Creating Platforms for the Proteomics and Membrane Proteins

为蛋白质组学和膜蛋白创建平台

• 批准号: 7580724
• 负责人: Paul Cremer
• 金额: $ 29.83万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7580724
• 关键词: ATP phosphohydrolase; Acrylamides; Antibodies; Architecture; Arts; Attention; Biological Assay; Blood Clot; Blood coagulation; Buffers; Caliber; Cells; Cellular Membrane; Cellulose; Charge; Chemistry; Dimensions; Drug Delivery Systems; E coli inner membrane protein; Electrodes; Electrolyses; Electrophoresis; Ensure; Environment; Escherichia coli; Film; Gel; Genome; Glass; Goals; Heating; Hemolysin; Integral Membrane Protein; Ion Channel; Ions; Lipid Bilayers; Lipids; Liposomes; Liquid substance; Measurement; Measures; Membrane; Membrane Microdomains; Membrane Proteins; Methods; Modeling; Monitor; Open Reading Frames; Organism; Pattern; Peripheral; Phospholipids; Polymers; Pore Proteins; Post-Translational Protein Processing; Procedures; Process; Property; Protein translocation; Proteins; Proteomics; Relative (related person); Resolution; Rest; Sepharose; Side; Solid; Structure; Surface; Techniques; Temperature; Testing; Vesicle; Water; Work; annexin A5; design; fluidity; nanopore; novel; periplasm; prevent; protein aggregation; public health relevance; two-dimensional; voltage

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.

描述（由申请人提供）：该项目的长期目标是设计色谱方法，以隔离和鉴定膜蛋白，以帮助其蛋白质组学分析。目前，将膜蛋白从其天然生物膜环境中取出，以便在凝胶中进行电泳分离。由于蛋白质聚集，这可能不合适。实际上，据信膜蛋白被认为是蛋白质的一部分，通常可以从细胞中与其可溶性对应物相比。为了解决这个问题，膜蛋白和相关的带电脂质将通过固体支持的脂质双层中的电泳分离。在整个色谱过程中，这将使这些生物大分子保持在更本地的环境中。脂质双层代表了一种新的分离材料，因此，有必要优化用作分离矩阵的双层组件。具体而言，由于蛋白质分配到液体膨胀和液体冷凝域的能力不同，因此将在此过程中利用微域结构（例如脂质筏）。除了膜化学外，还将调整施加的电压，温度，缓冲条件和图案过程。还应该有可能获得二维分离。这项工作的第二个目的是开发支持，几乎所有跨膜蛋白都可以在平面双层环境中横向移动。跨膜蛋白会从支撑双层的下部小叶伸出，可以与潜在的无机支撑相互作用并变得不动。将采取两个步骤来解决这个问题。首先，双层将放在富含水的聚合物垫上，该垫子将在非常接近脂质囊泡中发现的环境中保持膜蛋白。其次，聚合物垫将通过钝化的蛋白质膜与底物解耦。该提案的第三个目的是通过离子通道测量值使分离的蛋白质可用于询问。为此，将在直径约100至500 nm的玻璃纳米电极（GNE）上审问孤立的蛋白质带。最终的具体目的是将蛋白质与大肠杆菌的内膜分离。将特别注意监测SEC和TAT易位设备的跨膜物种。我们将监视这些蛋白质的离子通道特性。有必要确保在分离过程中所有膜蛋白在高度方向上取向。公共卫生相关性：通常认为大多数生物体基因组中有15％至30％的开放式阅读帧编码膜蛋白。此外，3个药物靶标中有2个是嵌入细胞膜中的蛋白质。因此，开发色谱分析以识别这些物种，其表达水平以及遵循翻译后修饰至关重要。