串联质谱寡糖结构解析方法评介

糖组学的研究与发展对生命科学及生物医药的发展具有重要的推动作用.寡糖结构的解析是糖组学中重要的研究课题之一.串联质谱分析技术以其具有高特异性及高灵敏度的特点成为了广为使用的寡糖结构解析方法.本文首先概述了串联质谱寡糖结构解析的研究背景;然后介绍了现有的寡糖结构解析策略及基于每种策略的经典解析方法,并对所列方法的原理和算法进行逐一分析讨论;最后,总结现有方法的优缺点,对串联质谱寡糖结构研究领域进行了研究展望.     

寡糖的构象分析

糖在生物体内具有极为重要的生物学功能,其在细胞及分子识别等许多过程中的作用已日益得到重视,糖的结构特征已成为研究识别过程的重要依据.集中介绍了有关寡糖结构的基本概念、理论研究热点及现状,并对有关研究方法进行了评述．寡糖结构理论研究的发展方向之一是研究糖与蛋白质的相互作用以及糖蛋白中糖链的结构与功能.     

人乳寡糖的结构及其分离分析

母乳中存在的人乳寡糖（HMOs）是一类结构高度复杂的低聚糖，对婴儿的肠道菌群、免疫屏障、大脑发育发挥积极作用。由于母乳中基质复杂，寡糖的种类繁多，丰度跨度大，存在众多异构体，这都使得检测面临诸多挑战。现已有多种技术用于HMOs的分析，发现了200多种HMOs，液相色谱和毛细管电泳在分离HMOs方面效果显著，核磁共振、质谱、红外多光子解离光谱推动了对HMOs结构的全面解析。本文回顾了对HMOs实现高灵敏度和高特异性分析的多种技术方法，比较了不同技术的优缺点，还重点介绍了质谱以及不同技术联用在推动HMOs解析和测定方面的突破，为探究寡糖的结构-功能关系、深入理解HMOs的生物学功能提供了全面的技术支持。     

研究: 奶牛与水牛初乳中乳寡糖组分比较研究

乳寡糖是由乳汁中含量丰富的固体物质组成．研究结果表明,乳寡糖有提高免疫、益生元及抗感染等作用,已发现与婴儿肠道发育、神经智力发育等多方面关系密切．水牛奶是除牛奶外的第二大奶源,国际上公认其为营养含量高、口感好的优质乳制品,但目前针对水牛乳寡糖的研究多以美洲水牛为研究对象,尚无中国水牛的相关研究．本研究利用固相萃取对已脱脂和除去蛋白质的广西水牛初乳乳汁样品进行纯化,并采用苯胺 (aniline,Bn)衍生化试剂对其进行衍生化处理,通过UPLC-ESI-Q-TOF-MS液相质谱进行优化后,对水牛初乳中的寡糖组分进行测定并与牛乳进行了对比,最终测得奶牛初乳中19种及水牛初乳中的9种乳寡糖组分,并对二者的种类及含量进行比较,发现在两种初乳的乳寡糖中,中性糖二糖m/z 385.15和中性糖三糖m/z 547.21以及酸性糖m/z 635.23均为其主要寡糖成分,与其他乳寡糖相比含量相对较高．总体而言水牛初乳中的中性寡糖占比比奶牛初乳高,二者中性糖占乳寡糖总量的比例分别为88.88%和63.16%．     

对氨基苯甲酸乙酯衍生法测定IgM中的糖链结构

糖蛋白中痕量的完整寡糖链结构可用现代质谱方法测定。对寡糖的对氨基苯甲酸乙酯衍生物的液态二次离子质谱进行了研究,测定了基质效应,比较了正、负离子谱,使得麦芽七糖衍生物的最小检测量达到4p mol。应用氘标记类似物及高分辨质谱数据解释了IgM中N-连接的寡糖链分子结构。     

人乳寡糖的分离分析

糖是一类重要的生命活性物质,它不仅是细胞能量代谢的源泉,还常作为信号分子参与细胞的各种活动。人乳寡糖（human milk oligosaccharides,HMOs）在人乳干物质中的含量仅次于乳糖和脂肪,高于蛋白质,对婴儿的发育和健康具有重要作用。为了更好的理解人乳寡糖的生物功能和结构的关系,对其组成和结构开展分析研究是必不可少的。对人乳寡糖进行了简要的介绍,并就其预处理方法、分离分析和结构表征方法进行了综述,以期为人乳寡糖的深入研究提供参考。     

中药中寡糖分离分析方法研究进展CSCD

糖链结构与功能研究是未来糖生物学研究的重要方向,必将为人类疾病的研究和治疗带来新的希望。随着对糖类研究的不断深入,寡糖因其所具有的多种药理活性受到了国内外学者的广泛关注。寡糖结构受连接位点、糖环结构和分支等因素的影响而呈现复杂性和多样性,这为其提取,纯化、分离分析以及结构表征带来巨大了挑战。本文综述了近年来国内外在中药寡糖提取纯化、分离分析以及结构表征这三方面的研究内容,以期为寡糖的现代药物研究和临床应用以及功效产品创制提供科学参考。     

