CORE--Peptidomics, RIA, and Proteomics

核心——肽组学、放射免疫分析和蛋白质组学

• 批准号: 7415064
• 负责人: JOSEPH R REEVE
• 金额: $ 5.26万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-12-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7415064
• 关键词: Agonist; Amino Acid Sequence Databases; Amino Acids; Antibodies; Arts; Atmospheric Pressure; Biochemical; Blood capillaries; Capillary Electrophoresis; Caring; Cells; Chemicals; Chromatography; Computers; Condition; Electrons; Electrospray Ionization; Enzyme-Linked Immunosorbent Assay; Equipment; Evaluation; Funding; Gel; High Pressure Liquid Chromatography; Human Resources; Individual; Informatics; Investigation; Ions; Label; Laboratories; Liquid Chromatography; Magnetism; Mass Chromatography; Mass Spectrum Analysis; Methods; Modification; Numbers; Pattern; Peptides; Performance; Physiological; Proteins; Proteome; Proteomics; Protocols documentation; Radioimmunoassay; Radiolabeled; Reagent; Research; Research Activity; Research Personnel; Resources; Robotics; Sampling; Services; Signal Transduction; Solutions; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Speed; Structure; Support System; System; Techniques; Testing; Time; Training; base; capillary; enzyme substrate; gel electrophoresis; instrument; instrumentation; investigator training; ion source; mass spectrometer; radiotracer; research study; skills; synthetic peptide

The four main objectives of the Pepfidomics, RIA and Proteomics (PRP) Core are to: 1) train CURE: DDRCC investigators in the use of peptide, radioimmunoassay and proteomic techniques for their own research activities; 2) perform services for CURE: DDRCC investigators for studies requiring expensive equipment or requiring too much skill to be performed in individual laboratories (i.e. mass spectral analysis, microsequence analysis, amino acid analysis, etc.); 3) provide instrumentation for studies requiring expensive equipment and simple protocols where CURE: DDRCC investigators and staff can be trained to perform experiments (i.e. radioimmunoassay of large numbers of samples, HPLC studies using the same method repeatedly); 4) provide unique reagents necessary for CURE: DDRCC investigators to conduct their own research (i.e. radiolabels, synthetic peptides, fluorescent labeled synthetic peptides). Many physiological, pharmacological, and biochemical studies performed at CURE: DDRCC utilize peptide reagents, including biologically active agonists and antagonists, cell permeable mimics of intracellular proteins that interfere with signal transduction steps, enzyme substrates, etc. Proper care and evaluation of these reagents are necessary for valid results. CURE: DDRCC investigators are trained in how to make, store and evaluate stock solutions, how to dilute peptides properly, and how to evaluate the loss or modification of peptides under experimental conditions. The PRP will also provide services and equipment for CURE: DDRCC investigators to acquire state of the art characterization of peptides. A wealth of antibodies has been produced by the Antibody Core during the past funding period, and these continue to be an integral component of several studies performed at CURE: DDRCC. These antibodies will be tested and distributed bythe PRP Core. The personnel of the Core will continue to enable investigators to perform radioimmunoassay and ELISA techniques. The Shared Proteomics Facility is a new addition to the CURE: DDRCC. Purification and characterization of proteins by high performance capillary electrophoresis, high performance liquid chromatography, microsequence analysis, and mass spectral analysis require instrumentation far too costly for individual laboratories and expertise to perform complicated techniques properly and efficiently. Many of the chromatography techniques used for years by Dr. Reeve and his staff (1) are directly applicable to proteomics. This is especially true for low abundance proteins of a cell because 2D gels are limited in volume of sample that can be applied. Liquid chromatography also offers the ability to perform 3D or 4D separations so proteins can be highly purified, resulting in cleaner subsequent mass spectral patterns. The PRP will train CURE: DDRCC investigators in proteomic techniques, perform liquid chromatography, mass spectral analysis and informatics necessary to characterize the proteomes under investigation. The Proteomic facilities include a variety of high performance mass spectrometers capable of providing detailed information on the structure of biomolecules, such as time-of-flight, magnetic sector, quadrupole, triple quadrupole, quadrupole ion trap and quadrupole-time-of-flight (QTOF) tandem mass spectrometers (i.e., MS/MS). These instruments are equipped with conventional electron impact (EI), chemical (CI), atmospheric pressure chemical (APCI) and fast atom bombardment (FAB) ion sources, in addition to electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) sources for the sensitive analysis of peptides and proteins. On-line separation methods such as capillary HPLC are interfaced to ESI-MS systems to characterize low abundance level peptide and protein samples. The Resource also has access to supporting systems for proteomics research, including gel electrophoresis, automated protein digesters, and a robotic MALDI sample spotter. A computer server network used for high speed protein sequence database searching is dedicated for mass spectrometry-based protein identification.

