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Non-Enzymatic Cell Lysis/Protein Digestion Proteomics

非酶细胞裂解/蛋白质消化蛋白质组学

基本信息

批准号：
6956253
负责人：
FRANCO BASILE
金额：
$ 21.08万
依托单位：
UNIVERSITY OF WYOMING
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-07-01 至 2009-06-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The goal of this proposal is to develop and characterize non-enzymatic based methods for single-step cell lysis and site-specific protein digestion. It is expected that the developed techniques will simplify sample preparation and increase sample throughput in proteomic related measurements. Moreover, the ability to prepare the sample by non-enzymatic means has the potential to yield simpler and faster methodology that can be incorporated into proteomic-based lab-on-a-chip devices. Toward these goals, two methodologies will be developed in this work, and include electrochemical and microwave-heating chemically induced reactions at specific amino acids. In this proposal, the development and testing of these methodologies will be carried out with a focus on the detection and identification of microorganisms (i.e., pathogens). To achieve this, the following studies will be carried out: (i) Characterization and optimization of digestion parameters for each method studied using model peptides and proteins for site specificity and possible side reactions, (ii) Test each methodology with model microorganisms for their ability to lyse the cell and produce peptides suitable for MS/MS analyses, and (iii) Develop a prototype lab-on-a-chip device (electrochemical method only) to conduct the lysis/digestion step on-line with MS/MS detection. Specifically, each method will be investigated for its ability to induce cell lysis and digestion of its proteins, all in a single step. The long term goal of this project is to develop and characterize novel protein chemistry that can be incorporated into simple lab-on-a-chip devices for sample pre-processing prior to MS-based proteomic measurements. Advantages anticipated from these studies include the ability to perform high throughput sample preparation for proteomic measurements and its direct effect on the rapid diagnosis of microbial related diseases.

描述（由申请人提供）：本提案的目标是开发和表征用于单步细胞裂解和位点特异性蛋白质消化的非酶基方法。期望开发的技术将简化样品制备和提高样品通量在蛋白质组学相关的测量。此外，通过非酶手段制备样品的能力有可能产生更简单、更快速的方法，可以纳入基于蛋白质组学的芯片实验室设备。为了实现这些目标，在这项工作中将发展两种方法，包括电化学和微波加热化学诱导特定氨基酸的反应。在本提案中，这些方法的开发和测试将重点放在微生物（即病原体）的检测和鉴定上。为此，将进行下列研究：(i)使用模型肽和蛋白质对每种方法的酶解参数进行表征和优化，以确定位点特异性和可能的副反应；（ii）使用模型微生物测试每种方法裂解细胞和产生适合MS/MS分析的肽的能力；（iii）开发一个原型芯片实验室设备（仅限电化学方法），通过MS/MS检测在线进行酶解/酶解步骤。具体来说，每种方法都将被研究其诱导细胞裂解和蛋白质消化的能力，所有这些都在一个步骤中完成。该项目的长期目标是开发和表征新的蛋白质化学，可以整合到简单的芯片实验室设备中，在基于ms的蛋白质组学测量之前进行样品预处理。这些研究的预期优势包括为蛋白质组学测量进行高通量样品制备的能力及其对微生物相关疾病的快速诊断的直接影响。