High-performance MALDI-TOF-TOF for Proteomics

描述（由申请人提供）：本项目的目标是开发一种新的高性能MALDI-TOF-TOF仪器，以消除限制蛋白质组学进展的主要瓶颈。新的TOF-TOF产生高质量的MS-MS光谱，比当前的MS-MS仪器快几个数量级。基本上所有产生的光谱都以相应的速度进行解释。在第一阶段设计并构建了一个原型TOF-TOF仪器，用于证明高性能MALDI仪器的可行性，该仪器可提供高通量蛋白质组学所需的速度、灵敏度和动态范围。这一新仪器的基本组成部分包括一个新的简化MALDI离子源，设计用于用5 μ m激光器24/7进行常规操作;一个高分辨率第一级MS，提供至少m/z 4000的单一同位素前体选择，离子传输没有可测量的损失，相邻质量的传输小于1%;一种新的TOF-TOF分析仪，它结合了高分辨率的前体选择和多路复用操作，允许在每次激光发射后选择和分析多种前体;以及高分辨率第二级MS，其结合了碎片光谱的自动内部校准，提供所有碎片质量的高质量准确度。第一阶段的进度报告说明了这些组成部分的设计和性能。 该项目第二阶段的目标是完成一个功能齐全的、商业上可行的MS-MS系统的开发，包括所有这些元素，并证明其在生物样品应用中的性能。余下的工作不仅需要最后完成整个系统的设计和建造，包括电子和自动控制，而且还需要证明所产生的元件和系统的长期可靠性和坚固性。第二阶段开发的原型的最后测试将在我们的合作者的实验室进行，用于需要以高灵敏度和宽动态范围快速可靠地分析生物样品的应用。 在第三阶段，具有上述属性的高通量、高分辨率、多路复用MALDI TOF-TOF质谱仪将被商业化，使所有以前的MALDI MS-MS仪器过时。在相关项目中开发的分离接口和自动化的增加提供了集成系统，可以在许多应用中取代电喷雾LC-MS-MS系统。 公共卫生相关性：人们广泛认识到，需要串联质谱与有效分离的组合来接近生物流体（例如血浆、血清、尿液和CSF）的全局分析所需的灵敏度和动态范围。近端流体和组织样品的分析要求稍低，因为所需的动态范围基本上较小，但是可用的总样品的量可能更有限，使得需要相等的最终灵敏度。多维分离结合质谱法可以提供所需的动态范围，但需要大量的级分，并且目前没有MS-MS技术达到所需速度和灵敏度的数量级。

DESCRIPTION (provided by applicant): The goal of this project is to develop a new high-performance MALDI-TOF-TOF instrument that removes a major bottleneck limiting progress in proteomics. The new TOF-TOF produces high-quality MS-MS spectra orders of magnitude faster than current MS-MS instruments. Essentially all of the spectra produced are interpreted with corresponding speed. A prototype TOF-TOF instrument for demonstrating feasibility of a high-performance MALDI instrument that provides the speed, sensitivity, and dynamic range required for high-throughput proteomics was designed and constructed in phase I. The essential elements of this new instrument include a new simplified MALDI ion source designed to operate routinely 24/7 with 5 khz laser; a high-resolution first stage MS that provides mono-isotopic precursor selection up to at least m/z 4000 with no measurable loss in ion transmission and less than 1% transmission of adjacent masses; a new TOF-TOF analyzer incorporating high-resolution precursor selection and multiplex operation that allows multiple precursors to be selected and analyzed following each laser shot; and a high resolution second stage MS that incorporates automatic internal calibration of fragment spectra providing high mass accuracy on all fragment masses. Design and performance of these elements are described in the progress report for phase I. The goal of phase II of this project is to complete the development of a fully functional, commercially viable MS-MS system including all of these elements and to demonstrate the performance in applications to biological samples. The remaining work requires not only finalizing the design and construction of the complete system, including electronics and automatic controls, but also demonstrating the long-term reliability and ruggedness of the resulting elements and system. Final testing of the prototypes developed in phase II will be carried out in laboratories of our collaborators on applications requiring rapid and reliable analysis of biological samples with high sensitivity and broad dynamic range. In phase III a high-throughput, high-resolution, multiplexed MALDI TOF-TOF mass spectrometer with the attributes described above will be commercialized, rendering all previous MALDI MS-MS instruments obsolete. Addition of separations interfaces and automation developed in related projects provide integrated systems that may replace electrospray LC-MS-MS systems for many applications. PUBLIC HEALTH RELEVANCE: It is widely recognized that a combination of tandem mass spectrometry with efficient separation is required to approach the sensitivity and dynamic range required for global analysis of biological fluids such as plasma, serum, urine, and CSF. Analysis of proximal fluids and tissue samples is somewhat less demanding in that the dynamic range required is substantially smaller, but the amount of total sample available may be more limited so that equal ultimate sensitivity is required. Multi-dimensional separation combined with mass spectrometry can provide the required dynamic range, but a large number of fractions are required and no current MS-MS technique comes within an order of magnitude of the required speed and sensitivity.