New Materials for High Efficiency Protein Separations

用于高效蛋白质分离的新材料

• 批准号: 7526980
• 负责人: MARY J. WIRTH
• 金额: $ 29.69万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-03 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7526980
• 关键词: Address; Adsorption; Biological Markers; Detection; Diagnosis; Digestion; Dimensions; Disease; Drug effect disorder; Fluorescence; Gel; Goals; Grant; Height; Hour; Hybrids; Isoelectric Focusing; Kinetics; Length; Medical Research; Membrane Proteins; Miniaturization; Onset of illness; Prevention; Proteins; Proteomics; Public Health; Range; Research; Science; Silicon Dioxide; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Speed; Staging; Today; Translating; Travel; Two-Dimensional Gel Electrophoresis; Two-Dimensional Polyacrylamide Gel Electrophoresis; Vision; Work; base; electric field; gel electrophoresis; improved; mass spectrometer; size; voltage

DESCRIPTION (provided by applicant): Proteomic analyses today are very slow because each of the separation steps is slow. The separation speeds are limited by the materials. Presently, the workhorse of proteomics is 2D polyacrylamide gel electrophoresis (PAGE). These separations require many hours at low voltages to achieve efficient separations, and loss of protein on the gel reduces sensitivity, which is an especially severe problem for membrane proteins. We propose to address these problems with the use of colloidal crystals for 2D gel electrophoresis. Our goal is to achieve a 100-fold increase in speed of 2D separations of the most highly resolving gels, 20 cm in length, by using silica colloidal crystals of only 4 cm in length. In addition to the higher speed accrued from five-fold shorter travel distances, silica based materials allow more than an order of magnitude higher electric field. The combined miniaturization and higher field promises a combined 100-fold increase in speed along each dimension of the separation to give 2D separations in a few minutes rather than in many hours. The progress in the previous grant period addressed the materials science that demonstrated the promise, and the newly proposed work will carry the research from the materials stage to the separation stage. We propose to investigate transport in the media to understand how to maximize the overall speed of the separation. We propose to achieve on-chip integration of 2D separations: isoelectric focusing followed by sieving separation in the same colloidal crystal. We propose to improve the materials further by investigating inverse opal media, which have a larger free volume, yet the same pore size. We propose to study the sensitivity and dynamic range of fluorescence detection in differential gel electrophoresis for biomarker discovery. We propose to achieve mass spectrometric detection of protein from the colloidal crystals, investigating both MALDI and electrokinetic transfer of the protein from the colloidal crystal. The relevance of the research to public health is that a two order of magnitude increase in proteomic analyses would have an enormous impact on medical research, including determining mechanisms of drug action, the isolation of biomarkers to diagnose diseases, and an understanding of the onset of diseases for their prevention. PUBLIC HEALTH RELEVANCE The relevance of the research to public health is that a two order of magnitude increase in proteomic analyses would have an enormous impact on medical research, including determining mechanisms of drug action, the isolation of biomarkers to diagnose diseases, and an understanding of the onset of diseases for their prevention.

描述（由申请人提供）：当今的蛋白质组学分析非常缓慢，因为每个分离步骤都很慢。分离速度受到材料的限制。目前，蛋白质组学的主力是2D聚丙烯酰胺凝胶电泳（PAGE）。这些分离需要在低电压下进行数小时才能实现有效的分离，并且凝胶上蛋白质的损失会降低灵敏度，这对于膜蛋白来说是一个特别严重的问题。我们建议使用胶体晶体进行2D凝胶电泳来解决这些问题。我们的目标是通过使用长度仅为4 cm的二氧化硅胶体晶体，使最高分辨率的凝胶（长度为20 cm）的2D分离速度提高100倍。除了从五倍更短的行进距离获得更高的速度之外，基于二氧化硅的材料允许超过一个数量级的更高电场。结合小型化和更高的领域承诺在分离的每个维度上的速度增加100倍，以在几分钟内而不是在许多小时内提供2D分离。上一个资助期的进展解决了材料科学，证明了这一承诺，而新提出的工作将把研究从材料阶段带到分离阶段。我们建议调查运输媒体了解如何最大限度地提高整体速度的分离。我们建议实现2D分离的片上集成：等电聚焦，然后在同一胶体晶体中筛分分离。我们建议通过研究反蛋白石介质，它具有更大的自由体积，但相同的孔径，以进一步改善材料。我们建议研究差异凝胶电泳中荧光检测的灵敏度和动态范围，用于生物标志物的发现。我们建议实现从胶体晶体的蛋白质的质谱检测，调查MALDI和电动转移的蛋白质从胶体晶体。这项研究与公共卫生的相关性在于，蛋白质组学分析的两个数量级的增加将对医学研究产生巨大影响，包括确定药物作用机制，分离生物标志物以诊断疾病，以及了解疾病的发病情况以预防疾病。公共卫生研究与公共卫生的相关性在于，蛋白质组学分析的两个数量级的增加将对医学研究产生巨大影响，包括确定药物作用机制，分离生物标志物以诊断疾病，以及了解疾病的发病情况以预防疾病。