酶与质谱的糖链结构分析方法

位点特异性糖链结构的解析,是糖蛋白质结构分析面临的巨大挑战。Pronase E蛋白水解酶,能够降解糖蛋白或糖肽中大部分的氨基酸序列,而保留糖链与少量氨基酸的序列,与色谱、质谱分析等联用,可以实现糖链结构的鉴定,同时,保留的氨基酸序列可以辅助实现修饰位点的识别,两者结合,可以获取位点特异性糖链结构的信息。但Pronase E酶解的缺点是,酶解效率较低,常需要较高浓度的蛋白酶。本实验将Pronase E固定化在基质上,固定化后的Pronase E具备较高的局部浓度,从而实现目标糖蛋白的快速高效酶解。采用核糖核酸酶B作为标准糖蛋白,优化了Pronase E酶切的方案,包括酶切时蛋白与酶的用量比、酶切时间、固定化Pronase E酶的有效贮存时间等;同时优化选择了糖链的富集方法,并对于基质辅助激光解吸飞行时间质谱分析中,糖链适合的基质进行对比选择,从而获得更好的糖链谱图及更为丰富的糖链结构信息。     

Combination of two hydrophilic interaction chromatography methods that facilitates identification of 2-aminobenzamide-labeled oligosaccharides

Hydrophilic interaction chromatography (HILIC) is used to separate 2-aminobenzamide- (2-AB) labeled N-linked oligosaccharides. The glycans of the model protein, bovine fetuin, are identified following comparison of elution patterns of seven 2-AB-labeled glycan standards, of which two are of the high-mannose type and five are of the complex type. The combination of two HILIC methods, using an Amide-80 column, having different resolutions and selectivities, markedly facilitates the identification of the fetuin glycans. These HILIC methods are suitable for obtaining glycan profiles of complex mixtures.     

Capillary electrophoresis: a superior miniaturized tool for analysis of the mono-, di-, and oligosaccharide constituents of glycan moieties in proteoglycans

Proteoglycans are a major family of glycoconjugates which participate in and regulate several cellular events and biological functions. Their glycan chains determine their physicochemical and biological properties. Capillary electrophoresis, because of its high resolving power and sensitivity, has been successfully used for the analysis of carbohydrates. The monosaccharide constituents, the disaccharide sulfation pattern, and the uronic acid distribution within glycan chains of proteoglycans determine their interactions with matrix effectors and are responsible for numerous effects. Determination of the chemical composition and identification of key structural components and domains of glycans are, therefore, essential in understanding the biological functions of proteoglycans. In this report an overview of the capillary electrophoresis methods used to analyze and characterize the structure of the glycan chains of proteoglycans is presented.     

BOLD--a biological O-linked glycan database

Glycans can be O-linked to proteins via the hydroxyl group of serine, threonine, tyrosine, hydroxylysine or hydroxyproline. Sometimes the glycan is O-linked to the hydroxyl group via a phosphodiester bond. The core monosaccharide residue may be N-acetylgalactosamine, N-acetylglucosamine, galactose, glucose, fucose, mannose, xylose or arabinose. These O-linked glycans can remain as a monosaccharide, but often a complex structure is built up by stepwise addition of monosaccharides. Monosaccharides known to be added include galactose, N-acetylglucosamine, fucose, N-acetylneuraminic acid, N-glycolylneuraminic acid and 2-keto-3-deoxynonulosonic acid. O-linked glycans can also contain sulfate and phosphate residues. This leads to the possibility of the existence of numerous O-glycan structures. The biological O-linked database (BOLD) is a relational database that contains information on O-linked glycan structures, their biological sources (with a link to the SWISS-PROT protein database), the references in which the glycan was described (with a link to MEDLINE), and the methods used to determine the glycan structure. The database provides a valuable resource for glycobiology researchers interested in O-linked oligosaccharide structures that have been previously described on proteins from different species and tissues.     

