Protein and metal analysis core

项目总结（见说明）： 瓦伦丁计划项目旨在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 淀粉样蛋白的，四个项目提出了新的实验，以了解ALS的毒性性质。 分析核心（核心A; PI：Whitelegge）提供物理、化学和蛋白质组学分析服务 SOD1-多聚体和来自人细胞和转基因植物的其它提取物。 具体目标1。金属分析：SOD1的不完全或不适当的金属化对金属的性质有明显的影响。 蛋白质的特性，因此准确测量铜和锌的水平至关重要 在对项目1、2、3和4重要的样本中。电感耦合等离子体质谱法（ICP-MS） 由于在线尺寸排阻色谱法，ICP-MS将用于以极高的灵敏度测量金属水平 以及在整个组织、分离的细胞器和分离的蛋白质中的再现性。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（与丽莎米勒，布鲁克海文国家实验室）。 具体目标2。蛋白质多聚体分析：非天然形式的SOD1，包括单体和多聚体 物种似乎是毒性的关键。SOD1在不同条件下可以获得的结构的多样性 条件，以及影响其动力学的因素，将被描述为了解 毒性途径。核心A将使用色谱法、质谱法（包括氢氘 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。可溶性低聚物和 体外产生的原纤维（Proj. 1和4），来自人胚胎干细胞运动神经元（HESCMN; 项目。2)和转基因小鼠（Proj. 3)将被分析。 第三章.一般蛋白质组学和靶向蛋白质组学： 项目1、2、3和4中建议的工作所需的材料。建立了用于多种质谱分析的质谱分析方案。 的测量。蛋白质组学将用于测量HESC-MN被植入后发生的早期细胞事件。 暴露于确定的SOD1制剂（项目1和2），以确定毒性的分子基础， 失败。我们将使用自上而下的高分辨率傅里叶变换质谱实验， 建立选择的反应监测方案，用于精确定量WT与突变体SOD 1， 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).