凝胶色谱法制备寡糖

采用已知结构的多糖,控制其水解条件,使之产生所需寡糖片断。用聚丙烯酰胺凝胶色谱(BiO-6el P-4)分离,对中性糖可以分离到含十一个糖残基的寡糖。对氨基糖可分离到含七个糖残基的寡糖。用薄层色谱和快原子轰击质谱鉴定了它们的纯度。     

基于质谱的磷酸化蛋白质组学: 富集、检测、鉴定和定量

磷酸化是一种调控生命活动的重要翻译后修饰,调控生物的生长发育、信号转导、以及疾病的发生发展．从上世纪80年代开始,质谱应用于蛋白质磷酸化的检测中,极大地推动了磷酸化蛋白质组学的发展．质谱检测拥有高灵敏度、高通量的特点,更重要的是具有位点分辨率,因此基于质谱的磷酸化蛋白质组检测方法得到不断的发展和推广．常见的磷酸化蛋白质组研究,首先对磷酸化肽段进行富集,然后进行串联质谱分析,最后通过搜索引擎对修饰位点进行鉴定和定量．本文从这个三个基本方面,对磷酸化蛋白质组研究进行综述,并对未来研究发展方向进行讨论．     

综述: 基于质谱技术的糖链结构解析研究进展

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．     

利用串联质谱鉴定氨基酸突变的生物信息学算法

氨基酸突变能够改变蛋白的结构和功能,影响生物体的生命过程.基于串联质谱的鸟枪法蛋白质组学是目前大规模研究蛋白质组学的主要方法,但是现有的质谱数据鉴定流程为了提高鉴定结果的灵敏度往往会有意压缩数据库中的氨基酸突变信息.因此,如何挖掘数据中的氨基酸突变信息成为当前质谱数据鉴定的一个重要部分.当前应用于氨基酸突变鉴定的串联质谱鉴定方法大致可以分为3大类:基于序列数据库搜索的方法、基于序列标签搜索的算法以及基于图谱库搜索的算法.本文首先详细介绍了这3种氨基酸突变鉴定算法,并分析了各种方法的特点和不足,然后介绍了氨基酸突变鉴定的研究现状和发展方向.随着基于串联质谱的蛋白质组学的不断发展,蛋白序列中的氨基酸突变信息将被更好地解析出来,从而得以深入探讨由氨基酸突变引起的蛋白结构和功能改变,为揭示氨基酸突变的生物学意义奠定基础.     

Structural analysis of the lipopolysaccharide from Pasteurella multocida genome strain Pm70 and identification of the putative lipopolysaccharide glycosyltransferases

Pasteurella multocida is an important multispecies veterinary pathogen. The cell surface lipopolysaccharide (LPS) is an important virulence factor and forms the basis of the serotyping scheme, although little structural information about it is known. The structure of the LPS from the Pasteurella multocida genome strain Pm70 was elucidated in this study. The LPS was subjected to a variety of degradative procedures. The structures of the purified products were established by monosaccharide and methylation analyses, NMR spectroscopy, and mass spectrometry. The structure of the core oligosaccharide was determined on the basis of the combined data from these experiments. Identification of the core oligosaccharide structure enabled a search for glycosyltransferase homologs in the Pm70 genome and revealed a clustering of the genes putatively responsible for outer core oligosaccharide biosynthesis.     

N-糖肽的规模化质谱解析方法进展

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.     

Structural analysis of the core oligosaccharide from Pasteurella multocida strain X73

The structure of the core oligosaccharide region of the lipopolysaccharide from the Pasteurella multocida strain X73 was elucidated. The lipopolysaccharide was subjected to a variety of degradative procedures. The structure of the purified oligosaccharide was established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structure illustrates a similar structure to the recently identified oligosaccharide from another P. multocida strain VP161, but with additional symmetrical substitution of the terminal galactose residues with phosphoethanolamine moieties, where based on the NMR data all sugars were found in pyranose ring forms and Kdo is 3-deoxy-alpha-D-manno-2-oct-2-ulosonic acid, l,D-alpha-Hep is l-glycero-D-manno-heptose, PEtn is phosphoethanolamine and PCho is phosphocholine.     

The complete structure of the core carbohydrate backbone from the LPS of marine halophilic bacterium Pseudoalteromonas carrageenovora type strain IAM 12662T

The complete novel structure of the components of the core oligosaccharide fraction from the LOS of the halophilic marine bacterium Pseudoalteromonas carrageenovora was characterized. The fully de-acylated lipooligosaccharide was studied by means of compositional analysis, matrix-assisted laser desorption/ionization mass spectrometry and complete (1)H and (13)C and (31)P NMR spectroscopy. The core oligosaccharide is composed by a mixture of species differing for the length of the sugar chain and the phosphorylation pattern: [carbohydrate structure]; see text. All sugars are D-pyranoses. Hep is L-glycero-D-manno-heptose, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, P is phosphate, residues and substituents in italic are not stoichiometrically linked.     

