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).

项目概述（见说明）：