Identification and Structural Elucidation of Proteins in MALDI-TOF-TOF MS-MS

MALDI-TOF-TOF MS-MS 中蛋白质的鉴定和结构解析

• 批准号: 8332393
• 负责人: MARVIN L VESTAL
• 金额: $ 10万
• 依托单位: VIRGIN INSTRUMENTS CORPORATION
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2012-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8332393
• 关键词: Acceleration; Affinity; Biological; Caliber; Charge; Chemicals; Chemistry; Complex; Complex Mixtures; Computer software; Corticotropin; Cross Reactions; Cytochromes; Data; Detection; Development; Dissociation; Electron Transport; Electronics; Electrons; Elements; Engineering; Evaluation; Genes; Goals; Immunoglobulin G; Indium; Insulin; Ions; Laboratories; Lasers; Liquid substance; Measurement; Molecular Weight; Monitor; Myoglobin; Normal Statistical Distribution; Optics; Peptides; Performance; Phase; Physiologic pulse; Post-Translational Protein Processing; Posttranslational Amino Acid Modification; Process; Production; Protein Analysis; Proteins; Proteomics; Protocols documentation; Protons; Reaction; Reagent; Relative (related person); Reliability of Results; Resolution; SHFM1 gene; Sampling; Schedule; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Speed; System; Techniques; Technology; Testing; Thioredoxin; Time; Ubiquitin; Validation; Width; Work; base; cost; design; design and construction; discount; improved; instrument; instrumentation; ion source; ionization; molecular mass; novel; operation; price lists; programs; protein aminoacid sequence; prototype; public health relevance; voltage

DESCRIPTION (provided by applicant): This project is focused on developing improved instrumentation and protocols for identification and structural elucidation of large peptides and intact proteins. The proposed instrument is optimized for application to doubly charged positive ions and provides unique capabilities for MS-MS of intact proteins ionized by MALDI. Fragmentation mechanisms available include unimolecular fragmentation following excitation in the MALDI ion source (PSD), and electron transfer dissociation (ETD) from reactions of small negative ions with doubly charged intact proteins. The approach is based on recent advances in MALDI-TOF and TOF-TOF technology but requires addition of a second pulsed ion source and a new TOF analyzer for fragment ions that features up to 45 kV acceleration to achieve high performance on high mass, high energy ions. The ion optics are designed so that beams from the two sources can be merged within the collision region with the pulses accurately synchronized in time, and with the relative velocity of the beams accurately determined. For collisions between positive and negative ions the relative velocity can be set very close to zero, providing sufficient time for efficient ion chemistry to occur. A novel element in this instrument is an additional pulsed accelerator in the field-free space adjacent to the ion source that effectively reduces the velocity spread of selected ions and allows high resolution precursor selection simultaneously with high resolution measurements of fragment spectra. The first product resulting from this work is a new TOF-TOF that produces high-quality MS-MS spectra on intact proteins. This instrument generates very high quality MS-MS spectra at unprecedented speed and with very high sensitivity. In combination with a new LC interface with MALDI being developed in a separate project, an integrated LC-MS-MS system incorporating the new TOF-TOF will provide throughput more than an order of magnitude higher than any existing LC-MS-MS system, whether electrospray or MALDI, and will confidently identify many more proteins at low concentrations in complex biological samples. Because this instrument is designed for analysis of intact proteins, it will be particularly valuable for monitoring post-translational modifications that are frequently missed using bottom-up approaches for protein analysis. PUBLIC HEALTH RELEVANCE: Functional proteomics requires characterization of proteins at the phenotypic level rather than merely gene level identifications. It is necessary to characterize the entire peptide sequence of a protein and post-translational modifications must be fully detected. The MALDI-TOF-TOF with ETD developed in this project provides accurate molecular weight and complete, unambiguous sequence, including unusual amino acids and post-translational modifications, on proteins and large peptides present at trace levels in complex mixtures. Speed, sensitivity, and dynamic range are substantially superior to the performance presently possible with other technologies.

描述（由申请人提供）：该项目的重点是开发改进的仪器和协议，用于大肽和完整蛋白质的鉴定和结构阐明。所提议的仪器针对双电荷正离子的应用进行了优化，并为 MALDI 电离的完整蛋白质的 MS-MS 提供了独特的功能。可用的断裂机制包括 MALDI 离子源 (PSD) 激发后的单分子断裂，以及小负离子与带双电荷的完整蛋白质反应产生的电子转移解离 (ETD)。该方法基于 MALDI-TOF 和 TOF-TOF 技术的最新进展，但需要添加第二个脉冲离子源和新的碎片离子 TOF 分析仪，该分析仪具有高达 45 kV 的加速能力，以实现高质量、高能量的高性能离子。离子光学器件的设计使得来自两个源的光束可以在碰撞区域内合并，脉冲在时间上精确同步，并且光束的相对速度精确确定。对于正离子和负离子之间的碰撞，相对速度可以设置为非常接近零，为有效的离子化学发生提供足够的时间。该仪器的一个新颖元件是在离子源附近的无场空间中的附加脉冲加速器，它有效地降低了所选离子的速度扩散，并允许在高分辨率碎片光谱测量的同时进行高分辨率母体选择。 这项工作的第一个产品是一种新的 TOF-TOF，它可以对完整蛋白质产生高质量的 MS-MS 谱图。该仪器以前所未有的速度和非常高的灵敏度生成非常高质量的 MS-MS 谱图。结合在单独项目中开发的带有 MALDI 的新型 LC 接口，采用新型 TOF-TOF 的集成 LC-MS-MS 系统将提供比任何现有 LC-MS-MS 系统高出一个数量级以上的通量，无论是电喷雾还是 MALDI，都可以自信地识别复杂生物样品中低浓度的更多蛋白质。由于该仪器专为分析完整蛋白质而设计，因此它对于监测翻译后修饰特别有价值，而使用自下而上的蛋白质分析方法经常会遗漏这些修饰。 公共健康相关性：功能蛋白质组学需要在表型水平上表征蛋白质，而不仅仅是基因水平鉴定。有必要表征蛋白质的整个肽序列，并且必须充分检测翻译后修饰。该项目中开发的带有 ETD 的 MALDI-TOF-TOF 可为复杂混合物中痕量水平的蛋白质和大肽提供准确的分子量和完整、明确的序列，包括不常见的氨基酸和翻译后修饰。速度、灵敏度和动态范围远远优于目前其他技术的性能。