USE OF SULFATE RADICAL ANION IN PROTEIN FOOTPRINTING

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们开发了一种利用过氧化氢和紫外光产生羟基自由基来绘制蛋白质溶剂可降解表面的方法。虽然早期的工作是使用连续光源，但我们最近超越了连续光源，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达的表面。这种方法能够跟踪100纳秒甚至更慢的反应时间--比任何已知的蛋白质展开事件都要快。具体地说，该方法避免了与OH(蛋白质氧化)反应可能导致蛋白质展开和氧化天然蛋白质中无法访问的位置的问题，给出了误导性的结果。我们使用248 nm KrF准分子激光在低浓度(15 mM，0.04%)下裂解过氧化氢，从而避免了不必要的氧化，提供了在不到一微秒内修饰蛋白质的羟基自由基。在清除剂的存在下，自由基寿命减少到1微秒，但反应时间足以提供蛋白质的显著氧化。可以说，这些时间比超二级蛋白质结构由于修饰而展开的速度更快。自由基形成步骤发生在纳升流动池中，因此每个样品块只有一个激光脉冲照射。使用标准分析蛋白质组学来定位氧化位点，只需要不到一纳摩尔的蛋白质。 新试剂的开发是本研究的一个新方向。一种是硫酸盐自由基阴离子，在过硫酸盐的光解过程中产生。

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.