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: 6478919
• 负责人: Catherine E. Costello
• 金额: $ 5.36万
• 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/6478919
• 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.

结构识别的主要难点 通过质谱分析得到的碳水化合物低聚物是大量的 由于两种不同物质的存在而产生的结构异构体 单体残基之间的连接位置和可能性 与单个残基的多个连接。 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 表征氨基或所需的残基 核酸寡聚物。 多个链接还允许多个 连接拓扑和生物系统中的碳水化合物和 糖复合物通常表现出支链和线性 结构。 在过去的几十年里，基于质谱技术 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（选择）设置中，串联质量 光谱尝试能够提供彻底的拓扑 有关糖苷键的表征和广泛信息。 在更一般的情况下，来自 MS/MS 的信息会受到损害。 这 碳水化合物低聚物中原子种类范围相对较窄 导致同量异位离子碎片。 不稳定糖苷共存 键与耐用的吡喃糖环结构导致 较大离子的碎裂途径几乎完全涉及 糖苷键的断裂。 开环途径用于 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 片段还具有代际层次结构等特征 表征可以识别结构特征。 按顺序 解离分子，离子碎片数量减少 自由度已准备好，它们的解离反过来导致 增强非糖苷键的解离。 实用的 MSn 用于碳水化合物分析的潜力取决于性能 离子捕获装置的各个方面，即碰撞能量 可以获得连续的离子碎片保留 离子解离的世代。 实施碳水化合物 MSn FT ICR 质谱仪因分析需求而受到影响 保留并解离相对较小的离子碎片 高活化能。 磁控管膨胀和离子的动力学 典型碳水化合物 MSn 实验中的加热计算公式为 结合非线性捕获的详细数值模拟 势和三维离子中性散射。 在分析中 这些实验，离子运动的动力学不稳定性和 内部加热被证明是一个严重的问题。 我们的主要焦点 因此，当前 FT-ICR MS 研究的重点是离子动力学 重点关注实施重测和MSn的问题。 最近的工作分为理论/数值研究 离子动力学和与相关的仪器修改 实施回旋加速器轴向旋转以冷却离子 平移运动。 数字目标是： a) 开发 桥代码，使电极电势由商业产生 可以使用SIMION包进行轨迹模拟； b) 实施 轨迹中的随机、三维排斥散射 模拟； c) 允许宽带激励； d) 实施库仑 离子对之间的耦合； e) 在MIDAS中输出时域文件 兼容的格式，即让模拟生成“光谱”； f） 构建一套程序来反映不同的用户目标和 操作环境。 最后一个目标包括开发 与图形用户界面相结合的程序（基于 CVI LabWindows、National Instruments）在 Windows 95/NT 和 shell 下运行 脚本控制的程序在 UNIX 中作为后台进程运行 （Linux）环境。 回旋加速器的仪器改造 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。