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，以识别和结构化大肽和完整蛋白质。提出的仪器被优化，以应用于双电荷阳性离子，并为MALDI电离的完整蛋白质提供了独特的功能。可用的碎裂机制包括在MALDI离子源（PSD）激发后的单分子碎片，以及来自小离子与双电荷完整蛋白的反应的电子转移解离（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TOF分析仪，用于碎片离子，该碎片离子具有高达45 kV的加速度，以在高质量，高能离子上实现高性能。设计了离子光学元件，以便可以在碰撞区域中合并两个源的光束与脉冲准确地同步，并与光束的相对速度准确确定。对于正离子和负离子之间的碰撞，相对速度可以非常接近零，从而为有效的离子化学物质提供了足够的时间。该仪器中的一个新元素是与离子源相邻的无现场空间中的附加脉冲加速器，可有效地降低所选离子的速度扩散，并允许使用碎片光谱的高分辨率测量同时选择高分辨率的前体选择。 这项工作产生的第一个产品是一种新的TOF-TOF，可在完整蛋白上产生高质量的MS-MS光谱。该仪器以前所未有的速度产生非常高质量的MS-MS光谱，并且具有很高的灵敏度。结合与单独的项目中开发的新的LC接口，结合新TOF-TOF的集成LC-MS-MS系统将比任何现有的LC-MS-MS系统（无论是电气报纸还是MALDI）高的量级，无论是在复杂的生物学样本中，低浓度的蛋白质都会比任何现有的LC-MS-MS系统高。由于该仪器设计用于完整蛋白质的分析，因此对于监测翻译后修饰特别有价值，这些修饰通常使用自下而上的方法进行蛋白质分析。 公共卫生相关性：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。必须表征蛋白质的整个肽序列，并且必须完全检测到翻译后修饰。该项目中开发的具有ETD的MALDI-TOF-TOF提供了准确的分子量和完整的，明确的序列，包括异常的氨基酸和翻译后修饰，对蛋白质和大型肽在复杂混合物中的痕量水平上存在。速度，灵敏度和动态范围基本上优于其他技术目前的性能。