Structure elucidation of glycosphingolipids and gangliosides using high-performance tandem mass spectrometry

Glycosphingolipids and gangliosides have been investigated by using fast atom bombardment high-performance tandem mass spectrometry (FABMS/MS). Homologous compounds have been investigated in order to ascertain the fragmentation. Collision-induced dissociation spectra of the molecular species in the positive ion mode mainly afford information on the ceramide constitution (aglycon as a whole, N-acyl residue, and long-chain base), whereas negative ion spectra show fragments informative of the sugar sequence and the degree of branching of the carbohydrate. In the case of gangliosides carrying a complex oligosaccharide moiety, collision spectra of fragment ions have been performed in order to gain additional structural data. The advantage of tandem mass spectrometry over conventional fast atom bombardment mass spectrometry (FABMS) consists in the fact that collision spectra of the individual components from mixtures, as usually encountered with these kinds of samples, can be recorded. Furthermore, the exclusion of most of the interfering signals from the matrix allows the identification of pertinent fragments at low mass.     

Microanalysis of N-linked oligosaccharides in a glycoprotein by capillary liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry

We have studied rapid and simple sugar mapping using liquid chromatography/electrospray ionization mass spectrometry (LC/MS) equipped with a graphitized carbon column. The oligosaccharide mixture was separated on the basis of the sequence, branching structure, and linkage, and each oligosaccharide was characterized based on its molecular mass. In this study we demonstrated the usefulness of capillary LC/MS (CapLC/MS) and capillary liquid chromatography/tandem mass spectrometry (CapLC/MS/MS) as sensitive means for accomplishing the structural analysis of oligosaccharides in a low-abundance glycoprotein. The carbohydrate heterogeneity and molecular mass information of each oligosaccharide can be readily obtained from CapLC/MS of a small amount of glycoprotein. CapLC/MS/MS provided b-ion series, which is informative with regard to monosaccharide sequence. Exoglycosidase digestion followed by CapLC/MS elucidated a carbohydrate residue linkage. Using this method, we characterized N-linked oligosaccharides in hepatocyte growth factor produced in mouse myeloma NS0 cells as the complex-type bi-, tri-, and tetraantennary terminated with N-glycolylneuraminic acids and alpha-linked galactose residues. Sugar mapping with CapLC/MS and CapLC/MS/MS is useful for monitoring glycosylation patterns and for structural analysis of carbohydrates in a low-abundance glycoprotein and thus will become a powerful tool in biological, pharmaceutical, and clinical studies.     

Faster mass spectrometry-based protein inference: junction trees are more efficient than sampling and marginalization by enumeration

The problem of identifying the proteins in a complex mixture using tandem mass spectrometry can be framed as an inference problem on a graph that connects peptides to proteins. Several existing protein identification methods make use of statistical inference methods for graphical models, including expectation maximization, Markov chain Monte Carlo, and full marginalization coupled with approximation heuristics. We show that, for this problem, the majority of the cost of inference usually comes from a few highly connected subgraphs. Furthermore, we evaluate three different statistical inference methods using a common graphical model, and we demonstrate that junction tree inference substantially improves rates of convergence compared to existing methods. The python code used for this paper is available at http://noble.gs.washington.edu/proj/fido.     

Site-specific glycosylation analysis of human apolipoprotein B100 using LC/ESI MS/MS

Human apolipoprotein B100 (apoB100) has 19 potential N-glycosylation sites, and 16 asparagine residues were reported to be occupied by high-mannose type, hybrid type, and monoantennary and biantennary complex type oligosaccharides. In the present study, a site-specific glycosylation analysis of apoB100 was carried out using reversed-phase high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI MS/MS). ApoB100 was reduced, carboxymethylated, and then digested by trypsin or chymotrypsin. The complex mixture of peptides and glycopeptides was subjected to LC/ESI MS/MS, where product ion spectra of the molecular ions were acquired data-dependently. The glycopeptide ions were extracted and confirmed by the presence of carbohydrate-specific fragment ions, such as m/z 204 (HexNAc) and 366 (HexHexNAc), in the product ion spectra. The peptide moiety of glycopeptide was determined by the presence of the b- and y-series ions derived from its amino acid sequence in the product ion spectrum, and the oligosaccharide moiety was deduced from the calculated molecular mass of the oligosaccharide. The heterogeneity of carbohydrate structures at 17 glycosylation sites was determined using this methodology. Our data showed that Asn2212, not previously identified as a site of glycosylation, could be glycosylated. It was also revealed that Asn158, 1341, 1350, 3309, and 3331 were occupied by high-mannose type oligosaccharides, and Asn 956, 1496, 2212, 2752, 2955, 3074, 3197, 3438, 3868, 4210, and 4404 were predominantly occupied by mono- or disialylated oligosaccharides. Asn3384, the nearest N-glycosylation site to the LDL-receptor binding site (amino acids 3359-3369), was occupied by a variety of oligosaccharides, including high-mannose, hybrid, and complex types. These results are useful for understanding the structure of LDL particles and oligosaccharide function in LDL-receptor ligand binding.