Integrated Low Abundance Gel Protein Analysis Platform

描述（由申请人提供）：该项目的第一阶段旨在展示创新技术的组合使用，这些技术提供了2-D PAGE和MALDI-MS之间急需的联系，即在集成和高通量平台中的电子蛋白质转移和纳米级蛋白水解消化。我们的研究工作将开发和验证有效地从聚丙烯酰胺凝胶中提取带负电荷的SDS-蛋白质复合物到熔融石英毛细管中的技术基础，然后进行超快速原位胰蛋白酶消化和沉积到MALDI靶标上进行蛋白质鉴定。我们的研究目标是通过在蛋白质提取步骤期间实现场扩增蛋白质堆积/浓缩并进行毛细管内蛋白水解消化来提供识别低丰度蛋白质的能力，从而有效地避免分析物损失和稀释。新开发的胰蛋白酶膜反应器能够在几秒钟内而不是几小时内进行全面的蛋白水解消化，同时提供毛细管接口，用于在线组合凝胶蛋白提取和将蛋白消化物直接沉积到MALDI靶标上。为了实现这些技术在商业仪器平台的自动化和高通量分析，一些系统级的问题将在本研究的第二阶段得到解决。

DESCRIPTION (provided by applicant): The Phase I of this project aims to demonstrate the combined usage of innovative technologies which provide the critically needed link between 2-D PAGE and MALDI-MS, namely electronic protein transfer and nanoscale proteolytic digestion in an integrated and high throughput platform. Our research efforts will develop and validate the technical basis for effectively extracting negatively charged SDS-protein complexes from polyacrylamide gel into fused-silica capillary, followed by ultrafast in situ tryptic digestion and deposition onto the MALDI target for protein identification. Our research objectives are to provide the capability to identify low abundance proteins by achieving field-amplification protein stacking/concentration during the protein extraction step and performing in-capillary proteolytic digestion, effectively avoiding analyte loss and dilution. The newly developed trypsin membrane reactor enables comprehensive proteolytic digestion in seconds instead of hours, while offering capillary interfaces for on-line combination with gel protein extraction and direct deposition of protein digest onto the MALDI target. In order to implement these technologies in a commercial instrumentation platform for automated and high throughput analysis, a number of system-level issues will be addressed in the Phase II of this research.