Multiplexing N‐glycan analysis by DNA analyzer

Analysis of N‐glycan structures has been gaining attentions over the years due to their critical importance to biopharma‐based applications and growing roles in biological research. Glycan profiling is also critical to the development of biosimilar drugs. The detailed characterization of N‐glycosylation is mandatory because it is a nontemplate driven process and that significantly influences critical properties such as bio‐safety and bio‐activity. The ability to comprehensively characterize highly complex mixtures of N‐glycans has been analytically challenging and stimulating because of the difficulties in both the structure complexity and time‐consuming sample pretreatment procedures. CE‐LIF is one of the typical techniques for N‐glycan analysis due to its high separation efficiency. In this paper, a 16‐capillary DNA analyzer was coupled with a magnetic bead glycan purification method to accelerate the sample preparation procedure and therefore increase N‐glycan assay throughput. Routinely, the labeling dye used for CE‐LIF is 8‐aminopyrene‐1,3,6‐trisulfonic acid, while the typical identification method involves matching migration times with database entries. Two new fluorescent dyes were used to either cross‐validate and increase the glycan identification precision or simplify sample preparation steps. Exoglycosidase studies were carried out using neuramididase, galactosidase, and fucosidase to confirm the results of three dye cross‐validation. The optimized method combines the parallel separation capacity of multiple‐capillary separation with three labeling dyes, magnetic bead assisted preparation, and exoglycosidase treatment to allow rapid and accurate analysis of N‐glycans. These new methods provided enough useful structural information to permit N‐glycan structure elucidation with only one sample injection.     

Glycopeptide analysis by mass spectrometry

Glycosylation is one of the most important post-translational modifications found in nature. Identifying and characterizing glycans is an important step in correlating glycosylation structure to the glycan's function, both in normal glycoproteins and those that are modified in a disease state. Glycans on a protein can be characterized by a variety of methods. This review focuses on the mass spectral analysis of glycopeptides, after subjecting the glycoprotein to proteolysis. This analytical approach is useful in characterizing glycan heterogeneity and correlating glycan compositions to their attachment sites on the protein. The information obtained from this approach can serve as the foundation for understanding how glycan compositions affect protein function, in both normal and aberrant glycoproteins.     

Comparison of hydrophilic-interaction, reversed-phase and porous graphitic carbon chromatography for glycan analysis

Hydrophilic-interaction liquid chromatography (HILIC), reversed-phase chromatography (RPC) and porous graphitic carbon (PGC) chromatography are typically applied for liquid chromatographic separations of protein N-glycans. Hence the performances of these chromatography modes for the separation of fluorescently labeled standard glycan samples (monoclonal antibody, fetuin, ribonuclease-B) covering high-mannose and a broad range of complex type glycans were investigated. In RPC the retention of sialylated glycans was enhanced by adding an ion-pairing agent to the mobile phase, resulting in improved peak shapes for sialylated glycans compared to methods recently reported in literature. For ion pairing RPC (IP-RPC) and HILIC ultra-high performance stationary phases were utilized to maximize the peak capacity and thus the resolution. But due to the shallow gradient in RPC the peak capacity was lower than on PGC. Retention times in HILIC and IP-RPC could be correlated to the monosaccharide compositions of the glycans by multiple linear regression, whereas no adequate model was obtained for PGC chromatography, indicating the significance of the three-dimensional structure of the analytes for retention in this method. Generally low correlations were observed between the chromatography methods, indicating their orthogonality. The high selectivities, as well as the commercial availability of ultra-high performance stationary phases render HILIC the chromatography method of choice for the analysis of glycans. Even though for complete characterization of complex glycan samples a combination of chromatography methods may be necessary.     

Online enzymatic sequencing of glycans from Trastuzumab by phospholipid-assisted capillary electrophoresis

CE separations of glycans taken from the cancer drug, Trastuzumab (Herceptin(?)), were accomplished using phospholipid additives. Glycans were labeled with 1-aminopyrene-3,6,8-trisulfonic acid and were separated with efficiencies as high as 510000 theoretical plates in a 60.2 cm 25 μm id fused-silica capillary. The thermally tunable phospholipid was loaded into the capillary when it possessed a viscosity similar to that of water. The temperature was increased, and the separations were performed when the material exhibited higher viscosity. Enzymes were integrated into the separation with the phospholipid additive. Neuraminidase, β1-4 galactosidase, and β-N-acetylglucosaminidase were injected into the capillary without covalent modification and used for enzyme hydrolysis. Exoglycosidase enzymes cleaved the terminal glycan residues. The glycan sequence could be verified based on enzyme specificity. Neuraminidase was used to determine total glycan content of the low-abundance glycans containing sialic acid. β1-4 Galactosidase and β-N-acetylglucosaminidase were used sequentially in-capillary, to determine the structure of the high-abundance glycans.     

