CAREER: A novel approach for deciphering cellular metabolic phenotypes using tandem mass spectrometry

这项提案的总体研究目标是更好地了解细胞代谢，以便有目的地操纵细胞行为。代谢是活细胞将营养物质转化为产物的基本过程。由于难以精确和准确地测量细胞内代谢通量，目前关于代谢在细胞行为中的作用的知识水平是有限的。该项目将通过使用新型测量技术串联质谱法以更高的精度和准确度量化细胞的代谢状态，为微生物代谢和细胞生理学的知识做出贡献。本文的工作将通过以下几个方面增加对代谢通量分析的科学认识：（1）介绍一种新的代谢通量分析方法（MFA）;（2）提供验证通量结果和识别潜在误差来源的改进技术;（3）开发新的算法和易于使用的MFA工具;（4）提高对两种重要模式微生物代谢网络运作的认识，本建议的教育目标是促进定量系统生物学原理的教学，使其成为化学工程教育的一个组成部分，并提高代表性不足的群体的参与，生物学研究具体而言，教育方法是：（1）提供暑期研究机会，少数民族本科生从特拉华州州立大学，当地首屈一指的HBCU;和（2）开发新的课程材料和刺激的顶点实验室课程生物蛋白质生产，回收和纯化在特拉华州大学。该项目还为学生在跨学科环境中的教育和培训提供了独特的机会。学生将获得微生物细胞培养，代谢工程，代谢组学，通量分析，质谱和定量建模的经验。这些经验将使学生处于代谢工程和定量系统生物技术新兴领域的最前沿。

The overall research objective of this proposal is to develop better understanding of cellular metabolism to allow purposeful manipulation of cell behavior. Metabolism is the fundamental process by which living cells convert nutrients into products. The current state-of-knowledge regarding the role of metabolism in cellular behavior is limited due to difficulties in measuring intracellular metabolic fluxes precisely and accurately. This project will contribute to the knowledge of microbial metabolism and cell physiology by quantifying the metabolic state of cells at a much higher level of precision and accuracy using a novel measurement technique, tandem mass spectrometry. The proposed work will add to the scientific understanding by: (1) introducing a new method of metabolic flux analysis (MFA); (2) providing improved techniques for validating flux results and identifying potential sources of errors; (3) developing new algorithms and easy to use tools for MFA; and (4) improving knowledge of metabolic network operation for two important model microbes, Escherichia coli and Saccharomyces cerevisiae.The educational objective of this proposal is to promote teaching of quantitative systems biology principles as an integral part of chemical engineering education and to enhance the participation of underrepresented groups in biological research. Specifically, the educational approach is to: (1) provide summer research opportunities to minority undergraduate students from Delaware State University, a local premier HBCU; and (2) develop new course material and a stimulating capstone laboratory course on biological protein production, recovery and purification at the University of Delaware. This project also provides unique opportunities for student education and training in an interdisciplinary environment. Students will gain experience in microbial cell culture, metabolic engineering, metabolomics, flux analysis, mass spectrometry and quantitative modeling. These experiences will place the students at the forefront of metabolic engineering and the emerging field of quantitative systems biotechnology.