Hybrid Chip Device for Automated 2-D Protein Separation

描述（由申请人提供）：蛋白质组学的主要限制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中蛋白质分离的主要工具。然而，它远没有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，自动化是具有挑战性的。由于2-DE涉及许多手工过程，凝胶对凝胶的重现性很差，这使得2-DE测定方法的标准化变得困难。传统的2-DE检测限（LOD）不足以检测低丰度蛋白质，其线性动态范围（LDR）不足以处理细胞内蛋白质浓度的极端差异。此外，目前2-DE的分离速度和通量也较低，这限制了其在大规模蛋白质组学研究中的应用。此外，该技术每次检测通常需要几十微克的样品，这限制了它在样品稀缺时的使用。我们提出了一种将毛细管IEF （CIEF）与毛细管凝胶电泳（CGE）相结合的混合装置，用于蛋白质自动分离。CIEF将在微芯片通道中进行。CIEF后，微芯片通道被分割成200个较短的通道，这些通道内的样品被同时分别注入200个毛细管中进行平行CGE分离。我们将开发一种三色激光诱导荧光共聚焦扫描仪用于混合装置，以提高蛋白质检测灵敏度和动态范围。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，我们将通过将该平台应用于分离、检测和定量已经在脑脊液（CSF）和血液中鉴定出的四种疾病相关蛋白的同种异构体来验证该平台的实际适用性。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，提高再现性、降低LOD、增加LDR、缩短分离时间和提高吞吐量。

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.