New Materials for High Efficiency Protein Separations

描述（由申请人提供）：今天的蛋白质组学分析非常缓慢，因为每个分离步骤都很慢。分离速度受物料的限制。目前，蛋白质组学的主力是二维聚丙烯酰胺凝胶电泳（PAGE）。这些分离需要在低电压下进行许多小时才能实现有效的分离，并且凝胶上蛋白质的损失降低了灵敏度，这对于膜蛋白来说是一个特别严重的问题。我们建议用胶体晶体进行二维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，将长度为20厘米的高分辨率凝胶的2D分离速度提高100倍。除了从五倍缩短的行进距离中积累的更高速度外，二氧化硅基材料允许超过一个数量级的高电场。小型化和高场的结合保证了沿着每个分离维度的速度增加100倍，在几分钟内而不是几个小时内实现二维分离。在之前的资助期内，材料科学的进展证明了这一前景，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运输，以了解如何最大限度地提高分离的总体速度。我们建议实现二维分离的片上集成：等电聚焦，然后在同一胶体晶体中筛分分离。我们建议通过研究具有更大自由体积和相同孔径的反蛋白石介质来进一步改进材料。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，以发现生物标志物。我们建议实现胶体晶体中蛋白质的质谱检测，研究蛋白质从胶体晶体中的MALDI和电动转移。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、分离生物标记物以诊断疾病，以及了解疾病的发病以预防疾病。该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，分离生物标记物以诊断疾病，以及了解疾病的发病以预防疾病。

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.