ANALYTICAL PROTEIN PROFILING

蛋白质分析

• 批准号: 7561415
• 负责人: MICHAEL D POWELL
• 金额: $ 24.42万
• 依托单位: MOREHOUSE SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7561415
• 关键词: AA Spectrophotometry; Acids; Ampholytes; Analytical Chemistry; Area; Biochemistry; Biological; Biological Assay; Blood capillaries; Boxing; Cations; Cells; Charge; Color; Complex; Computer Retrieval of Information on Scientific Projects Database; Computer software; Condition; Coupled; Data; Databases; Digestion; Dimensions; Dyes; Electrophoresis; Elements; Environmental Monitoring; Equipment; Fee Schedules; Fees; Flame Ionization; Fluorescence; Food Analysis; Funding; Gases; Gel; Gold; Grant; High Pressure Liquid Chromatography; Hour; Housing; Human Resources; Image; Immunoassay; Immunology; Injection of therapeutic agent; Institution; Label; Laboratories; Lasers; Maintenance; Measurement; Measures; Microbiology; Modeling; Modification; Molecular; Monitor; Natural graphite; Nitrogen; Pattern; Peptides; Phase; Phosphorus; Phosphorylation; Post-Translational Protein Processing; Preparation; Process; Protein Analysis; Proteins; Proteome; Proteomics; Publications; Range; Reader; Reading; Reagent; Research; Research Personnel; Resources; Running; Sampling; Scanning; Series; Services; Solvents; Source; Specimen; Spectrophotometry; Spottings; Standards of Weights and Measures; Structure; System; Time; Trace metal; Trypsin; Ubiquitination; United States National Institutes of Health; Work; absorption; analog; base; capillary; cost; detector; digital; drug testing; instrument; liquid chromatography mass spectrometry; mass spectrometer; microwave electromagnetic radiation; nitration; reversed phase chromatography; selected ion monitoring; size; surface enhanced laser desorption ionization; synthetic polymer Bioplex

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. II. DESCRIPTION Proteomics Lab  Michael Powell, Leader The Proteomics core lab houses a Surface Enhanced Laser Desorption-Ionization (SELDI) mass spectrometer, and a Thermo-Finnigan LTQ LC/MS/MS system. In December of 2006 we upgraded the software on the LTQ to allow the user to do differential expression using ITRAQ labels (Applied Biosystems). ITRAQ labels allow the quantitation of up to four different sample conditions in the same LC/MS/MS run. By slightly modifying our micro-capillary HPLC setup we also have added the ability to fractionate tryptic peptides from complex samples (such as cell lysates) using strong cation exchange (SCX) prior to further separation by C-18 reverse phase chromatography. We can now divide samples into 10 SCX fractions prior to reverse phase separation using the same sample injection in a single LC/MS/MS run. This level of separation, in combination with ITRAQ labels, should allow the maximum amount of information to be gathered from biological samples. In 2007 we have also added a BioPlex bead sorter, which allows the analysis of multiplex assays such as ELISAs from a single, small volume sample. This instrument was provided by the Microbiology, Biochemistry, and Immunology department and will be housed in the Proteomics facility. This will allow us to provide this service to our users in addition to our other Proteomics services. Together the equipment housed in our facility will allow us to offer a wider range of services to investigators seeking protein analysis. Analytical Chemistry Lab  Daniel Von Deutch, Leader The MSM ACL is equipped with two Agilent (HP) Gas Chromatographs (GC) for the detection of drugs and their metabolites. These include an Agilent (HP) 6890 Series capillary GC with Flame Ionization Detector (FID) and Nitrogen Phosphorus Detector (NPD) and an Agilent (HP) 5890 Series