PROTEASE DIGEST CONDITIONS FOR MEMBRANE PROTEIN PROTEOMICS

膜蛋白蛋白质组学的蛋白酶消化条件

• 批准号: 8364949
• 负责人: KELVIN H LEE
• 金额: $ 6.39万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8364949
• 关键词: Bacteriorhodopsins; Cell Wall; Centers of Research Excellence; Chinese Hamster Ovary Cell; Detergents; Enzymes; Escherichia coli; Faculty; Funding; Goals; Grant; Mass Spectrum Analysis; Membrane Proteins; Methanol; Methods; Modeling; Motivation; National Center for Research Resources; Peptide Hydrolases; Phospholipase A2; Plasmodesmata; Principal Investigator; Production; Proteins; Proteomics; Protocols documentation; Research; Research Infrastructure; Resources; Sampling; Solvents; Source; Temperature; Testing; Time; Trypsin; United States National Institutes of Health; Work; base; chymotrypsin; cost; improved; protein E

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The objective of this project is to establish a set of protease digest conditions suitable for proteomic and mass spectrometry (MS) based analysis of membrane proteins. The motivation for pursuing this work is not only the tremendous importance of membrane proteins, but also the growing application of proteomics methods for the identification, quantitation, and characterization of proteins in general. To achieve the objective we are pursuing two aims. Aim 1 tests protease digest conditions using two types of model membrane proteins (bacteriorhodopsin and phospholipase A2) with the goal of identifying an optimized set of conditions by varying methanol solvent concentration, temperature, enzyme (chymotrypsin, trypsin, or both), detergent additives, and time. The determination of an improved protocol will be based on a Mascot score (spectral assignment) and on sequence coverage. In Aim 2 the two resulting optimized protocols will be tested against proteins from collaborators who are active COBRE faculty working on membrane proteins. We have already been working with these collaborators on membrane protein studies and will apply the new methods to their samples. These samples include work on membrane associated proteins, cell wall proteins, plasmodesmata associated proteins, and others. If Aims 1 and 2 are completed in a timely manner, then we will pursue an extension to the project that involves the proteomic analysis, using the newly developed methods, of membrane proteins from E. coli and from CHO cells.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 该项目的目的是建立一组适合基于蛋白质组学和质谱法（MS）的膜蛋白分析的蛋白酶消化条件。追求这项工作的动机不仅是膜蛋白的巨大重要性，而且是蛋白质组学方法对蛋白质的鉴定，定量和表征的日益增长的应用。为了实现目标，我们正在追求两个目标。 AIM 1使用两种类型的模型膜蛋白（细菌紫红素和磷脂酶A2）测试蛋白酶消化条件，目的是通过改变甲醇溶剂浓度，温度，酶（chymotrypsin，therpsin，ter），，洗涤剂添加剂和时间和时间。改进协议的确定将基于吉祥物得分（光谱分配）和序列覆盖率。在AIM 2中，将对两种优化的方案进行测试，这些方案将针对活跃的柯布雷教师从事膜蛋白的合作者的蛋白质进行测试。我们已经与这些合作者合作进行了膜蛋白研究，并将将新方法应用于其样品。这些样品包括有关膜相关蛋白，细胞壁蛋白，质量肿块相关蛋白等的工作。如果目标1和2及时完成，那么我们将使用来自大肠杆菌和CHO细胞的膜蛋白的蛋白质组学分析的项目进行扩展。