CORE--SOLID PHASE PEPTIDE SYNTHESIS AND HPLC/CZE

核心--固相肽合成与HPLC/CZE

• 批准号: 6577255
• 负责人: JEAN E RIVIER
• 金额: $ 19.74万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6577255
• 关键词: biomedical facility; capillary electrophoresis; circular dichroism; high performance liquid chromatography; inhibin; ion exchange chromatography; mass spectrometry; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; peptide hormone; peptide hormone analog; protein purification; protein structure function; synthetic peptide; zonal electrophoresis

This Core will synthesize and characterize peptides, and develop new HPLC- and CZE-based analytical systems to improve on resolution and efficiency. Additionally, this Core will perform circular dichroism (CD) experiments as requested. Using the Merrifield automated SPPS, Core will synthesize, each year, a total of approximately 20 peptides (10 to 50 residues in length; linear, cyclic, phosphorylated). Using the latest techniques for the preparative purification of peptide/proteins, we will provide 10 to 100 mg of purified peptides/proteins to HPLC, ion exchange chromatography on FPLC and capillary zone electrophoresis (CZE). Characterization will use mass spectroscopy (LSIMS) and Edman degradation (Core C) when necessary. These peptides will support most projects will Core C will use CZE. Peptides will be used to carry out functional and structural studies as well as to raise specific polyclonal antibodies. Finally, we intend to use some of the resources of this Core to develop new analytical techniques and explore new applications. This Core will take advantage of the P.,I.'s experience (12% effort) and that of his technical assistant as well as his fully functional laboratory for SPPS and characterization. Laboratory space equipped with hoods and other small equipment is available. Major instruments available include an LSI Mass Spectrometer Model HX-110 and a 60 MHz NMR from JEOL, a Beckman P/ACE model 2050 CZE, three peptide synthesizers from Beckman, analytical and preparative HPLCs and lyophilizers. The P.I. will chair the users' committee responsible for setting priorities. It is expected that Core D will be able to operate on a first come, first served basis. In cases where priority will have to be determined, the decision reached by the majority of the users' committee (excluding the chair) will be implemented. Synthesis and purification of peptides will be carried out by M. Odrowaz-Sypniewski (30% effort). Quality control and method development using HPLC, FPLC and CZE will be carried out by D. Kirby (15% effort). MS analyses (15% effort) will be carried out by Dr. A. Craig (15% effort). With the consent of the concerned P.I.s, excess peptides will be distributed free of charge to any NIH sponsored researcher as we have done in the past. Recently, there has been a significant need for circular dichroism spectroscopy. We have been responsible for the instrument for more than ten years and will provide analyses to investigators on this Program Project.

该核心将合成和表征肽，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，该核心还将根据要求进行圆二色性 (CD) 实验。使用 Merrifield 自动化 SPPS，Core 每年将合成总共约 20 种肽（长度为 10 至 50 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，我们将向 HPLC、FPLC 离子交换层析和毛细管区带电泳 (CZE) 提供 10 至 100 mg 纯化的肽/蛋白质。必要时，表征将使用质谱 (LSIMS) 和 Edman 降解 (Core C)。这些肽将支持 Core C 将使用 CZE 的大多数项目。肽将用于进行功能和结构研究以及产生特异性多克隆抗体。最后，我们打算利用该核心的一些资源来开发新的分析技术并探索新的应用。该核心将利用 P.,I. 的经验（12% 的努力）及其技术助理的经验以及他用于 SPPS 和表征的功能齐全的实验室。提供配有通风罩和其他小型设备的实验室空间。可用的主要仪器包括 JEOL 的 HX-110 型 LSI 质谱仪和 60 MHz NMR、Beckman P/ACE 2050 CZE 型、Beckman 的三种肽合成仪、分析型和制备型 HPLC 以及冻干机。 P.I.将主持用户委员会，负责确定优先事项。预计 Core D 将能够按照先到先得的原则运行。在需要确定优先级的情况下，将执行用户委员会大多数成员（不包括主席）达成的决定。肽的合成和纯化将由 M. Odrowaz-Sypniewski 进行（30% 的工作量）。 D. Kirby 将使用 HPLC、FPLC 和 CZE 进行质量控制和方法开发（15% 的工作量）。 MS 分析（15% 的工作量）将由 A. Craig 博士（15% 的工作量）进行。经相关 P.I. 同意，多余的肽将像我们过去所做的那样免费分发给任何 NIH 资助的研究人员。最近，对圆二色光谱的需求非常大。我们负责该仪器已有十多年，并将为该计划项目的研究人员提供分析。