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

使用可控电渗透流进行快速、高分辨率的蛋白质分离

• 批准号: 7739628
• 负责人: Frank Jahnke
• 金额: $ 33.1万
• 依托单位: SONATA BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-15 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7739628
• 关键词: Address; Adsorption; Antibodies; Arizona; Attention; Biological; Blood capillaries; Capillary Electrophoresis; Cells; Chromatography; Complement; Coupled; Coupling; DNA analysis; Deposition; Devices; Diagnosis; Diagnostic; Electrodes; Electrophoresis; Enzymes; Escherichia coli; Extravasation; Film; Fluorescence; Generations; Goals; Heating; Hormones; Hour; Human; Ionic Strengths; Isoelectric Focusing; Lasers; Length; Life; Liquid substance; Mass Spectrum Analysis; Measurement; Measures; Methods; Microfluidic Microchips; Modeling; Molecular Profiling; Molecular Weight; Operating System; Organ; Performance; Pharmacologic Substance; Phase; Plague; Process; Proteins; Proteome; Proteomics; Quartz; Reference Standards; Resolution; Sampling; Shapes; Silicon Dioxide; Solutions; Spectrometry, Mass, Electrospray Ionization; Speed; Staging; Surface; Technology; Thick; Time; Tissues; Toxic effect; Two-Dimensional Polyacrylamide Gel Electrophoresis; Universities; Visible Radiation; Width; Zaleplon; base; capillary; density; dielectric property; electric field; human disease; improved; mass spectrometer; novel; numb protein; physical property; polyacrylamide; polymerization; process optimization; protein complex; protein expression; public health relevance; research study; silicon carbide; tool; two-dimensional; voltage

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.

描述（由申请人提供）：本I阶段建议的目的是证明新型微流体设备的性能，该器能在传统的二维聚丙烯酰胺凝胶电泳（2D-PAGE）的性能后提高100倍，用于分离复杂蛋白质混合物。具体的目标是达到25,000的设备峰值容量（相比之下，为2d页）和1.5小时的周期时间（相比之下，2D页3天）。新设备还提高了十个因子，即在现有2D色谱，毛细管电泳和微流体设备的峰值上的峰值上提高了一个因子。周期时间大致可比。新设备连接到ESI质谱法，使其能够确定自上而下的蛋白质表达曲线。提出的微流体设备将毛细管等电聚焦（CIEF）与单个快速毛细管区电泳（CZE）相结合。仔细注意通过抑制聚焦期间的电渗透流量来减少原发性和次要原因，当动员这些峰时保持聚焦的CIEF峰形状，并仔细将CIEF与CZE耦合。选择较大值的CZE流速。然后，通过ESI界面去除分离的蛋白质到质谱仪。提案活动的重点是展示证明设备可行性的关键步骤。该设备将主要在亚利桑那州立大学的纳米牛B制造；过程优化将用于创建透明材料并具有其他理想的物理特性。该设备孔将用高密度聚丙烯酰胺衍生化，以最大程度地减少蛋白质的吸附。 CIEF和CZE分离的性能将在一系列系统工作条件下使用参考蛋白混合物进行量化。聚焦或分离的蛋白质将进一步从其分离区域动员，以确定设备耦合期间峰值的扩散。将研究2D分离，从已知的蛋白质混合物到大肠杆菌裂解物提取物。最后，通过将其与ESI质量规格相结合来验证设备性能。该设备应在靶向发现，确定人类疾病的机制和进展，发现药物化合物对已知靶标或这些化合物对人体器官的毒性以及鉴定蛋白质诊断标记的活性。公共卫生相关性：确定细胞或组织表达哪些蛋白质是对DNA分析的有力补充。当产生异常的蛋白质（包括酶，抗体和激素）时，许多人类疾病是引起的，并且直接测量这些异常蛋白质对它们的诊断和创建新药物非常有帮助。本提案中开发的工具将以极大的精度确定和量化，并加快以方便使用的格式表达的蛋白质。它的性能将通过增加一个实验确定的蛋白质数量增加十倍，并将分析时间减少十倍，从而提高当前最佳技术的性能。新工具应为靶向发现，确定人类疾病的机制和进展，发现药物化合物对已知靶标的活性或这些化合物对人体器官的毒性以及鉴定蛋白质诊断标记的活性。