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 将为该计划项目的项目提供基本功能。其中包括生物和生化研究项目所需的肽的合成。此外，核心将通过质谱和基于埃德曼的化学测序来表征合成、天然或修饰的肽和蛋白质。 Core C 将合成和表征肽，并开发新的基于 HPLC 和 CZE 的分析系统，以提高分辨率和效率。此外，该核心将表现出圆二色性 按要求进行实验。使用 Merrifield 自动化 SPPS，Core C 每年将合成总共约 10-15 个肽（长度为 20 至 40 个残基；线性、环状、磷酸化）。使用最新的肽/蛋白质制备纯化技术，我们将提供 10 至 100 毫克高度纯化的肽来支持大多数项目中进行的研究。所有肽都将通过核心可用的技术进行广泛表征，包括 HPLC、CZE、MS 和 Edman 降解。参与神经内分泌调节的肽和蛋白质的蛋白质化学表征是该计划的重要组成部分。核心提供最先进的仪器和训练有素的研究人员和支持人员来进行这些表征。核心将表征肽、蛋白质及其翻译后修饰。特别是，二硫键排列 重组和天然存在的 CRFR 的可溶形式将被确定。此外，我们将确认该项目生成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肽并通过质谱和埃德曼降解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。将设计亲和标记的受体和受体片段并将其转染到细胞中。将分离复合物并通过质谱法鉴定其蛋白质成分。