CHARACTERIZATION OF GLYCOPROTEIN PERMETHYLATED OLIGOSACCHARIDES BY FT ICR MS

通过 FT ICR MS 表征糖蛋白全甲基化低聚糖

• 批准号: 6345195
• 负责人: Catherine E. Costello
• 金额: $ 3.38万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6345195
• 关键词: biomedical equipment development; biomedical resource; carbohydrates; proteins; spectrometry; structural biology; technology /technique

A principal difficulty in the structural identification of carbohydrate oligomers by mass spectrometry is the large number of structural isomers that arise from the existence of both different linkage positions between monomer residues and the possibility of multiple linkages to a single residue. Hence, for a straight-chain oligomer, structural identification requires determination of the sequence of glycosyl linkage types, including the linkage positions and the anomeric configurations, as well as the sequence of monomer residues that would be required for characterization of amino or nucleic acid oligomers. Multiple linkages also allow for multiple connection topologies and in biological systems carbohydrates and glycoconjugates commonly exhibit branched as well as linear structures. In the past decades, mass spectral techniques based on ion fragmentation have played a major role in the analysis of carbohydrate structures. In some (select) settings, tandem mass spectrome try is able to provide both a thorough topological characterization and extensive information about glycosidic linkages. In more general settings, information from MS/MS is compromised. The relatively narrow range of atomic species in carbohydrate oligomers leads to isobaric ion fragments. The coexistence of labile glycosidic bonds along with durable pyranose ring structures leads to fragmentation pathways in larger ions that involve almost exclusively the breaking of glycosidic bonds. Ring-opening pathways used in linkage assignments are suppressed. MSn techniques employing ion trap mass spectrometers can address both of these limits. With MSn, ion fragments are also characterized by a generational hierarchy and such characterization can identify structural features. By sequentially dissociating a molecule, ion fragments with a reduced number of degrees of freedom are prepared and their dissociation, in turn, leads to enhanced dissociation of non-glycosidic bonds. The practical potent ial of MSn for carbohydrate analysis hinges on performance aspects of the ion trapping device, i.e., the collision energies that can be obtained and the ion fragment retention in successive generations of ion dissociation. Implementing carbohydrate MSn with FT ICR mass spectrometers is compromised by the analytical need to retain and dissociate relatively small ion fragments with relatively high activation energies. Kinetics of magnetron expansion and ion heating in prototypical carbohydrate MSn experiments are calculated by detailed numerical simulations incorporating both nonlinear-trapping potentials and three-dimensional ion-neutral scattering. In analyzing these experiments, both dynamical instabilities of the ion motion and internal heating are shown to be significant problems. Our main focus of current research in FT-ICR MS is therefore ion dynamics with an emphasis on the problems of implementing remeasurement and MSn. Recent effort has been divided into theoretical/numerical studies of ion dynamics and instrumental modifications associated with implementing cyclotron to axial rotation for the cooling of the ion's translational motion. The numerical objectives are: a) develop bridge code so that electrode potentials generated by a commercial package (SIMION) could be used for trajectory simulation; b) implement stochastic, three dimensional repulsive scattering in the trajectory simulations; c) allow for broadband excitation; d) implement Coulomb coupling between ion pairs; e) output time domain files in a MIDAS compatible format, i.e., have the simulations generate 'spectra'; f) construct a suite of programs to reflect different user objectives and operating environments. The last objective includes developing programs coupled with a graphical user interfa ce (based on CVI LabWindows, National Instruments) to run under Windows 95/NT and shell script controlled programs to run as background processes in a UNIX (Linux) environment. The instrumental modifications for cyclotron to axial rotation have involved the computer design and numerical analysis of various cell and excitation geometries.

结构鉴定的一个主要困难 碳水化合物低聚物通过质谱是大量的 结构异构体，产生于两种不同的 单体残基之间的连接位置和 多个键合到单个残基上。 因此，对于直链 低聚物，结构鉴定需要确定 糖基连接类型的序列，包括连接位置 和异头构型，以及单体的序列 这些残基是表征氨基或 核酸寡聚体。 多重链接还允许多重 连接拓扑结构和生物系统中的碳水化合物， 糖缀合物通常表现出支链以及直链 结构. 在过去的几十年里，基于质谱技术的质谱仪 离子碎裂在分析中发挥了重要作用， 碳水化合物结构 在某些（选定）设置中，串联质量 spectrometry能够提供彻底拓扑 特征和有关糖苷键的广泛信息。 在更一般的设置中，来自MS/MS的信息受到损害。 的 碳水化合物低聚物中原子种类的范围相对较窄 导致同量异位素离子碎片。 不稳定糖苷的共存 键沿着与持久的吡喃糖环结构导致 大离子的碎裂路径几乎完全 糖苷键的断裂。 开环通路用于 链接分配被抑制。 采用离子阱的MSn技术 质谱仪可以解决这两个限制。 与MSn，离子 片段也具有世代等级的特征， 表征可以识别结构特征。 通过顺序 解离分子、离子碎片，减少离子数量 自由度是准备好的，它们的解离反过来又导致 增强了非糖苷键的解离。 实际 MSn用于碳水化合物分析的有效性取决于性能 离子捕获装置的方面，即，碰撞能量 可以获得和离子碎片保留在连续 离子解离的世代。 实施碳水化合物MSn， FT ICR质谱仪的分析需求受到影响， 保留和解离相对小的离子碎片， 高活化能。 磁控管膨胀和离子动力学 典型碳水化合物MSn实验中的加热计算如下： 详细的数值模拟结合非线性捕获 电势和三维离子中性散射。 在分析 这些实验，离子运动的动力学不稳定性和 内部加热被证明是重要的问题。 我们的主要重点 因此，FT-ICR MS目前研究的主要内容是离子动力学， 重点讨论了实施复测和MSn的问题。 最近的努力已分为理论/数值研究， 离子动力学和仪器的修改与 实施回旋加速器到轴向旋转以冷却离子的 平移运动 数字目标是：a）发展 桥接代码，使得由商业信号产生的电极电势 SIMION软件包可用于弹道仿真; B）实现 弹道中的随机三维排斥散射 模拟; c）允许宽带激发; d）实施库仑 离子对之间的耦合; e）在MIDAS中输出时域文件 兼容格式，即，使模拟生成“光谱”; f） 构建一套程序，以反映不同的用户目标， 操作环境。 最后一个目标是发展 与图形用户界面（基于CVI）耦合的程序 LabWindows，National Instruments）在Windows 95/NT和shell下运行 脚本控制的程序作为UNIX中的后台进程运行 （Linux）环境。 回旋加速器的仪器改进 轴向旋转涉及计算机设计和数值计算 分析各种细胞和激发几何形状。