Fast, High-Resolution Protein Separations using Controllable Electro-Osmotic Flow

描述（由申请人提供）：本阶段I提案的目的是证明一种新型微流体装置的性能，该装置的性能将比用于分离复杂蛋白质混合物的传统二维聚丙烯酰胺凝胶电泳（2D-PAGE）的性能提高100倍。具体目标是实现25，000的装置峰值容量（相比2D-PAGE的3，000）和1.5小时的循环时间（相比2D-PAGE的3天）。新设备还将现有2D色谱、毛细管电泳和微流体设备的峰值容量提高了10倍;循环时间大致相当。该新设备与ESI质谱仪连接，使其能够确定自上而下的蛋白质表达谱。所提出的微流控装置结合了毛细管等电聚焦（cIEF）与单一快速毛细管区带电泳（CZE）。通过在聚焦过程中抑制电渗流、在这些峰移动时保持聚焦的cIEF峰形状以及cIEF与CZE的小心偶联，仔细注意减少分辨率损失的主要和次要原因。CZE流速选择为大值。分离的蛋白质然后通过ESI接口被移除到质谱仪。提案活动侧重于证明器械可行性的关键步骤。该设备将主要在亚利桑那州立大学的纳米工厂制造;工艺优化将用于制造透明并具有其他理想物理特性的材料。器械孔将用高密度聚丙烯酰胺衍生化，以尽量减少蛋白质吸附。将在一系列系统操作条件下使用参比蛋白混合物定量cIEF和CZE分离的性能。聚焦或分离的蛋白质将进一步从其分离区域移动，以确定在装置耦合期间峰如何扩散。将研究从已知蛋白质混合物到E.大肠杆菌裂解物提取物。最后，通过将其与ESI质谱相结合来验证设备性能。该设备应该在靶点发现、确定人类疾病的机制和进展、发现药物化合物对已知靶点的活性或这些化合物对人体器官的毒性以及识别蛋白质诊断标记物方面找到广泛的应用。公共卫生相关性：确定细胞或组织表达的蛋白质是对DNA分析的有力补充。许多人类疾病都是由异常蛋白质产生引起的，包括酶、抗体和激素，直接测量这些异常蛋白质对诊断和新药开发非常有帮助。本提案中开发的工具将以极高的精度和速度确定和量化以方便使用的格式表达的蛋白质。它的性能将比目前最好的技术提高十倍，可以从一个实验中确定的蛋白质的数量，并减少分析时间的一个因素。这些新工具应该广泛应用于靶点发现，确定人类疾病的机制和进展，发现药物化合物对已知靶点的活性或这些化合物对人体器官的毒性，以及确定蛋白质诊断标志物。

DESCRIPTION (provided by applicant): The objective of this Phase I proposal is to demonstrate the performance of a novel microfluidic device that will improve by a factor of 100 upon the performance of traditional two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) used to separation of complex protein mixtures. Specific goals are to achieve a device peak capacity of 25,000 (compared to 3,000 for 2D-PAGE) and a cycle time of 1.5 hours (compared to 3 days for 2D-PAGE). The new device also improves by a factor ot ten on the peak capacities of existing 2D chromatography, capillary electrophoresis and microfluidic devices; the cycle time is roughly comparable. The new device is interfaced to ESI mass spectrometry, enabling it to determine top-down protein expression profiles. The proposed microfluidic device combines capillary isoelectric focusing (cIEF) with a single fast capillary zone electrophoresis (CZE). Careful attention is paid to reducing primary and secondary causes of resolution loss by suppressing electro-osmotic flow during focusing, preserving focused cIEF peak shapes when those peaks are mobilized, and careful coupling of cIEF with CZE. CZE flow rates are selected for large values. Separated proteins are then be removed through an ESI interface to a mass spectrometer. Proposal activities focus on demonstrating the key steps to demonstrate device feasibility. The device will be fabricated primarily at Arizona State University's Nanofab; process optimization will be used to create materials that are transparent and have other desirable physical properties. The device pores will be derivatized with high-density polyacrylamide to minimize protein adsorption. Performance of cIEF and CZE separations will be quantified using reference protein mixtures over a range of system operating conditions. Focused or separated proteins will further be mobilized from their separation regions to determine how peaks spread during device coupling. 2D separations will be studied, from known proteins mixtures to an E. coli lysate extract. Finally, device performance is verified by combining it with ESI mass spec. The device should find broad application to target discovery, determining the mechanism and progression of human disease, finding the activity of pharmaceutical compounds to known targets or the toxicity of these compounds to human organs, and identifying protein diagnostic markers. PUBLIC HEALTH RELEVANCE: Determining what proteins a cell or tissue expresses is a powerful complement to DNA analysis. Many human diseases are caused when abnormal proteins are produced, including enzymes, antibodies and hormones, and measuring these abnormal proteins directly is extremely helpful to their diagnosis and to creating of new pharmaceuticals. The tools developed in this proposal will determine and quantify with great precision and speed the proteins that are expressed in a format that is convenient to use. Its performance will improve on the current best technology by increasing by ten times the number of proteins that can be determined from one experiment, and reduce the analysis time by a factor of ten. The new tools should find broad application to target discovery, determining the mechanism and progression of human disease, finding the activity of pharmaceutical compounds to known targets or the toxicity of these compounds to human organs, and identifying protein diagnostic markers.