倍率组学，RIA和蛋白质组学（PRP）核心的四个主要目标是：1）火车治疗：DDRCC研究人员在使用肽，放射免疫测定和蛋白质组学技术方面用于其自己的研究活动； 2）为治疗提供服务：DDRCC调查人员需要昂贵的设备或需要过多技能以无法在单个实验室中执行的研究（即质谱分析，微序列分析，氨基酸分析等）； 3）为需要昂贵的设备和简单协议提供的研究提供仪器：可以培训DDRCC调查人员和员工以执行实验（即大量样本的放射免疫测定，而使用相同方法的HPLC研究）； 4）提供治疗所需的独特试剂：DDRCC研究人员进行自己的研究（即放射性标记，合成肽，荧光标记的合成肽）。在治疗中进行的许多生理，药理和生化研究：DDRCC使用 肽试剂，包括生物活性激动剂和拮抗剂，细胞内细胞内蛋白的可渗透模仿，这些模仿会干扰信号转导步骤，酶底物等。适当的护理和对这些试剂的适当护理和评估是有效结果的。治疗：DDRCC研究人员接受了如何制造，存储和评估库存溶液，如何正确稀释肽以及如何评估实验条件下肽的损失或修饰的培训。 PRP还将为CURE提供服务和设备：DDRCC调查人员以获取肽的最先进状态。在过去的资金期间，抗体核心已经产生了大量的抗体，这些抗体仍然是在CURE上进行的几项研究的组成部分：DDRCC。这些抗体将由PRP核测试和分布。核心人员将继续使调查人员能够 执行放射免疫分析和ELISA技术。共享的蛋白质组学设施是治疗方法的新补充：DDRCC。通过高性能毛细管电泳，高性能液相色谱，微序列分析和质谱分析对蛋白质的纯化和表征，需要仪器成本太高，对于单个实验室和专业知识而言，无法正确，有效地执行复杂的技术。 Reeve博士及其员工（1）使用多年的许多色谱技术直接适用于蛋白质组学。对于细胞的低丰度蛋白而言，尤其如此，因为2D凝胶在可以应用的样品体积上受到限制。 Liquid chromatography also offers the ability to perform 3D or 4D separations so proteins can be highly purified, resulting in cleaner subsequent mass spectral patterns. PRP将训练治疗：DDRCC研究人员的蛋白质组学技术，进行液相色谱，质谱分析和所需的信息，以表征正在研究的蛋白质组织。 The Proteomic facilities include a variety of high performance mass spectrometers capable of providing detailed information on the structure of biomolecules, such as time-of-flight, magnetic sector, quadrupole, triple quadrupole, quadrupole ion trap and quadrupole-time-of-flight (QTOF) tandem mass spectrometers (i.e., MS/MS).这些仪器配备了传统的电子撞击（EI），化学（CI），大气压化学（APCI）和快速原子轰击（Fab）离子源（ESI）除了电喷雾电离（ESI）以及基质辅助激光剂量吸收/电离（MALDI）来源（MALDI）来源 用于对肽和蛋白质的敏感分析。在线分离方法（例如毛细血管HPLC）与ESI-MS系统连接，以表征低丰度水平的肽和蛋白质样品。该资源还可以访问用于蛋白质组学研究的支持系统，包括凝胶电泳，自动化蛋白质消化器和机器人MALDI样本摄影剂。用于高速蛋白质序列数据库搜索的计算机服务器网络专门用于基于质谱的蛋白质识别。