CORE--SOLID PHASE PEPTIDE SYNTHESIS AND HPLC/CZE

使用梅里菲尔德自动化固相方法合成肽 核心D将每年综合总共约20个 肽（长度为10至50个残基;线性、环状、磷酸化）。 使用最新的制备技术（大规模：mg至g 量）纯化肽/蛋白质，我们建议提供 相对大量（10至100 mg）的纯化肽/蛋白质 支持在大多数项目中开展的调查。使用最新的 分析技术，我们将确定所有的纯度和身份 合成肽和蛋白质，包括所用的肽的合成肽和蛋白质 临床上将通过反相HPLC法测定纯度， 离子交换色谱FPLC和毛细管区带电泳 （捷克共和国）表征将使用氨基酸分析（AAA），质量分析 光谱（LSIMS）和Edman降解（核心C）。的 在这个核心的庇护下合成的肽将支持大多数 Core C将使用AAA和CZE。肽将用于 进行功能和结构研究，并提出具体的 多克隆抗体（见项目I、II、IV、V）。最后，我们打算 利用该核心的一些资源开发新的分析 技术，探索新的应用。这个核心会利用 PI的经验（7%的努力）和他的技术 助理以及他的SPPS功能齐全的实验室， 特征化实验室空间配备有通风橱和其他小型 设备可用。可用的主要仪器包括LSI质量 HX-110型光谱仪和来自JEOL的60 MHz NMR，Beckman P/ACE 型号2050 CZE，Perkin Elmer AAA，四台肽合成仪，来自 Beckman，分析型和制备型HPLC和冻干机。PI将主持 负责确定优先事项的用户委员会。预计 核心D将能够按照先到先得的原则运作。 在必须确定优先顺序的情况下， 由大多数用户委员会（不包括主席） 切实贯彻将进行肽的合成和纯化 在G-C。Jiang（100%的努力）。质量控制和方法开发 使用HPLC、FPLC和CZE将由C.米勒（30%的努力）。 AAA（10%工作量）和MS分析（15%工作量）将由以下人员进行： D.在A.克雷格（8%的努力）。与 经相关PI同意，多余的肽将免费分发 像我们过去所做的那样，对任何NIH资助的研究人员收取费用。

Using the Merrifield automated solid phase approach to peptide synthesis (SPPS), Core D 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 (large scale: mg to gram quantities) purification of peptides/proteins, we propose to provide relatively large quantities (10 to 100 mg) of purified peptides/proteins to support investigations carried out in most Projects. Using the latest analytical techniques we will ascertain the purity and identity of all synthetic peptides and proteins including that of peptides to be used clinically. Determination of purity will be done by reverse phase-HPLC, ion exchange chromatography on FPLC and capillary zone electrophoresis (CZE). Characterization will use amino acid analysis (AAA), mass spectroscopy (LSIMS) and Edman degradation (Core C) when necessary. The peptides synthesized under the aegis of this core will support most projects while Core C will use AAA and CZE. Peptides will be used to carry out functional and structural studies as well as to raise specific polyclonal antibodies (See Projects I, II, IV, V). 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 PI's experience (7% effort) and of that of his technical assistants as well as of his fully functional laboratory for SPPS and characterization. Laboratory space equipped with hoods and other small equipment is available. Major instruments available include a LSI Mass Spectrometer Model HX-110 and a 60 MHz NMR from JEOL, a Beckman P/ACE model 2050 CZE, a Perkin Elmer AAA, four peptide synthesizers from Beckman, analytical and preparative HPLCs and lyophilizers. PI 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 G-C. Jiang (100 % effort). Quality control and method development using HPLC, FPLC and CZE will be carried out by C. Miller (30 % effort). AAA (10 % effort) and MS analysis (15 % effort) will be carried out by D. Pantoja under the supervision of A. Craig (8 % effort). With the consent of the concerned PIs, excess peptides will be distributed free of charge to any NIH sponsored researcher as we have done in the past.