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分离和鉴定膜蛋白的色谱方法，以帮助其蛋白质组学分析。目前，为了在凝胶中进行电泳分离，膜蛋白被从其天然生物膜环境中去除。由于蛋白质聚集，这可能不合适。事实上，与可溶蛋白相比，膜蛋白被认为是可从细胞中典型识别的蛋白质的一部分。为了解决这个问题，膜蛋白和相关的带电脂质将在固体支持的脂质双层中通过电泳分离。这将使这些生物大分子在整个色谱过程中保持在一个更自然的环境中。脂质双分子层是一种新型的分离材料，因此，有必要对用作分离基质的双分子层组分进行优化。具体地说，微结构域，如脂筏，将在这一过程中被利用，因为蛋白质划分为液体膨胀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，和图案过程将被调整。它也应该可以获得二维分离。这项工作的第二个目标是开发支持，使几乎所有的跨膜蛋白在平面双层环境中保持横向移动。跨膜蛋白，从支持双分子层的下层小叶突出，可以与下面的无机支持相互作用而变得不动。解决这个问题将采取两个步骤。首先，双分子层将建立在一个水合良好的聚合物垫上，这将使膜蛋白保持在一个非常接近于脂质囊泡的环境中。其次，聚合物缓冲层将通过钝化蛋白膜与底物分离。这个提议的第三个目的是使分离的蛋白质可以通过离子通道测量来检测。为此，分离的蛋白质带将在直径约100至500纳米的玻璃纳米孔电极（GNE）上进行检测。最后的具体目标是从大肠杆菌的内膜中分离蛋白质。将特别注意监测跨膜物种从SEC和Tat易位装置。我们将监测这些蛋白质的离子通道特性。这将是必要的，以确保所有的膜蛋白在分离过程中高度矢量定向。公共卫生相关性：一般认为，大多数生物体基因组中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，开发色谱分析来鉴定这些物种，它们的表达水平，以及遵循翻译后修饰是至关重要的。