Microfluidic System for Cancer Proteome Analysis

用于癌症蛋白质组分析的微流体系统

• 批准号: 6852656
• 负责人: DANIEL R. KNAPP
• 金额: $ 25.31万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6852656
• 关键词: analytical chemistry; biomedical equipment development; clinical research; high throughput technology; human tissue; liquid chromatography mass spectrometry; miniature biomedical equipment; protein purification; proteomics

DESCRIPTION (provided by applicant): This proposal is a combined R21/R33 application to develop an integrated multidimensional analysis system for characterization of complex mixtures of proteins, i.e. proteome analysis. The system will employ a microfluidic implementation of a 2D chromatography separation system directly interfaced via electrospray ionization to a mass spectrometer. The analytical approach is to digest the protein mixture prior to separation and then separate and analyze the resulting fragment peptides. Protein identification is done by computer matching of tandem mass spectra with predicted data from sequence databases. The separation system, which will employ strong cation exchange and reversed phase partition chromatography, is based upon a published capillary system which has been shown to be superior to conventional two dimensional gel electrophoresis methods for proteomic studies in yeast. The microchip implementation will enable improvements over the published approach to yield a more powerful, yet simpler to use and less expensive system. The ultimate implementation of the system will replace 2D gel separation with a relatively inexpensive, disposable injection molded plastic microfluidic device which interfaces directly to the mass spectrometer. The prototype devices will be produced using recently developed soft lithography methods which obviate the need for complex microfabrication facilities and yield designs directly transferable to large scale production by injection molding. This approach will facilitate the application of proteome analysis in cancer research by significantly reducing the labor and art required compared to 2D electrophoresis and eliminating the need for manual transfer and processing of gel for mass spectrometric analysis. The system will facilitate the molecular analysis of cancer by providing a readily accessible approach to determining the expression patterns of proteins in normal and neoplastic tissues as well as characterizing the protein content of clinical specimens. The fact that the system will be implemented as a microfluidic device makes it amenable to very small sample sizes and is potentially extendable to single cell analysis.

描述（由申请人提供）： 该提案是 R21/R33 的组合应用，旨在开发集成的 用于表征复杂混合物的多维分析系统 蛋白质，即蛋白质组分析。该系统将采用微流体 直接接口的二维色谱分离系统的实施 通过电喷雾电离至质谱仪。分析方法是 在分离之前消化蛋白质混合物，然后分离和 分析所得片段肽。蛋白质鉴定是通过 串联质谱与序列预测数据的计算机匹配 数据库。该分离系统将采用强阳离子交换和 反相分配色谱法，基于已发表的毛细管 该系统已被证明优于传统的二维凝胶 用于酵母蛋白质组学研究的电泳方法。微芯片 实施将能够改进已发布的方法，以产生 更强大、更易于使用且更便宜的系统。终极 该系统的实施将取代二维凝胶分离 廉价的一次性注塑塑料微流体装置 直接连接至质谱仪。原型设备将是 使用最近开发的软光刻方法生产，避免了 需要复杂的微加工设施和直接产量设计 可通过注塑转移到大规模生产。这种做法 将促进蛋白质组分析在癌症研究中的应用 与 2D 相比，显着减少了所需的劳动力和艺术 电泳，无需手动转移和处理 用于质谱分析的凝胶。该系统将促进分子 通过提供易于获取的方法来确定癌症分析 正常组织和肿瘤组织中蛋白质的表达模式 表征临床标本的蛋白质含量。事实是 系统将作为微流体装置实现，使其非常适合 样本量小，有可能扩展到单细胞分析。