USING FPOP WITH TOP DOWN AND BOTTOM UP MS TO CHARACTERIZE PROTEIN CONFORMATION

使用 FPOP 结合自上而下和自下而上 MS 来表征蛋白质构象

• 批准号: 8361431
• 负责人: MICHAEL L GROSS
• 金额: $ 0.94万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361431
• 关键词: Amino Acids; Anions; Development; Funding; Grant; Hydrogen Peroxide; Hydroxyl Radical; Inorganic Sulfates; Label; Mass Spectrum Analysis; Methods; Molecular Conformation; National Center for Research Resources; Principal Investigator; Protein Conformation; Proteins; Proteolysis; Reagent; Reporting; Research; Research Infrastructure; Resources; Source; Time; Trypsin; United States National Institutes of Health; Unspecified or Sulfate Ion Sulfates; biomedical resource; cost; interest; liquid chromatography mass spectrometry; oxidation; photolysis; tool

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. We reported the FPOP (fast photochemical oxidation of proteins) method whereby the exposed amino-acid residues are covalently labeled by oxidation with hydroxyl radical produced by photolysis of hydrogen peroxide. Modified residues can be detected and quantified by standard trypsin proteolysis followed by LC/MS/MS analysis. It has been well established by the Chance group that 14 out of 20 amino acids can be modified by hydroxyl radical for the purpose of footprinting, thus potentially covering ~65% of the sequence of a typical protein. Since FPOP occurs in a time scale faster than protein conformational change, only the native conformation is labeled. Recently, sulfate radical anion as a new FPOP reagent was developed in our group. Being also a nonspecific labeling reagent, sulfate radical anion differs from OH radical in selectivity and reactivity, and is thus another possible tool for FPOP. Development of new FPOP reagents to label proteins in a more specific way is of great interests to us.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 我们报道了 FPOP（蛋白质快速光化学氧化）方法，通过过氧化氢光解产生的羟基自由基氧化，暴露的氨基酸残基被共价标记。修饰残基可以通过标准胰蛋白酶蛋白水解进行检测和定量，然后进行 LC/MS/MS 分析。 Chance 小组已经确定，出于足迹追踪的目的，20 个氨基酸中有 14 个可以被羟基自由基修饰，因此可能覆盖典型蛋白质序列的约 65%。由于 FPOP 发生的时间尺度比蛋白质构象变化快，因此仅标记天然构象。最近，我们课题组开发出了硫酸根阴离子作为一种新型的FPOP试剂。 硫酸根阴离子也是一种非特异性标记试剂，其选择性和反应性与 OH 自由基不同，因此是 FPOP 的另一种可能的工具。我们对开发新的 FPOP 试剂以更特异的方式标记蛋白质非常感兴趣。