CAREER: Quantification of Prion Protein Sorption to Soil Components

这项NSF CAREER提案的主要目标是：(1)开发一种高灵敏度、定量的朊病毒蛋白测量方法；(2)定量描述朊病毒蛋白与表面反应性土壤矿物质的关联，并研究影响这些颗粒附着和脱离的因素。目前缺乏朊病毒蛋白的定量分析方法。因此，PI打算利用蛋白质组学的最新进展，开发基于特征肽方法的朊病毒蛋白定量的液相色谱-串联质谱（LC-MS/MS）方法。第二个目标将通过使用新开发的LC-MS/MS方法进行定量的一系列批量附着和分离实验来解决。朊病毒蛋白与粘土矿物和金属氧化物的结合将作为pH值的函数进行研究，以确定朊病毒和矿物表面电荷对附着的影响。由于pH变化和表面活性剂和溶解有机物的存在，朊病毒蛋白附着的可逆性将在批量分离实验中进行研究。朊病毒和矿物表面之间的静电和疏水相互作用的重要性将被明确审查。实验结果将在扩展的Derjaguin-Landau-Verwey-Overbeek理论框架内解释。PI将使用原子力显微镜来测量朊病毒蛋白和一部分矿物表面之间的相互作用力。从这些实验中，PI将得出粘附效率，以促进预测朊病毒在多孔介质中的运输。更广泛的影响：预计LC-MS方法的用途将远远超出本提案中概述的环境命运研究。该方法有可能对医学和兽医研究以及传染性海绵状脑病的诊断产生重大影响。基于我们的研究结果，对朊病毒附着在土壤组分上的理解的提高自然会导致工程应用的发展。这一职业发展计划的教育部分侧重于将研究和教学结合起来，并促进环境工程和科学领域的跨学科发展。学院将开设一门关于环境胶体化学的新研究生课程，并为环境专业人员开设一门关于处理和处置生物威胁剂的短期课程。通过加强K-12教育也将实现更广泛的影响。该项目将扩大与一名中学科学教师的现有合作，设计、实施和评估围绕探究和发现为基础的学习练习组织的环境工程和科学教学单元。除了在主要期刊上发表研究成果外，有关环境中朊病毒的信息将通过威斯康星大学麦迪逊分校矿物和分子虚拟博物馆的“展示”传播给更广泛的受众。在项目进行期间，PI将为本科生提供参与研究的机会，并特别考虑代表性不足的少数族裔申请人。PI将利用现有资源来实现他的教育目标，如西澳大学nsf资助的三角洲研究、教学和学习项目，为研究生提供在课堂教学、编写教学材料、非正式教育、面向不同学生群体的教学、技术教学和实习方面发展技能的机会。

Pedersen 0547484The main objectives of this NSF CAREER proposal are (1) to develop a highly sensitive, quantitative method for prion protein measurement, and (2) to quantitatively describe the association of prion proteins with surface-reactive soil minerals and to examine the factors influencing attachment to and detachment from these particles. Quantitative analytical methods for prion proteins are currently lacking. The PI therefore intends to exploit recent advances in proteomics to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for prion protein quantitation based on a signature peptide approach. The second objective will be addressed in a series of batch attachment and detachment experiments using the newly developed LC-MS/MS method for quantitation. The association of prion proteins to clay minerals and metal oxides will be examined as a function of pH to determine the influence of prion and mineral surface charge on attachment. Reversibility of prion protein attachment due to shifts in pH and the presence of surfactants and dissolved organic matter will be investigated in batch detachment experiments. The importance of electrostatic and hydrophobic interactions between prions and mineral surfaces will be explicitly examined. Experimental results will be interpreted within the framework of extended Derjaguin-Landau-Verwey-Overbeek theory. The PI will use atomic force microscopy to measure interaction forces between prion protein and a subset of mineral surfaces. From these experiments, the PI will derive sticking efficiencies to facilitate prediction of prion transport in porous media.Broader impacts: The LC-MS method is expected to have utility well beyond the environmental fate studies outlined in this proposal. The method has the potential to significantly impact medical and veterinary research and the diagnosis of transmissible spongiform encephalopathies. Improved understanding of prion attachment to soil components will naturally lead to the development of engineering applications based on our findings. The educational component of this career development plan focuses on integrating research and instruction and fostering increased interdisciplinarity in environmental engineering and science. The PI will develop a new graduate course on environmental colloid chemistry and a short course for practicing environmental professionals on the treatment and disposal of biological threat agents. Broader impacts will also be realized through enhanced K-12 education. The PI will extend an existing collaboration with a middle school science teacher to design, implement and evaluate an instructional unit on environmental engineering and science organized around inquiry- and discovery-based learning exercises. In addition to publishing research findings in leading journals, information about prions in the environment will be disseminated to a wider audience through a "display" in the UW Madison Virtual Museum of Minerals and Molecules. During the duration of the project, the PI will provide opportunities for undergraduate students to participate in research, with special consideration given to underrepresented minority applicants. The PI will leverage existing resources to reach his educational objectives such as UW NSF-funded Delta Program in Research, Teaching and Learning to provide opportunities for graduate students to develop skills in classroom teaching, preparation of instructional materials, informal education, teaching to diverse student audiences, teaching with technology and internships.