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

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解析。拟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。可用的片段化机制包括在MALDI离子源（PSD）中激发后的单分子片段化，以及来自小负离子与双电荷完整蛋白质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，但需要增加第二个脉冲离子源和新的TOF分析仪，用于碎片离子，其具有高达45 kV的加速，以实现高质量，高能量离子的高性能。离子光学器件被设计成使得来自两个源的射束可以在碰撞区域内合并，其中脉冲在时间上精确同步，并且射束的相对速度被精确确定。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零，从而为有效的离子化学反应提供足够的时间。该仪器中的一个新元素是在邻近离子源的无场空间中的附加脉冲加速器，其有效地降低了所选离子的速度扩散，并允许高分辨率前体选择同时进行碎片光谱的高分辨率测量。 这项工作产生的第一个产品是一种新的TOF-TOF，它可以在完整的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生成非常高质量的MS-MS光谱。结合一个新的LC接口与MALDI正在开发中的一个单独的项目，集成LC-MS-MS系统纳入新的TOF-TOF将提供吞吐量超过一个数量级高于任何现有的LC-MS-MS系统，无论是电喷雾或MALDI，并将有信心地确定更多的蛋白质在低浓度的复杂生物样品。由于该仪器是专为分析完整的蛋白质，它将是特别有价值的监测翻译后修饰，经常错过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的鉴定。有必要表征蛋白质的整个肽序列，并且必须完全检测翻译后修饰。本项目开发的MALDI-TOF-TOF with ETD提供了精确的分子量和完整、明确的序列，包括复杂混合物中痕量水平的蛋白质和大肽的不寻常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.