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

描述(由申请人提供)：该项目专注于开发改进的仪器和方案，用于鉴定和鉴定大肽和完整蛋白质的结构。该仪器针对双电荷阳离子的应用进行了优化，并为MALDI电离的完整蛋白质的MS-MS提供了独特的能力。可用的碎裂机制包括在MALDI离子源(PSD)中激发后的单分子碎裂，以及小负离子与双电荷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，但需要增加第二个脉冲离子源和新的TOF碎片离子分析仪，其特点是加速高达45千伏，以实现对高质量、高能离子的高性能。离子光学的设计使得来自两个源的光束可以在对撞区内合并，使脉冲在时间上精确同步，并精确地确定光束的相对速度。对于正离子和负离子之间的碰撞，可以将相对速度设置为非常接近于零，从而为高效的离子化学提供足够的时间。该仪器中的一个新元件是在离子源附近的无场空间中额外的脉冲加速器，它有效地减少了所选离子的速度扩散，并允许在进行高分辨率碎片光谱测量的同时进行高分辨率前体选择。这项工作产生的第一个产品是一种新的TOF-TOF，它可以在完整蛋白质上产生高质量的MS-MS光谱。该仪器以前所未有的速度和非常高的灵敏度产生非常高质量的MS-MS光谱。与另一个单独项目中正在开发的与MALDI的新LC接口相结合，集成了新的TOF-TOF的LC-MS-MS系统将提供比任何现有的LC-MS-MS系统高出一个数量级以上的吞吐量，无论是电喷雾还是MALDI，并将自信地在复杂的生物样品中识别更多低浓度的蛋白质。由于该仪器是为完整蛋白质的分析而设计的，因此对于使用自下而上的蛋白质分析方法经常遗漏的翻译后修饰的监测将特别有价值。 公共卫生相关性：功能蛋白质组学要求在表型水平上对蛋白质进行表征，而不仅仅是基因水平的鉴定。有必要对蛋白质的整个肽序列进行表征，并且必须完全检测到翻译后修饰。本项目开发的带有ETD的MALDI-TOF-TOF在复杂混合物中以微量存在的蛋白质和大肽上提供了准确的分子量和完整、明确的序列，包括不寻常的氨基酸和翻译后修饰。速度、灵敏度和动态范围大大优于目前其他技术所能达到的性能。

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.