Protein and metal analysis core

项目总结(见说明)： 瓦伦丁计划项目试图确定肌萎缩侧索硬化症(ALS)的分子基础 由超氧化物歧化酶1(SOD1)突变引起。已经确定SOD1蛋白是 淀粉样变性，四个项目提出了新的实验，以了解ALS毒性的本质。 分析核心(核心A；PI：Whitelege)提供物理、化学和蛋白质组分析服务 从人类细胞和转基因植物中提取的SODl多聚体和其他提取物。 具体目标1.金属分析：未完全或不适当地对硫化物进行金属化会对 蛋白质的性质，因此准确测量铜和锌的水平是至关重要的 在对项目1、2、3和4重要的样品中也包括电感耦合等离子体质谱(ICPMS) 作为在线尺寸排除色谱，电感耦合等离子体质谱将被用于高灵敏度的金属含量的测量 在整个组织、分离的细胞器和分离的蛋白质中具有重复性。金属浓度， 分布和氧化状态将使用同步加速器X射线在组织切片上进行空间成像 荧光显微镜(与布鲁克海文国家实验室的丽莎·米勒一起)。 特定目的2.蛋白质多聚体分析：SOD1的非天然形式，包括单体和多聚体 物种似乎是FALS毒性的关键。SOD在不同条件下所能达到的结构多样性 条件，以及影响其动力学的因素，将被表征为理解 致毒途径。堆芯A将使用层析、质谱学(包括氢-氚) 交换和羟基自由基足迹)、同步加速器FTIR成像和其他技术 表征多聚体制剂的大小和结构及其异质性。可溶低聚物和 在体外产生的纤维(项目。1和4)、人胚胎干细胞运动神经元多聚体(HESCMns； 项目。2)和转基因小鼠(Proj.3)将被分析。 特殊目标3.一般和靶向蛋白质组学：蛋白质同一性和数量的测量是 项目1、2、3和4中建议的工作所需。为各种类型建立了质谱学协议 测量的结果。蛋白质组学将用于测量HESC-MNS术后发生的早期细胞事件 暴露于定义的SODl制剂(项目1和2)，以确定毒性的分子基础 法国佬。我们将使用自上而下的高分辨率傅里叶变换质谱学实验和 建立精选的反应监测方案，以准确定量WT与突变的SOD1. 混合表达实验(项目2和3)。

PROJECT SUMMARY (See instructions): The Valentine program project seeks to determine the molecular basis of amyotrophic lateral sclerosis (ALS) caused by mutations in superoxide dismutase 1 (SODl). Having established that SODl proteins are amyloidogenic, four Projects propose novel experiments toward luiderstanding the nature of toxicity in ALS. The analytical core (Core A; PI: Whitelegge) provides services for physical, chemical and proteomic analysis of SODl-multimers and other extracts from human cells and transgenic arumals. Specific Aim 1. Metal analysis: Incomplete or improper metallation of SODl has profotind effects upon the protein's properties and it is therefore critically important that levels of Cu and Zn are measured accurately in samples important to projects 1, 2, 3 & 4. Inductively coupled plasma mass spectrometry (ICP-MS) as well as online size-exclusion chromatography ICP-MS will be used to measure metal levels with great sensitivity and reproducibility in whole tissues, isolated organelles and isolated proteins. Metal concentration, distribution, and oxidation state will be spatially imaged across tissue slices using synchrotron X-ray fluorescence microscopy (with Lisa Miller, Brookhaven National Laboratory). Specific Aim 2. Protein multimer analysis: Non-native forms of SODl including monomeric and multimeric species appear key to toxicity in FALS. The diversity of structures that SODl can attain under different conditions, and the factors that influence its kinetics, will be characterized toward understanding the pathway to toxicity. Core A will use chromatography, mass spectrometry (including hydrogen-deuterium exchange and hydroxyl radical footprinting), synchrotron FTIR imaging, and other techniques to characterize the size and structure of multimer preparations and their heterogeneity. Soluble oligomers and fibrils produced in vitro (Proj. 1 & 4), multimers from human embryoiuc stem cell motor neurons (HESCMNs; Proj. 2) and transgenic mice (Proj. 3) will be analyzed. Spedfic Aim 3. General and targeted proteomics: Measurements of protein identity and quantity are required for work proposed in Projects 1, 2,3 & 4. Mass spectrometry protocols are established for a variety of measurements. Proteomics will be used to measure early cellular events occurring after HESC-MNs are exposed to defined SODl preparations (Projects 1 & 2) toward defining the molecular basis of toxicity in FALS. We will use top-down high-resolution Fourier-transform mass spectrometry experiments and establish selected reaction monitoring protocols for accurate quantification of WT versus mutant SODl in mixed expression experiments (Projects 2 & 3).