Protein and metal analysis core

蛋白质和金属分析核心

• 批准号: 8452706
• 负责人: Julian P Whitelegge
• 金额: $ 17.26万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-11 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8452706
• 关键词: Adult; Amyotrophic Lateral Sclerosis; Cell Line; Cells; Chemicals; Chromatography; Coupled; Cuprozinc Superoxide Dismutase; Data; Deuterium; Enzymes; Event; Family; Fluorescence Microscopy; Fourier Transform; Heterogeneity; Human; Hydrogen; Hydroxyl Radical; Image; In Vitro; Instruction; Kinetics; Laboratories; Lead; Life; Link; Mass Spectrum Analysis; Measurement; Measures; Metals; Molecular; Molecular Conformation; Molecular Sieve Chromatography; Monitor; Motor Neurons; Mutation; Nature; Organelles; Pathway interactions; Plasma; Preparation; Process; Property; Proteins; Proteomics; Protocols documentation; Reaction; Reproducibility; Resolution; Roentgen Rays; Route; Sampling; Services; Slice; Spectroscopy, Fourier Transform Infrared; Stem cells; Structure; Synchrotrons; Techniques; Therapeutic Intervention; Tissues; Toxic effect; Transgenic Animals; Transgenic Mice; Transgenic Organisms; Work; base; human embryonic stem cell; insight; member; mutant; novel; oxidation; programs; research study; superoxide dismutase 1

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).

项目摘要（请参阅说明）： 情人节计划项目旨在确定肌萎缩性侧面硬化症（ALS）的分子基础 由超氧化物歧化酶1（SODL）中的突变引起。确定SODL蛋白是 淀粉样蛋白生成的四个项目提出了新的实验，以使ALS中毒性的性质进行震撼。 分析核心（核心A； PI：Whitelegge）为物理，化学和蛋白质组学分析提供服务 来自人类细胞和转基因芳香族的SODL培养物和其他提取物。 特定目标1。金属分析：SODL的不完整或不正确的金属化对 蛋白质的特性，因此，准确测量Cu和Zn的水平至关重要 在对项目1、2、3和4重要的样品中。还具有电感耦合等离子体质谱法（ICP-MS） 由于在线尺寸排斥色谱法ICP-MS将用于以极高的敏感性测量金属水平 以及整个组织，分离的细胞器和分离蛋白的可重复性。金属浓度， 分布和氧化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（Lisa Miller），布鲁克黑文国家实验室）。 特定目标2。蛋白质多聚体分析：SODL的非本地形式，包括单体和多聚体 物种似乎对毒性的毒性至关重要。 SODL可以在不同的情况下达到的结构的多样性 条件以及影响其动力学的因素将以理解 通往毒性的途径。核心A将使用色谱法，质谱法（包括氢欧洲 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 在体外产生的原纤维（Proj。1＆4），来自人类胚胎干细胞运动神经元（HESCMNS; Proj。 2）和转基因小鼠（Proj。3）将进行分析。 SPEDFIC AIM 3。一般和靶向蛋白质组学：蛋白质身份和数量的测量是 项目1、2、3和4中提出的工作所需的工作。建立了质谱协议 测量。蛋白质组学将用于测量hESC-MN之后发生的早期细胞事件 暴露于定义的SODL制剂（项目1和2），以定义毒性的分子基础 伪造。我们将使用自上而下的高分辨率傅立叶转换质谱实验和 建立选定的反应监测方案，以准确地量化WT与突变体SODL 混合表达实验（项目2和3）。