Core--Analytical and Peptide Synthesis

核心--分析与肽合成

• 批准号: 6956184
• 负责人: JEAN E RIVIER
• 金额: $ 24万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6956184
• 关键词: analytical chemistry; biomedical facility; mass spectrometry; peptide chemical synthesis; peptide structure; protein sequence; protein structure

This Core C will provide essential functions for the Projects of this Program Project. These include the synthesis of peptides that are required by the Projects for biological and biochemical investigations. In addition, the Core will characterize synthetic, natural or modified peptides and proteins by mass spectrometry and Edman based chemical sequencing. Core C will synthesize and characterize peptides, and develop new HPLC- and CZE-based analytical systems to improve resolution and efficiency. Additionally, this Core will perform circular dichroism experiments as requested. Using the Merrifield automated SPPS, Core C will synthesize, each year, a total of approximately 10-15 peptides (20 to 40 residues in length; linear, cyclic, phosphorylated). Using the latest techniques for the preparative purification of peptides/proteins, we will provide 10 to 100 mg of highly purified peptides to support investigations carried out in most Projects. All peptides will be extensively characterized by the techniques available to the Core, including HPLC, CZE, MS, and Edman degradation. The protein chemical characterization of peptides and proteins involved in neuroendocrine regulation is an essential part of this Program. The Core provides both state-of-the-art instrumentation and highly trained investigators and support personnel to carry out these characterizations. The Core will characterize peptides, proteins and their post-translational modifications. In particular, the disulfide arrangement of soluble forms of CRFRs, both recombinant and naturally occurring, will be determined. In addition, we will confirm the covalent structure of recombinant proteins that the Projects generate for use in biological studies. The processing of urocortins will be established by isolating the peptides from tissues or cell lines and determining their primary structure by mass spectrometry and Edman degradation. The Core will use its proteomics capability to define proteins interacting with the CRF receptor system. Affinity tagged receptors and receptor fragments will be designed and transfected into cells. Complexes will be isolated and their protein constituents will be identified by mass spectrometry.

该核心C语言将为本计划项目的项目提供必要的功能。其中包括生物和生化研究项目所需的多肽的合成。此外，Core还将通过质谱学和基于Edman的化学测序对合成的、天然的或修饰的多肽和蛋白质进行表征。Core C将合成和表征多肽，并开发基于高效液相和CZE的新分析系统，以提高分辨率和效率。此外，这个核心将执行圆形二色性 按要求进行实验。使用Merrifield自动SPPS，Core C每年将总共合成大约10-15个多肽(长度为20-40个残基；线性、环状、磷酸化)。利用最新的制备性纯化多肽/蛋白质的技术，我们将提供10至100毫克的高纯度多肽，以支持大多数项目的研究。所有的多肽都将通过Core可用的技术进行广泛的表征，包括高效液相色谱、CZE、MS和Edman降解。参与神经内分泌调节的多肽和蛋白质的蛋白质化学特征是该计划的重要组成部分。核心提供最先进的仪器以及训练有素的调查人员和支持人员来进行这些表征。核心将表征多肽、蛋白质及其翻译后修饰。特别是，二硫键的排列 将确定重组和自然发生的CRFR的可溶性形式。此外，我们将确认这些项目产生的用于生物学研究的重组蛋白的共价结构。尿皮质素的加工将通过从组织或细胞系中分离多肽，并通过质谱分析和Edman降解来确定其一级结构来建立。Core将利用其蛋白质组学能力来定义与CRF受体系统相互作用的蛋白质。亲和力标记的受体和受体片段将被设计并导入细胞。复合体将被分离，其蛋白质组分将通过质谱学进行鉴定。