New Materials for High Efficiency Protein Separations

描述(申请人提供)：今天的蛋白质组学分析非常慢，因为每个分离步骤都很慢。分离速度受材料的限制。目前，蛋白质组学的主流是双向凝胶电泳法(PAGE)。这些分离在低电压下需要数小时才能实现高效分离，而蛋白质在凝胶上的损失会降低灵敏度，这对膜蛋白质来说是一个特别严重的问题。我们建议通过使用胶体晶体进行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厘米的二氧化硅胶体晶体，将最高分辨率凝胶的2D分离速度提高100倍，长度为20厘米。除了旅行距离短五倍所带来的更高速度外，硅基材料还允许电场增加一个数量级以上。小型化和更高的磁场相结合，有望在分离的每个维度上将速度提高100倍，在几分钟内实现2D分离，而不是在许多小时内。前一批赠款期间的进展涉及展示了前景的材料科学，而新提出的工作将把研究从材料阶段推进到分离阶段。我们建议研究介质中的传输，以了解如何最大限度地提高分离的整体速度。我们提出了在同一胶体晶体中实现二维分离的片上集成：等电聚焦，然后筛分分离。我们建议通过研究反蛋白石介质来进一步改进材料，这种介质具有更大的自由体积，但孔径相同。我们建议研究差示凝胶电泳法中荧光检测的灵敏度和动态范围，以发现生物标志物。我们建议从胶体晶体中实现蛋白质的质谱学检测，研究MALDI和胶体晶体中蛋白质的电动转移。这项研究与公共卫生的相关性在于，蛋白质组分析增加两个数量级将对医学研究产生巨大影响，包括确定药物作用的机制，分离用于诊断疾病的生物标记物，以及了解疾病的发病情况以预防疾病。公共卫生相关性这项研究与公共卫生的相关性是，蛋白质组学分析增加两个数量级将对医学研究产生巨大影响，包括确定药物作用机制、分离用于诊断疾病的生物标记物以及了解疾病的发病情况以预防疾病。

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.