capillary GC coupled with 5972 Series Mass Spectrometer (MS - EI). Data can be acquired in the SCAN or Selected Ion Monitoring (SIM) modes. In addition, the ALC has: 1) an HP (Agilent) 1100 series HPLC equipped with a Diode Array Detector (DAD), a Variable Wavelength Detector (VWD) and a 1046A Fluorescence Detector (FD); 2) a Beckman Gold HPLC system equipped with a 125S Solvent Module, a 508 autosampler and a 168 diode array detector. In addition, the system is equipped with an SS420x analog/digital box to which fluorescence and electrochemical detectors are attached. These detectors include a high sensitivity Gilson fluorescence detector, an Agilent 1049A Electrochemical Detector (ECD) and an ESA Coulochem II ECD system outfitted with multiple Model 5011 high sensitivity two channel analytical cells; and 3) a Spectromax Plus is a UV/VIS variable wavelength microtiter plate reader with Softmax Pro software used for performing colorimetric spectrophotometry and immunoassays. In addition, the ACL has the capability to perform analysis of trace metals in biological specimens via Atomic Absorption (AA) spectrophotometry. Depending on the sample type and size, analysis can be performed on one of two AAs: 1) a Perkin-Elmer Model 3300 Flame Atomic Absorption (FAA) Spectrophotometer equipped with an autosampler. The FAA has sensitivity in the parts per million range and commonly used for analysis of food substances and for environmental testing; 2) a Perkin-Elmer Model 4100ZL electrothermal Graphite Furnace Atomic Absorption (GFAA) Spectrophotometer with Zeeman background correction and an AS90 autosampler. The GFAA provides the highest sensitivity (parts per billion) for most metallic elements and requires very small sample volumes; and 3) a CME MDS200 microwave oven for sample preparation that requires acid digestion. 2-D Gel Lab  Winston Thompson, Leader A range of electrophoresis equipment is available for 1D and 2D gel analyses including Bio-Rad IPG electro-focusing and SDS-PAGE apparatus for 7, 11 and 18 cm formats. We have also added the IPG Runner system from Invitrogen. This is a small format gel system that uses immobilized ampholyte strips to separate in the first dimension. An advantage of this system is the relatively short time necessary to run the first dimension ( 1 hour) instead of longer runs on other systems. Once a 1D or 2D gel is run, there are two options now available to quantitate and compare spots: 1) a BioRad gel imager and automated spot picker (Proteome Works) Bundled with this package is the latest version of PDQuest 8.0 for analysis of expression data from gels. Together, this package allows the user to image gels, compare the images, and do sophisticated analyses that allow a comparison of one or more images to a second group of images. 2) a second option available this quarter is the used of 2D DIGE on a BioRad laser scanner (Molecular imager FX). This system allows the comparison of two samples using different color Cy dyes. The samples are combined and run on the same gel. However, the imager allows the two different colors to be separately measured to allow a direct comparison. Acquired data will be processed with a Dell Optiplex GX260 (2.8 MHz P4, 2GB SDRAM) with a large (19/6) aspect ratio flat panel LCD monitor for displaying multiple gel images simultaneously. Pattern matching and quantitation is done using PDQuest software. Service Charges Proteomics Lab/2D Gel Lab We currently have a fee schedule for all services. For example, we charge by the LC/MS/MS run for analysis on the LTQ. This charge includes a digestion of the sample by trypsin, an LC/MS run, and the subsequent database searches. Database searches including post