High-performance MALDI-TOF-TOF for Proteomics

描述(申请人提供)：该项目的目标是开发一种新的高性能MALDI-TOF-TOF仪器，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光谱比目前的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。在第一阶段设计和建造了一台用于展示高性能MALDI仪器的可行性的TOF-TOF原型仪器，该仪器提供了高通量蛋白质组学所需的速度、灵敏度和动态范围。这台新仪器的基本部件包括一个新的简化的MALDI离子源，设计为以5 kHz激光全天候常规操作；高分辨率第一级MS，提供至少高达4000 m/z的单同位素前体选择，离子传输没有可测量的损失，相邻质量的透射率低于1%；新的飞行时间-飞行时间分析器结合了高分辨率前体选择和多路操作，允许在每次激光发射后选择和分析多个前体；高分辨率第二级MS，结合碎片光谱的自动内部校准，提供对所有碎片质量的高质量精度。第一阶段的进度报告介绍了这些要素的设计和性能。 该项目第二阶段的目标是完成包括所有这些元素的全功能、商业可行的MS-MS系统的开发，并展示其在生物样品应用中的性能。剩下的工作不仅需要最终完成整个系统的设计和建造，包括电子和自动控制，而且还需要展示所产生的元件和系统的长期可靠性和坚固性。在第二阶段开发的原型的最终测试将在我们的合作者的实验室中进行，这些应用需要对高灵敏度和宽动态范围的生物样本进行快速和可靠的分析。 在第三阶段，具有上述属性的高通量、高分辨率、多路复用的MALDI TOF-TOF质谱仪将被商业化，从而淘汰所有以前的MALDI MS-MS仪器。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，可在许多应用中取代电喷雾LC-MS-MS系统。 与公共卫生相关：人们普遍认为，串联质谱仪与有效分离相结合，才能接近对血浆、血清、尿液和脑脊液等生物液体进行全球分析所需的灵敏度和动态范围。对近端体液和组织样本的分析要求较低，因为所需的动态范围实质上较小，但可用的总样本量可能更有限，因此需要相同的最终灵敏度。多维分离与质谱学相结合可以提供所需的动态范围，但需要大量的级分，并且当前的MS-MS技术都没有达到所需的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.