Low-cost High-performance MALDI-TOF MS and MS-MS

描述（由申请人提供）：该项目的目标是开发一种新的低成本MALDI- tof质谱仪，该质谱仪优于MALDI目前使用的所有现有MS-MS技术。在所提出的仪器中，需要多个片段光谱段来生成完整的片段光谱，每个片段对应于片段质量与前体质量之比的特定范围；但与早期的后源衰变（PSD）仪器不同，精确的碎片离子质量可以同时测定由于光谱中许多前体离子而存在的碎片。因此，虽然可能需要10-15个片段来产生完整的片段谱，但可以在不牺牲灵敏度或质量准确性的情况下破碎100个或更多的前体。采用5 kHz的脉冲速率，允许比现有的TOF仪器更快地获取数据，通常限于200 hz或更低的速率。重点是MS-MS模式下的最佳性能，但反射式MS模式下的分辨率和质量精度可与目前商用仪器中最好的分辨率和质量精度相媲美。正离子或负离子都可以在MS和MS-MS模式下测量。该仪器的独特之处在于能够同时采集大量前驱体的碎片光谱，具有高分辨率和质量精度的快速灵敏前驱体和中性损失扫描，以及多路MS3光谱的采集。设计工作的重点是简单性、可靠性和与高性能一致的最低成本。采用模块化方法，其中许多主要组件对于一个系统家族是通用的。常用组件包括带运动控制的样品板处理系统，激光光学和控制，真空系统，数字化仪和计算机，以及几个电子模块。本文提出了一种新的碎片光谱校正方法，该方法基于对二级离子反射器中飞行时间方程的反演，得到了碎片质量与飞行时间之间的“精确”方程。一项重要的初步工作集中在验证这种方法并确定可以实现的碎片质量精度上。研究工作的主要部分是开发处理战斗时间数据所需的软件，以自动生成多个前体同时测量的碎片光谱。该软件将扩展到生成前体扫描、中性损失扫描和LC分离的多重反应监测，并解释MS3飞行时间数据。该系统将用于色氨酸的质谱联用蛋白鉴定以及复杂脂质样品的定量和定性测量。人类蛋白质组组织（Human Proteome Organization）已经启动了主要研究血浆、肝脏和大脑蛋白质组学的项目。这些倡议的前提是“全面、系统地表征健康和疾病中的循环蛋白，将极大地促进用于预防、诊断和治疗癌症和其他疾病的生物标志物的发展”。杨志刚，2004,4,1235-1240。在最近的HUPO会议（2006年长滩）上介绍了这些举措的进展情况。虽然提出了许多积极的结果，但由于不同实验室对相同样品的结果缺乏一致性，因此不可避免地得出结论，即目前的技术远远达不到实现既定目标所需的技术。这个项目，连同在一个单独的项目中开发的改进的分离接口，将为全面的蛋白质组学测量提供必要的工具。

DESCRIPTION (provided by applicant): The goal of this project is to develop a new low-cost MALDI-TOF mass spectrometer that outperforms all existing MS-MS techniques currently employed with MALDI. In the proposed instrument multiple segments of fragment spectra are required to generate a complete fragment spectrum, each segment corresponding to a particular range of the ratio of fragment mass to precursor mass; but unlike earlier post-source decay (PSD) instruments, accurate fragment ion masses may be determined simultaneously for fragments present due to many of the precursor ions in the spectrum. Thus although 10-15 segments may be required to generate a complete fragment spectrum, 100 or more precursors can be fragmented without sacrificing sensitivity or mass accuracy. A pulse rate of 5 kHz is employed, allowing data to be acquired much faster than in existing TOF instruments typically limited to rates of 200 hz or less. The emphasis is on optimum performance in MS-MS mode, but resolving power and mass accuracy in reflector MS mode are comparable to the best currently available in commercial instruments. Either positive or negative ions can be measured in both MS and MS-MS modes. The unique features of the proposed instrument include simultaneous acquisition of fragment spectra from a large number of precursors, rapid and sensitive precursor and neutral loss scans with high resolving power and mass accuracy, and acquisition of multiplexed MS3 spectra. The design effort is focused on simplicity, reliability, and minimum cost consistent with high performance. A modular approach is employed where many of the major components are common to a family of systems. Common components include the sample plate handling system with motion control, the laser optics and controls, vacuum system, digitizer and computer, and several electronics modules. A novel method for calibrating fragment spectra is described based on inversion of the equation describing the flight time in a two-stage ion reflector to yield an "exact" equation relating fragment mass to flight time. An important initial effort is focused on validating this approach and determining the fragment mass accuracy that can be achieved. A major portion of the research effort is directed toward developing the software required for processing time-of-fight data to automatically generate fragment spectra from simultaneous measurements on multiple precursors. The software will be extended to generating precursor scans, neutral loss scans, and multiple reaction monitoring with LC separation, and to interpreting MS3 time-of-flight data. The system will be validated for applications to protein identification by MS-MS on tryptic peptides and for quantitative and qualitative measurements on complex lipid samples. The Human Proteome Organization has launched major initiatives focused on plasma, liver, and brain proteomics. The premise of these initiatives is that "A comprehensive, systematic characterization of circulating proteins in health and disease will greatly facilitate development of biomarkers for prevention, diagnosis, and therapy of cancer and other diseases". Omenn, Proteomics 2004, 4, 1235-1240. Progress on these initiatives was presented at the most recent HUPO meeting (Long Beach 2006). While many positive results were presented, an inescapable conclusion based on the lack of agreement among results from different laboratories on identical samples must be that present technology falls woefully short of that required to achieve the stated goals. This project, together with improved separations interfaces being developed in a separate project, will provide essential tools for comprehensive proteomic measurements.