New Separations for MALDI-TOF Mass Spectrometry

描述（由申请人提供）：总结本研究的最终目标是提供集成系统，使复杂的生物样品进行分析，具有高灵敏度和宽的动态范围。这些系统适用于蛋白质、DNA和RNA寡聚体以及来自体液和组织的代谢物。拟议的项目是专门专注于开发新的样品板采用准直孔结构，允许各种分离技术，以有效地耦合到MALDI-TOF和TOF-TOF质谱。拟议的研究的第一个具体目标是一个改进的接口之间的HPLC和MALDI MS-MS是兼容的1毫米ID列，在流速高达50？L/min，而不牺牲对痕量组分的灵敏度。这将允许更大的样品加载量，并预计将动态范围增加至少五十倍，并允许更快，高分辨率的分离。第二个具体目标是开发平板凝胶电泳和MALDI-TOF-TOF之间的直接接口，允许在MALDI板上提取、消化和浓缩样品，样品损失最小，空间分辨率没有损失。第三个具体的目标是开发一个类似的接口直接与冷冻组织样品成像的完整蛋白质存在，并消化和识别的蛋白质MALDI-TOF MS-MS。该项目解决了一个主要的瓶颈限制的实用性质谱作为一个必要的工具，需要真正的全球生物系统的分析。现在人们普遍认识到，与高效分离技术相结合的质谱法对于功能蛋白质组学和代谢组学中复杂生物样品的大规模分析是必不可少的;但目前可用的系统是不够的。改进的MALDI-TOF和TOF-TOF质谱可以提供所需的MS性能，但改进的分离界面同样重要。该第一阶段项目将确定MALDI新方法的可行性，该方法允许在新型微流体系统中并行处理大量样品，以实现高通量和宽动态范围。未来的第二阶段工作将侧重于开发可供生物学家在各种研究应用中使用的实用系统。这些将与直接参与此类研究的合作者一起开发和评估。 MALDI-TOF和TOF-TOF技术的最新进展使该方法成为已建立的电喷雾LC-MS-MS系统的有吸引力的替代方案，用于定量和鉴定复杂生物样品中的蛋白质和肽。尽管取得了一些早期的成功，但采用速度缓慢，可能是因为潜在的优势尚未得到明确证明。这个项目，连同改进的MALDI-TOF-TOF仪器正在开发的一个单独的项目，将证明这种新方法的真实的优势。

DESCRIPTION (provided by applicant): Summary 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. The proposed 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. The first specific aim for the proposed research is an improved interface between HPLC and MALDI MS-MS that is compatible with 1 mm ID columns, operating at flow rates up to 50 ?L/min without sacrificing sensitivity for trace components. This will allow larger sample loadings and is expected to increase the dynamic range by at least a factor of fifty and allow faster, high-resolution separations. The second specific aim is to develop a direct interface between slab gel electrophoresis and MALDI-TOF-TOF that allows extraction, digestion, and concentration of samples on the MALDI plate with minimal sample loss and no loss in spatial resolution. A third specific aim is to develop a similar interface directly with frozen tissue samples both for imaging the intact proteins present, and for digesting and identifying the proteins by MALDI-TOF MS-MS. This project addresses a major bottleneck limiting the utility of mass spectrometry as an essential tool needed for truly global analyses of biological systems. It is now generally recognized that mass spectrometry interfaced with efficient separation technologies is essential for large-scale analyses of complex biological samples in functional proteomics and metabolomics; but currently available systems are inadequate. Improved MALDI-TOF and TOF-TOF mass spectrometry can provide the MS performance needed, but improved interfaces with separations are equally important. This Phase I project will establish the feasibility of a new approach to MALDI that allows parallel processing of large numbers of samples in a novel micro- fluidic system to achieve high throughput and broad dynamic range. A future Phase II effort will focus on developing practical systems that can be used by biologists in a variety of research applications. These will be developed and evaluated working with collaborators directly involved in such research. Recent advances in MALDI-TOF and TOF-TOF technology make this approach an attractive alternative to the established electrospray LC-MS-MS systems for quantitation and identification of proteins and peptides in complex biological samples. Despite some early success, adoption has been slow, probably because the potential advantages have not yet been clearly demonstrated. This project, together with improved MALDI-TOF-TOF instrumentation being developed in a separate project, will demonstrate the real advantages of this new approach.