New Separations for MALDI-TOF Mass Spectrometry

描述（由申请人提供）：本研究的最终目标是提供集成系统，使复杂生物样品能够进行高灵敏度和宽动态范围的分析。这些系统适用于蛋白质、DNA和RNA低聚物以及体液和组织中的代谢物。该项目特别专注于开发采用准直孔结构的新型样品板，使各种分离技术能够有效地与MALDI-TOF和TOF-TOF质谱相结合。第一阶段确定了新型MALDI板与几种广泛使用的分离技术（包括HPLC和平板凝胶电泳）以及冷冻组织切片直接成像的基本可行性。二期将生产新的MALDI样品板系列，针对这些应用和设备进行优化，用于与各种技术接口，以有效分离肽，蛋白质和代谢物。这些设备与正在并行项目中开发的新型MALDI-TOF质谱和MS-MS仪器相结合，将为复杂生物样品的全球分析能力提供数量级的提高。第二阶段的具体目标是专注于采用针对选定应用优化的准直孔结构的实用MALDI样品板，以及用于将MALDI MS和MS- MS与已建立的有效分离肽，蛋白质和代谢物的技术相连接的实用设备。用于这些应用的实用CHS板的定义是，它必须足够便宜，在单次使用后即可丢弃，或者必须有一个简单，可靠的程序来清洁板，没有可检测到的样品携带。这些新的MALDI样品板、平行消化板及其使用所需的仪器和试剂将为三期商业化提供重要的新产品。此外，将该项目的产出与维珍仪器公司同时进行的其他项目的成果相结合的完整集成系统将为产品提供更大的潜在市场。其中包括用于全球生物分析的全自动LC-MALDI MS-MS系统；自动“分子扫描仪”用于蛋白质分析，使用凝胶和MALDI MS和MS；一体化全球分析实验室；还有一个自动高速组织成像仪。公共卫生相关性：人类基因组计划已经建立了理解生物学的分子方法，但目前对生物体如何在分子水平上发挥作用的知识确实非常贫乏。功能分析必须进行，不仅在基因表达水平（转录组学），而且在蛋白质翻译和修饰水平（蛋白质组学），以及代谢物网络（代谢组学）。化学、分离科学、质谱和生物信息学的进一步改进将需要完成这场革命。分离和质谱之间的界面被认为是一个主要的瓶颈。这项研究将为克服这一障碍提供重要的新工具。

DESCRIPTION (provided by applicant): The ultimate goal of this research is to provide integrated systems that allow complex biological samples to be analyzed with high sensitivity and broad dynamic range. These systems are applicable to proteins, DNA and RNA oligomers, and metabolites from bodily fluids and tissues. This project is specifically focused on developing novel sample plates employing collimated hole structures that allow a variety of separation techniques to be efficiently coupled to MALDI-TOF and TOF-TOF mass spectrometry. Phase I established the basic feasibility of the new MALDI plates for interfacing with several widely used separation techniques including HPLC and slab gel electrophoresis, and with direct imaging of frozen tissue sections. Phase II will produce a new family of MALDI sample plates optimized for these applications and devices for interfacing with a variety of techniques for efficiently separating peptides, proteins, and metabolites. These devices integrated with new MALDI-TOF MS and MS-MS instruments being developed in parallel projects will provide orders of magnitude improvements in capabilities for global analysis of complex biological samples. Specific aims for Phase II are focused on practical MALDI sample plates employing collimated hole structures optimized for selected applications, and on practical devices for interfacing MALDI MS and MS- MS with established techniques for efficiently separating peptides, proteins, and metabolites. The definition of a practical CHS plate for these applications is that it must be sufficiently inexpensive to be disposable after single use, or there must be a simple, reliable procedure for cleaning the plates with no detectable sample carry-over. These new MALDI sample plates, parallel digestion plates, and the apparatus and reagents required for their use will provide important new products for commercialization in Phase III. In addition, complete integrated systems combining output of this project with results from other projects being pursued concurrently at Virgin Instruments Corp. will provide products with much larger potential markets. These include a fully automated LC-MALDI MS-MS system for global biological analyses; an automated "Molecular Scanner" for protein analyses using gels and MALDI MS and MS-MS; integrated global analysis laboratory; and an automated high-speed tissue imager. PUBLIC HEALTH RELEVANCE: The human genome project has established the molecular approach to understanding biology, but present knowledge of how an organism functions at the molecular level is really very poor. Functional analyses must be carried out, not only at the level of gene expression (transcriptomics), but also at the level of protein translation and modification (proteomics), and the metabolite network (metabolomics). Further improvements in chemistry, separations science, mass spectrometry, and bio-informatics will be required to complete this revolution. The interface between separations and mass spectrometry is believed to be a major bottleneck. The proposed research will provide important new tools toward overcoming this obstacle.