USE OF SULFATE RADICAL ANION IN PROTEIN FOOTPRINTING

硫酸根阴离子在蛋白质足迹分析中的应用

• 批准号: 8168745
• 负责人: MICHAEL L GROSS
• 金额: $ 2.04万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-10 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168745
• 关键词: Anions; Bolus Infusion; Cells; Cleaved cell; Computer Retrieval of Information on Scientific Projects Database; Development; Event; Funding; Grant; Hydrogen Peroxide; Hydroxyl Radical; Inorganic Sulfates; Institution; Lasers; Light; Maps; Methods; Modification; Physiologic pulse; Protein Footprinting; Proteins; Proteomics; Reaction; Reaction Time; Reagent; Research; Research Personnel; Resources; Sampling; Secondary Protein Structure; Site; Solvents; Source; Surface; Time; Ultraviolet Rays; United States National Institutes of Health; Unspecified or Sulfate Ion Sulfates; nanolitre; nanosecond; oxidation; photolysis

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have developed a method to map protein solvent acessible surfaces using hydrogen peroxide and UV light to generate hydroxyl radicals. Although the early efforts made use of continuous light source, we recently moved beyond that and developed an ultrafast reaction method to probe protein solvent-accessible surfaces by using a pulsed laser and a quencher. This approach is capable of following reaction times on the order of 100 nanoseconds and slower--faster than any protein known unfolding event. Specifically, the method circumvents problems that reaction with OH (protein oxidation) could cause protein unfolding and oxidation of sites that are not accessible in the native protein, giving misleading results. We avoid unwanted oxidation by using a 248-nm KrF excimer laser to cleave hydrogen peroxide at low concentrations (15 mM, 0.04%), affording hydroxyl radicals that modify the protein in less than a microsecond. In the presence of a scavenger, the radical lifetimes decrease to 1 microsecond, yet the reaction timescales are sufficient to provide significant oxidation of the protein. These times are arguably faster than super-secondary protein structure can unfold as a result of the modification. The radical formation step takes place in a nanoliter flow cell so that only one laser pulse irradiates each bolus of sample. The oxidation sites are located using standard analytical proteomics, requiring less than a nanomole of protein. A new direction in this research is the development of new reagents. One is the sulfate radical anion, generated in the photolysis of persulfate.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们已经开发了一种方法来映射蛋白质溶剂acessible表面使用过氧化氢和紫外光产生羟基自由基。 虽然早期的努力利用了连续光源，但我们最近超越了这一点，开发了一种超快反应方法，通过使用脉冲激光和猝灭剂来探测蛋白质溶剂可及表面。 这种方法能够跟踪大约100纳秒的反应时间，并且比任何已知的蛋白质解折叠事件都要慢。 具体而言，该方法避免了与OH反应（蛋白质氧化）可能导致蛋白质解折叠和天然蛋白质中不可接近的位点氧化的问题，从而产生误导性结果。 我们避免不必要的氧化，使用248纳米KrF准分子激光切割过氧化氢在低浓度（15毫米，0.04%），提供羟基自由基，修改蛋白质在不到一微秒。在存在清除剂的情况下，自由基寿命降低至1微秒，但反应时间尺度足以提供蛋白质的显著氧化。这些时间可以说比超二级蛋白质结构由于修饰而展开的速度更快。自由基形成步骤在纳升流动池中进行，使得仅一个激光脉冲照射每个样品团。使用标准的分析蛋白质组学定位氧化位点，需要少于一纳摩尔的蛋白质。 该研究的一个新方向是开发新试剂。 一种是硫酸根阴离子，在过硫酸盐的光解中产生。