translational modifications incur an additional charge for typical modifications such as phosphorylation, nitration, ubiquitination, etc. The current charge per run is $200 per run. Other more complicated analysis are charged from an estimate of the time required billed at $40 per hour. We will be determining a fee structure for BioPlex measurements in the first quarter of 2007 we will ask users to purchase their own kits and allow them access to a processing area to run the assays. The plates will then be read by our personnel. Therefore, our costs for supplies should be minimal. In practice, we have tended to offer all of our services on a collaborative basis. In these cases, our intent is to try and produce preliminary data for a grant or publication where the user is not currently funded for a specific project. Analytical Chemistry Lab The Analytical Chemistry Laboratory has a standard fee schedule for services. The fee structure is based on the amount of time required to process the samples (extraction, derivatization, etc) and the cost of supplies and reagents. In addition, a general fee is requested to cover equipment maintenance.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 二. 描述 蛋白质组学实验室 Michael Powell，负责人 蛋白质组学核心实验室配备表面增强激光解吸电离 (SELDI) 质谱仪和 Thermo-Finnigan LTQ LC/MS/MS 系统。 2006 年 12 月，我们升级了 LTQ 上的软件，允许用户使用 ITRAQ 标签（Applied Biosystems）进行差异表达。 ITRAQ 标签允许在同一次 LC/MS/MS 运行中对多达四种不同的样品条件进行定量。 通过稍微修改我们的微毛细管 HPLC 设置，我们还增加了在通过 C-18 反相色谱进一步分离之前使用强阳离子交换 (SCX) 从复杂样品（例如细胞裂解物）中分离胰蛋白酶肽的能力。 现在，我们可以在单次 LC/MS/MS 运行中使用相同的样品注射，在反相分离之前将样品分成 10 个 SCX 级分。 这种分离水平与 ITRAQ 标签相结合，应该可以从生物样品中收集最大量的信息。 2007 年，我们还添加了 BioPlex 微珠分选仪，可对单个小体积样品进行多重检测（例如 ELISA）分析。 该仪器由微生物学、生物化学和免疫学系提供，将安装在蛋白质组学设施中。 这将使我们能够在其他蛋白质组学服务之外向用户提供这项服务。 我们设施中的设备将使我们能够为寻求蛋白质分析的研究人员提供更广泛的服务。 分析化学实验室 Daniel Von Deutch，负责人 MSM ACL 配备两台安捷伦 (HP) 气相色谱仪 (GC)，用于检测药物及其代谢物。其中包括配备火焰离子化检测器 (FID) 和氮磷检测器 (NPD) 的 Agilent (HP) 6890 系列毛细管 GC 以及配备 5972 系列质谱仪 (MS - EI) 的 Agilent (HP) 5890 系列毛细管 GC。可以在扫描或选择离子监测 (SIM) 模式下采集数据。此外，ALC还具有： 1) HP (Agilent) 1100系列HPLC，配备二极管阵列检测器(DAD)、可变波长检测器(VWD)和1046A荧光检测器(FD)； 2) Beckman Gold HPLC系统，配备125S溶剂模块、508自动进样器和168二极管阵列检测器。此外，该系统还配备了 SS420x 模拟/数字盒，上面连接有荧光和电化学检测器。这些检测器包括高灵敏度 Gilson 荧光检测器、Agilent 1049A 电化学检测器 (ECD) 和配备多个 5011 型高灵敏度双通道分析池的 ESA Coulochem II ECD 系统； 3) Spectromax Plus 是一款 UV/VIS 可变波长微量滴定板读数器，配有 Softmax Pro 软件，用于执行比色分光光度法和免疫测定。 此外，ACL 还能够通过原子吸收 (AA) 分光光度法对生物样本中的痕量金属进行分析。根据样品类型和大小，可以在两种 AA 之一上进行分析：1) 配备自动进样器的 Perkin-Elmer 3300 型火焰原子吸收 (FAA) 分光光度计。 FAA 的灵敏度达到百万分之一范围，通常用于食品物质分析和环境测试； 2) Perkin-Elmer 4100ZL 型电热石墨炉原子吸收 (GFAA) 分光光度计，带塞曼背景校正和 AS90 自动进样器。 GFAA 为大多数金属元素提供最高的灵敏度（十亿分之一），并且需要非常小的样品量； 3) 一台 CME MDS200 微波炉，用于需要酸消解的样品制备。 2-D 凝胶实验室 Winston Thompson，负责人 一系列电泳设备可用于 1D 和 2D 凝胶分析，包括适用于 7、11 和 18 cm 格式的 Bio-Rad IPG 电聚焦和 SDS-PAGE 装置。我们还添加了 Invitrogen 的 IPG Runner 系统。 这是一种小型凝胶系统，使用固定的两性电解质条在第一维进行分离。 该系统的优点是运行第一维所需的时间相对较短（1 小时），而不是在其他系统上运行较长的时间。 运行 1D 或 2D 凝胶后，现在有两种选项可用于定量和比较斑点：1) BioRad 凝胶成像仪和自动斑点选择器 (Proteome Works) 与此软件包捆绑在一起的是最新版本的 PDQuest 8.0，用于分析凝胶的表达数据。 总之，该软件包允许用户对凝胶进行成像、比较图像并进行复杂的分析，从而将一个或多个图像与第二组图像进行比较。 2) 本季度提供的第二个选项是在 BioRad 激光扫描仪（分子成像仪 FX）上使用 2D DIGE。该系统允许使用不同颜色 Cy 染料比较两个样品。 将样品合并并在同一凝胶上运行。 然而，成像仪允许单独测量两种不同的颜色，以便进行直接比较。 采集的数据将使用 Dell Optiplex GX260（2.8 MHz P4、2GB SDRAM）进行处理，该显示器具有大宽高比（19/6）平板液晶显示器，可同时显示多个凝胶图像。模式匹配和定量是使用 PDQuest 软件完成的。 服务费 蛋白质组学实验室/2D 凝胶实验室 目前，我们对所有服务都有收费表。 例如，我们按在 LTQ 上进行分析的 LC/MS/MS 运行收费。 该费用包括胰蛋白酶消化样品、LC/MS 运行以及随后的数据库搜索。 包括翻译后修饰在内的数据库搜索会对磷酸化、硝化、泛素化等典型修饰产生额外费用。目前每次运行的收费为 200 美元。 其他更复杂的分析根据估计所需时间收费，每小时 40 美元。 我们将在 2007 年第一季度确定 BioPlex 测量的费用结构，我们将要求用户购买自己的套件并允许他们进入处理区域来运行检测。 然后我们的人员将读取这些车牌。 因此，我们的供应成本应该是最低的。 在实践中，我们倾向于在协作的基础上提供所有服务。 在这些情况下，我们的目的是尝试为用户当前没有为特定项目提供资助的赠款或出版物生成初步数据。 分析化学实验室 分析化学实验室有标准的服务费用表。费用结构基于处理样品（提取、衍生化等）所需的时间以及耗材和试剂的成本。此外，还需要支付一般费用来支付设备维护费用。