An Integrated Microfluidic System for the Combining Top-Down and Bottom-Up Proteomics

项目摘要/摘要 系统生物学试图了解健康的生物系统是如何工作的，以及当 它们停止正常工作并患病，从而获得所有生化成分的整体视图 并对它们如何改变和互动进行建模。生物系统可以是细胞、组织、生物体，甚至 生物群落。蛋白质构成了细胞内几乎所有的机械，因此它们极大地 影响与疾病相关的正常生物功能和功能障碍。因此，通过改进 蛋白质组分析可以提高我们对生物系统正常功能和功能障碍的理解。 这项研究项目的长期目标是为蛋白质组分析提供改进的方法 增加可以从生物样本中分析的蛋白质的细节。也就是说， 鉴定的蛋白质形式将大大改进，并超过多肽鉴定的蛋白质数量 基于碎片离子光谱的方法。为了在蛋白质形式检测方面取得如此大的改进，一种 将开发新的微流控系统，将完整蛋白质和多肽鉴定策略整合在一起 以无缝和高度自动化的格式。拟议工作的具体目标将包括发展 高效二维分离及微流控快速进样系统的研制 正在处理。该装置的性能将通过对鲍曼不动杆菌的分析来确定 和人类Jurkat细胞，分别代表相对简单和复杂的蛋白质组。除了……之外 作为一个相对简单的模型，鲍曼不动杆菌具有临床意义，因为它会引起人类感染和 正在以惊人的速度获得抗生素耐药性。人类的Jurkat细胞参与人类的免疫 已经得到了高度的研究和表征。这种高度的Jurkat细胞特征将 提供了一个很好的基准来比较所提出的微流控蛋白质组系统的性能 反对其他常用的蛋白质组学方法。

Project Summary/Abstract Systems biology seeks to understand how healthy biological systems work, and what goes wrong when they stop working properly and become diseased gaining a holistic view of all of the biochemical components and modeling how they change and interact. The biological system can be a cell, tissue, organism, or even community of organisms. Proteins form nearly all of the machinery within a cell and consequently they greatly influence proper biological function and dysfunction that is associated with disease. Thus, by improving proteome analysis we can improve our understanding of proper function and dysfunction of biological systems. The long-term objective of this research project is to provide improved methods for proteome analysis that increase the detail of the proteins that can be analyzed from a biological sample. Namely, the number of proteoforms identified will be greatly improved and surpass the number of proteins identified with peptide fragment ion spectra based methods. To achieve this large improvement in the detection of proteoforms, a novel microfluidic system will be developed that will integrate intact protein and peptide identification strategies in a seamless and highly automated format. The specific aims of the proposed work will include development of a high-efficiency two-dimensional separation and development of a microfluidic system for rapid sample processing. The performance of the device will be determined through the analysis of Acinetobacter baumannii and human jurkat cells, which represent relatively simple and complex proteomes, respectively. In addition to being a relatively simple model, A. baumannii is clinically relevant because it causes infections in humans and is gaining antibiotic resistance at an alarming rate. Human jurkat cells are involved in the human immune response and have been highly studied and characterized. This high degree of jurkat cell characterization will provide an excellent benchmark to compare the performance of the proposed microfluidic proteomic system against other commonly used proteomics approaches.