PROTEASE DIGEST CONDITIONS FOR MEMBRANE PROTEIN PROTEOMICS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 本项目的目的是建立一套适合于膜蛋白蛋白质组学和质谱学分析的酶消化条件。从事这项工作的动机不仅是膜蛋白的巨大重要性，而且蛋白质组学方法在一般蛋白质的鉴定、定量和表征方面的应用也越来越多。为了实现这一目标，我们正在追求两个目标。目的1用两种模型膜蛋白(细菌视紫红质和磷脂酶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(凝乳酶、胰酶或两者)、洗涤剂添加剂和时间来确定一组优化的条件。改进协议的确定将基于吉祥物分数(频谱分配)和序列覆盖。在目标2中，所得到的两个优化方案将与来自在膜蛋白方面工作的活跃的Cobre教员的合作者的蛋白质进行测试。我们已经与这些合作者在膜蛋白研究方面进行了合作，并将把新方法应用于他们的样本。这些样本包括膜相关蛋白、细胞壁蛋白、胞间连丝相关蛋白等。如果目标1和目标2及时完成，那么我们将继续扩大该项目，包括使用新开发的方法对来自大肠杆菌和CHO细胞的膜蛋白进行蛋白质组分析。

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.