Creating Platforms for the Proteomics and Membrane Proteins

描述(申请人提供)：这个项目的长期目标是设计分离和鉴定膜蛋白的层析方法，以帮助他们的蛋白质组学分析。目前，为了在凝胶中进行电泳性分离，膜蛋白被从其天然的生物膜环境中移除。由于蛋白质聚集，这可能是不合适的。事实上，与可溶性蛋白相比，膜蛋白被认为是从细胞中通常可以识别的一部分蛋白的表达不足。为了解决这个问题，膜蛋白和相关的带电脂类将在固体支持的脂类双层中通过电泳法分离。这将使这些生物大分子在整个层析过程中保持在一个更自然的环境中。脂双分子膜代表了一种新的分离材料，因此，有必要对用作分离基质的双分子膜的组成进行优化。具体地说，在这一过程中将利用微域结构，如脂筏，因为蛋白质将其划分为液体扩展结构域和液体凝聚结构域的能力不同。除了膜化学，施加的电压、温度、缓冲条件和图案化过程也将进行调整。也应该有可能获得二维分离。这项工作的第二个目标是开发支撑物，使几乎所有的跨膜蛋白在平面双层环境中保持横向移动。从支持的双层的下部小叶突出的跨膜蛋白可以与下面的无机载体相互作用并变得不能移动。将采取两个步骤来解决这个问题。首先，双层将放置在水合良好的聚合物垫上，该垫将使膜蛋白保持在与脂泡中发现的环境非常接近的环境中。其次，聚合物垫将通过钝化蛋白质膜与基材解耦。这项提议的第三个目标是使分离的蛋白质可以通过离子通道测量进行询问。为此，分离的蛋白质条带将在直径约100至500 nm的玻璃纳米孔电极(GNE)上进行询问。最终的具体目标是从大肠杆菌的内膜中分离出蛋白质。将特别注意监测SEC和TAT转位装置的跨膜物种。我们将监测这些蛋白质的离子通道特性。在分离过程中，有必要确保所有的膜蛋白都是高度定向的。与公共卫生相关：一般认为，大多数生物基因组中15%至30%的开放阅读框编码膜蛋白。此外，3个药物靶点中有2个是嵌入细胞膜的蛋白质。因此，开发层析分析方法来识别这些物种、它们的表达水平以及随后的翻译后修饰是至关重要的。

DESCRIPTION (provided by applicant): The long-term objective of this project is to design chromatographic methods for the isolation and identification of membrane proteins in order to aid in their proteomic analysis. Presently, membrane proteins are removed from their native biomembrane environment in order to perform electrophoretic separation in gels. This can be unsuitable due to protein aggregation. Indeed, membrane proteins are believed to be underrepresented as a fraction of the proteins that can be typically identified from cells in comparison with their soluble counterparts. To remedy this problem, membrane proteins and associated charged lipids will be separated by electrophoresis in solid supported lipid bilayers. This will maintain these biomacromolecules in a much more native like environment during the entire chromatographic procedure. Lipid bilayers represent a new material for separation and, therefore, it will be necessary to optimize the components of the bilayer which are employed as the separation matrix. Specifically, microdomain structures such as lipid rafts will be exploited in the process because of the differing ability of proteins to partition into liquid expanded and liquid condensed domains. In addition to membrane chemistry, the applied voltage, temperature, buffer conditions, and patterning processes will be tuned. It should also be possible to obtain two-dimensional separation. The second aim of this work is to develop supports that allow virtually all transmembrane proteins to remain laterally mobile within the planar bilayer environment. Transmembrane proteins, which protrude from the lower leaflet of the supported bilayer, can interact with an underlying inorganic support and become immobile. Two steps will be taken to remedy this problem. First, the bilayer will rest on a well-hydrated polymer cushion that will act to keep membrane proteins in an environment very close to the one found in lipid vesicles. Second, the polymer cushion will be decoupled from the substrate by a passivating protein film. The third aim of this proposal is to make the separated proteins available for interrogation by ion channel measurements. For this purpose, the isolated proteins bands will be interrogated over a glass nanopore electrode (GNE) approximately 100 to 500 nm in diameter. The final specific aim is to separate proteins from the inner membrane of E. coli. Particular attention will be paid to monitoring transmembrane species from the SEC and Tat translocation apparatuses. We will monitor the ion channel properties of these proteins. It will be necessary to ensure that all membrane proteins are highly vectorially oriented during the separation. PUBLIC HEALTH RELEVANCE: It is generally believed that 15 to 30% of open reading frames in the genomes of most organisms encode membrane proteins. Moreover, 2 out of 3 drug targets are proteins embedded in cellular membranes. It is therefore vital to develop chromatographic assays to identify these species, their expression levels, as well as follow posttranslational modifications.