Hybrid Chip Device for Automated 2-D Protein Separation

用于自动二维蛋白质分离的混合芯片装置

• 批准号: 7129385
• 负责人: Shaorong Liu
• 金额: $ 32.01万
• 依托单位: TEXAS TECH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7129385
• 关键词: apolipoproteins; biomedical automation; biomedical equipment development; biotechnology; capillary electrophoresis; cerebrospinal fluid; ferroxidase; fluorescent dye /probe; high throughput technology; human tissue; isoelectric point; microarray technology; protein isoforms; protein purification; proteomics; transferrin

DESCRIPTION (provided by applicant): One of the principal limitations in proteomics is lack of efficient tools for protein analysis, although significant progress has been made. 2-DE was the first that was able to achieve proteomic analysis and it is still the major workhorse for separations of proteins for complex biological samples. However, it is far from dealing with the real complexity of proteomes. Because IEF and SDS-PAGE are two isolated separation steps, their operations are labor intensive and automation is challenging. Due to the numerous manual processes involved with 2-DE, gel-to-gel reproducibility is poor, which make the standardization of 2-DE assay methods difficult. The limit of detection (LOD) of traditional 2-DE is inadequate for detecting low abundance proteins and its linear dynamic range (LDR) is insufficient for handling the extremely divergent protein concentrations in cells. Additionally, the separation speed and throughput of current 2-DE are also low, which inhibits its applications for large-scale proteomic investigations. Furthermore, the technique usually requires tens of micrograms of sample for each assay, which restrains its use when samples are scarce. We propose to develop hybrid device to integrate capillary IEF (CIEF) with capillary gel electrophoresis (CGE) for automated protein separation. CIEF will be performed in a microchip channel. After CIEF, the microchip channel is segmented into 200 shorter channels and the samples inside these channels are simultaneously and respectively injected into 200 capillaries for parallel CGE separations. We will develop a three-color laser-induced fluorescence confocal scanner for the hybrid device to increase protein detection sensitivity and dynamic range. One color will be used to measure the CGE separated proteins, the second color will be used to monitor a set of pl markers, and the third color will be used to detect a set of size markers. We will then validate the practical applicability of the platform by applying it to separate, detect and quantitate the isoforms of four disease-associated proteins that have already been identified in cerebrospinal fluid (CSF) and blood. The platform will advance 2-DE technology by integrating CIEF and parallel CGE for automated 2-DE with improved reproducibility, reduced LOD, increased LDR, decreased separation time and enhanced throughput.

描述(申请人提供)：蛋白质组学的主要局限性之一是缺乏有效的蛋白质分析工具，尽管已经取得了重大进展。2-DE是第一个能够实现蛋白质组学分析的方法，它仍然是复杂生物样品蛋白质分离的主要工具。然而，它远远不能处理蛋白质组的真正复杂性。由于IEF和SDS-PAGE是两个独立的分离步骤，它们的操作是劳动密集型的，自动化具有挑战性。由于2-DE涉及的人工步骤繁多，凝胶对凝胶的重复性差，这使得2-DE分析方法的标准化变得困难。传统的双向凝胶电泳法的检测下限(LOD)不足以检测低丰度蛋白质，其线性动态范围(LDR)不足以处理细胞内极不同的蛋白质浓度。此外，目前的2-DE分离速度和吞吐量也较低，这限制了其在大规模蛋白质组研究中的应用。此外，这项技术通常每次化验需要数十微克的样本，这限制了它在样本稀缺时的使用。我们建议开发一种将毛细管电泳法(CIEF)和毛细管凝胶电泳法(CGE)相结合的混合设备，用于蛋白质的自动分离。CIEF将在微芯片通道中执行。在CIEF后，将芯片通道分割成200个较短的通道，并将这些通道内的样品同时分别注入200个毛细管中进行并行CGE分离。我们将为混合设备开发一种三色激光诱导荧光共聚焦扫描仪，以提高蛋白质检测的灵敏度和动态范围。一种颜色将用于测量CGE分离的蛋白质，第二种颜色将用于监测一组PL标记，第三种颜色将用于检测一组大小标记。然后，我们将通过应用该平台来分离、检测和定量已在脑脊液(CSF)和血液中发现的四种疾病相关蛋白的亚型，从而验证该平台的实用适用性。该平台将通过集成CIEF和并行CGE来推进2-DE技术，实现自动化2-DE，提高重复性，减少LOD，增加LDR，减少分离时间和提高吞吐量。