Enhanced Dynamic Range Proteomic Analysis: Phase II

描述（由申请人提供）：这项工作的长期目标是开发一种光学模块，该模块将减少成像系统中的杂散光，从而从数字图像中提供更精确的测量，并增加检测的动态范围，从而能够分析目前无法测量的物体。目前的II期提案旨在极大地加强蛋白质组学中荧光染料的多色光谱成像分析，以检测凝胶电泳中蛋白质水平的变化和蛋白质翻译后修饰。该光学模块的潜在未来应用将改善免疫组织化学中的多色荧光检测和细胞和组织中荧光蛋白的分析，微孔板读取和微流控分析，为多重诊断提供新的方法。所开发的系统将适用于几乎所有的光学成像系统，从而扩大应用范围，最终降低成本。具体目标是：(1)开发和表征降噪模块（NoRM）。该系统将记录初始图像，然后利用反馈回路“关闭”明亮像素和相关的杂散光，以更准确地测量图像内的区域；(2)在蛋白质组学应用中展示NoRM，这将验证该技术，并能够检测目前太弱而无法观察到的二维凝胶中的蛋白质和蛋白质模式（增强的蛋白质模式有望具有诊断价值），并指导增强的电洗脱/微流控消化/集成质谱分析；(3)在第二阶段工作结束时，为有限规模的发布准备生产原型规范。第二阶段的工作建立在成功的第一阶段工作的基础上，与目前使用的图像包围技术相比，第一阶段的工作展示了超过10倍的动态范围改进。提议的努力是跨学科的，需要在光学和机械设计、软件开发、生产工程、生物化学、蛋白质组学和系统生物学方面的专业知识。由此产生的技术将对蛋白质组学、显微镜和许多其他利用数码相机的技术有用。

DESCRIPTION (provided by applicant): The long-term goal of this effort is to develop an optical module that will reduce stray light within imaging systems, thereby providing more accurate measurements from digital images and increased dynamic range of detection to enable analysis of objects not currently measurable. The present Phase II proposal is aimed at greatly enhancing the analysis of multicolor spectral imaging of fluorescent dyes in proteomics to detect changes in protein levels and protein posttranslational modifications in gel electrophoresis. Potential future applications of the optical module will improve multicolor fluorescence detection in immunohistochemistry and analysis of fluorescent proteins in cells and tissues, microplate reading and microfluidic analysis for new methods of multiplex diagnostics. The system to be developed will be usable on nearly all optical imaging systems so as to broaden the scope of applications and ultimately reduce the cost. The specific aims are to: (1) Develop and characterize a Noise Reduction Module (NoRM). This system will record an initial image, then utilize a feedback loop to "turn off" the bright pixels and associated stray-light to more accurately measure regions within an image; (2) Demonstrate the NoRM in proteomics applications, which will validate the technology, and enable detection of proteins and patterns of proteins in 2-D gels that are currently too weak to observe (the enhanced protein patterns are expected to have diagnostic value), and guide enhanced electroelution/microfluidic digestion/integrated mass spectral analysis ; and (3) Prepare a production prototype NoRM for a limited scale release by the end of the Phase II effort. This Phase II effort builds on a successful Phase I effort that demonstrated greater than factor of 10 improvement in dynamic range as compared to currently used image bracketing technology. The proposed effort is cross-disciplinary, with expertise required in optical and mechanical design, software development, production engineering, biochemistry, proteomics, and systems biology. The resulting technology will be useful for proteomics, microscopy, and many other technologies that utilize digital cameras. PUBLIC HEALTH RELEVANCE: The proposed technology will reduce the stray light noise for digital imaging systems, thereby expanding capabilities for proteomics, glycomics, cell biology, diagnostics and any biomedical application that utilizes digital cameras. During this effort, patterns of weakly-expressed proteins and changes in these proteins, whose signals are currently too weak to be identified in electrophoresis gels will be measured and identified to better understand biological mechanisms, improve development of more specific drugs, and enhance regenerative and preventative medicine.