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质谱计，其性能优于目前采用的所有现有的MALDI-MS技术。在所提出的仪器中，需要多段碎片光谱来产生完整的碎片光谱，每一段对应于碎片质量与前体质量之比的特定范围；但与早期的源后衰变(PSD)仪器不同，由于光谱中存在许多前体离子，因此可以同时确定存在的碎片的准确碎片离子质量。因此，虽然可能需要10-15个片段才能产生完整的片段光谱，但可以在不牺牲灵敏度或质量精度的情况下将100或更多的前体片段碎裂。采用5 kHz的脉冲频率，使得数据的采集速度远远快于现有的TOF仪器，通常限于200赫兹或更低的频率。重点是在MS-MS模式下的最佳性能，但反射器MS模式下的分辨率和质量精度可与目前商业仪器中最好的可用仪器相媲美。无论是正离子还是负离子，都可以在MS和MS-MS模式下测量。该仪器的独特之处包括同时获取大量前驱体的碎片光谱，高分辨率和高质量精度的快速灵敏的前体和中性损失扫描，以及多路MS3光谱的获取。设计的重点是简单性、可靠性和与高性能一致的最低成本。在许多主要组件都是一系列系统通用的情况下，采用模块化方法。常见的组件包括带有运动控制的样板处理系统、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。介绍了一种刻度碎片光谱的新方法，该方法通过对描述两级离子反射器飞行时间的方程进行逆运算，得到碎片质量与飞行时间之间的“精确”关系式。一项重要的初始工作集中在验证这种方法和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，以根据对多个前体的同时测量自动生成碎片光谱。该软件将扩展到生成前兆扫描、中性损失扫描和LC分离的多反应监测，并解释MS3飞行时间数据。该系统将被验证应用于胰酶多肽的MS-MS鉴定和复杂脂肪样品的定量和定性测量。人类蛋白质组组织发起了以血浆、肝脏和大脑蛋白质组学为重点的重大倡议。这些举措的前提是“对健康和疾病中的循环蛋白进行全面、系统的表征将极大地促进癌症和其他疾病的预防、诊断和治疗的生物标记物的开发”。《蛋白质组学》，2004年第4期，1235-1240页。在最近一次HuPo会议(长滩，2006年)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，但基于不同实验室对相同样本的结果不一致而得出的不可避免的结论是，目前的技术远远达不到实现所述目标所需的水平。该项目与另一个单独项目中正在开发的改进的分离界面一起，将为全面的蛋白质组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.