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。其中包括项目和生化研究所需的肽的合成。此外，核心将通过质谱和基于Edman的化学测序来表征合成，天然或改良的肽和蛋白质。 Core C将合成和表征肽，并开发新的HPLC和基于CZE的分析系统以提高分辨率和效率。此外，该核心将执行圆形二科运动 根据要求进行实验。使用Merrifield自动SPPS，Core C每年将合成大约10-15肽（长度为20至40个残基；线性，环状，磷酸化）。使用最新技术来进行肽/蛋白质的制备纯化，我们将提供10至100 mg高度纯化的肽，以支持大多数项目中进行的研究。所有肽将以可用于核心的技术（包括HPLC，CZE，MS和Edman降解）的技术进行广泛的特征。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程序的重​​要组成部分。核心既提供了最先进的仪器，也提供了训练有素的调查人员，也提供了支持人员来执行这些特征。核心将表征肽，蛋白质及其翻译后修饰。特别是，二硫键的布置 将确定重组和天然发生的CRFR的可溶性形式。此外，我们将确认项目用于生物学研究的重组蛋白的共价结构。将通过从组织或细胞系中分离肽并通过质谱和Edman降解来确定其主要结构来确定尿素素的加工。核心将使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转染到细胞中。复合物将被分离，其蛋白质成分将通过质谱鉴定。