Structural study on the carbohydrate moiety of calf intestinal alkaline phosphatase

Surprisingly alkaline phosphatase (AP) (EC 3.1.3.1) of calf intestine is found in large amounts, e.g. 80%, within chyme. Most of the enzyme is present as a mixture of four differently hydrophobic anchor-bearing forms and only the minor part is present as an anchorless enzyme. To investigate whether changes in the N-glycosylation pattern are signals responsible for large-scale liberation from mucosa into chyme, the glycans of the two potential glycosylation sites predicted from cDNA were investigated by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry in combination with exoglycosidase treatment after tryptic digestion and reversed-phase chromatography. The glycans linked to Asn249 are at least eight different, mainly non-fucosylated, biantennary or triantennary structures with a bisecting N-acetylglucosamine. For the most abundant glycopeptide (40%) the following glycan structure is proposed: [carbostructure: see text]. The glycans linked to Asn410 are a mixture of at least nine, mainly tetraantennary, fucosylated structures with a bisecting N-acetylglucosamine. For the most abundant glycopeptide (35%) the following glycan structure is proposed: [carbostructure: see text]. For the structures the linkage data were deduced from the reported specificities of the exoglycosidases used and the specificities of the transglycosidases active in biosynthesis. The majority of glycans are capped by alpha-galactose residues at their non-reducing termini. In contrast to the glycans linked to other AP isoenzymes, no sialylation was observed. Glycopeptide 'mass fingerprints' of both glycosylation sites and glycan contents do not differ between AP from mucosa and chyme. These results suggest that the observed large-scale liberation of vesicle-bound glycosylphosphatidylinositol (GPI)-anchored AP from mucosa into chyme is unlikely to be mediated by alteration of glycan structures of the AP investigated. Rather, the exocytotic vesicle formation seems to be mediated by the controlled organization of the raft structures embedding GPI-AP. (c) 2001 John Wiley & Sons, Ltd.     

Universal proteolysis and MSn for N‐ and O‐ glycan branching analysis

The continually growing list of critical glycosylation‐related processes has made analytical methodology for detailed glycan characterization an area of increasing interest. Glycosylation is a post translational modification of unsurpassed complexity due to the variety of compositions and linkages formed by these biopolymers. Structural characterization of glycan isomers has been achieved using ion trap mass spectrometry and MSⁿ of released, permethylated glycans. However, N‐ and O‐glycans require different sample preparation strategies; and release of the glycans may be hindered, result in degradation of the glycan, and/or produce limited yields of permethylated product. In the current report, we demonstrate universal proteolysis of both N‐ and O‐linked glycoproteins to individual glycoamino acids. These samples were shown to be directly amenable to permethylation and MSⁿ analysis for isomeric structural determination. Universal proteolysis and permethylation provides an identical sample preparation strategy for both classes of glycans that avoids potential pitfalls of commonly used release methods. This methodology should be applicable to all glycoproteins and serve as an alternative to glycan release for MSⁿ branching analysis. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.     

Harnessing glycomics technologies: integrating structure with function for glycan characterization

Glycans, or complex carbohydrates, are a ubiquitous class of biological molecule which impinge on a variety of physiological processes ranging from signal transduction to tissue development and microbial pathogenesis. In comparison to DNA and proteins, glycans present unique challenges to the study of their structure and function owing to their complex and heterogeneous structures and the dominant role played by multivalency in their sequence-specific biological interactions. Arising from these challenges, there is a need to integrate information from multiple complementary methods to decode structure-function relationships. Focusing on acidic glycans, we describe here key glycomics technologies for characterizing their structural attributes, including linkage, modifications, and topology, as well as for elucidating their role in biological processes. Two cases studies, one involving sialylated branched glycans and the other sulfated glycosaminoglycans, are used to highlight how integration of orthogonal information from diverse datasets enables rapid convergence of glycan characterization for development of robust structure-function relationships.