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，用于鉴定和结构阐明大肽和完整蛋白质。该仪器对双带正离子进行了优化，并为MALDI电离的完整蛋白提供了独特的质谱分析能力。现有的断裂机制包括MALDI离子源（PSD）激发后的单分子断裂，以及小负离子与带双重电荷的完整蛋白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一个新的碎片离子TOF分析仪，该分析仪具有高达45 kV的加速度，以实现高质量、高能量离子的高性能。设计了离子光学系统，使两个源的光束能够在碰撞区域内合并，脉冲在时间上精确同步，并精确确定光束的相对速度。对于正离子和负离子之间的碰撞，相对速度可以设置得非常接近于零，为有效的离子化学发生提供了足够的时间。该仪器的一个新元素是在离子源附近的无场空间中额外的脉冲加速器，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高分辨率碎片光谱测量。这项工作的第一个产物是一种新的TOF-TOF，它可以对完整的蛋白质产生高质量的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质谱。结合在一个单独的项目中开发的新的带有MALDI的LC接口，集成的LC- ms - ms系统将提供比任何现有的LC- ms - ms系统高一个数量级以上的通量，无论是电喷雾还是MALDI，并且可以自信地在复杂的生物样品中识别出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，因此它对于监测翻译后修饰特别有价值，这些修饰在使用自下而上的蛋白质分析方法时经常被遗漏。

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.