ANALYTICAL PROTEIN PROFILING

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 I.说明 蛋白质组学实验室负责人迈克尔·鲍威尔 蛋白质组学核心实验室拥有一台表面增强激光解吸电离(SELDI)质谱仪和一台Thermo-Finnigan LTQ LC/MS/MS系统。还可提供BioRad自动斑点拾取器(Proteome Works)和BioRad凝胶成像仪(分子成像仪FX)。与这个包捆绑在一起的是最新版本的PDQuest8.0，用于分析凝胶中的表达数据。2007年，我们还增加了一台BioPlex珠子分选机，它可以从单个小体积样本中进行多重分析，如ELISA。通过这些设备，我们可以提供蛋白质鉴定和蛋白质修饰分析等服务。我们还可以通过凝胶和LC分析提供差异蛋白质表达分析。 分析化学实验室负责人Daniel von Deutsch MSM ACL配备了两个安捷伦(HP)1100系列高效液相系统。完毕 在过去的一年里，我们从药理学和毒理学系的Ward Kirlin博士那里获得了第二个Agilent系统。ACL中的高效液相系统包括：1)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)的HP(Agilent)1100系列高效液相色谱仪；2)配备1200系列荧光检测器(FD)和高灵敏度Gilson荧光检测器的HP(Agilent)1100系列高效液相色谱仪；3)配备125S溶剂模块、508自动进样器和168二极管阵列检测器的Beckman Gold HPLC系统。此外，该系统还配备了SS420x模拟/数字盒子，上面连接了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、安捷伦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。除高效液相系统外，ACL还配备了一台Spectrmax Plus和两台安捷伦气相色谱仪，这是一种带有Softmax Pro软件的UV/VIS可变波长微滴定板读取器，用于进行比色光度分析和免疫分析 (GC)检测药物及其代谢物的系统。GC包括带有火焰电离检测器(FID)和氮磷检测器(NPD)的安捷伦(HP)6890系列毛细管气相色谱，以及配有5972系列质谱仪(MS-EI)的安捷伦(HP)5890系列毛细管气相色谱。可以在扫描或选定的离子监测(SIM)模式下获取数据。 服务费： 蛋白质组学实验室/二维凝胶实验室 我们目前对所有服务都有一个收费表。例如，我们按 LC/MS/MS联用，对LTQ进行分析。这一费用包括用胰酶消化样品，LC/MS运行，以及随后的数据库搜索。数据库搜索，包括翻译后修饰，对于典型的修饰，如磷酸化、硝化、泛素化等，会产生额外的费用。其他更复杂的分析，从所需时间的估计中收费。目前，我们提供BioPlex分析，方法是让用户购买自己的试剂盒，然后为他们提供一个工作站，其中包含分析所需的所有材料。我们正在制定一项使用费，以支付平台校准和验证所需的材料成本和时间。随着今年我们核心经理的加入，我们应该能够提高我们的 电荷结构。 分析化学实验室 分析化学实验室有一个标准的服务收费表。费用结构是根据处理样品所需的时间(提取、衍生等)以及用品和试剂的费用确定的。对于非NIH赞助的项目(MSM和外部机构和公司)，评估劳动力成本。此外，由于ACL没有从NIH或MSM那里获得运营预算，因此向所有项目收取额外费用，以帮助支付设备维护和其他实验室管理费用。成本会定期调整，以反映供应商价格的变化。

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. I. 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. Also available are a BioRad automated spot picker (Proteome Works) and BioRad gel imager (Molecular imager FX). Bundled with this package is the latest version of PDQuest 8.0 for analysis of expression data from gels. 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. Together this equipment allows us to offer services such as protein identification and analysis of protein modifications. We can also offer differential protein expression analysis by both gel and LC analysis. Analytical Chemistry Lab  Daniel von Deutsch, Leader The MSM ACL is equipped with two Agilent (HP) 1100 series high HPLC systems. Over that past year, we obtained a second Agilent system from Dr. Ward Kirlin in the Department of Pharmacology and Toxicology. The HPLC systems in the ACL include: 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) an HP (Agilent) 1100 series HPLC equipped with a 1200 series Fluorescence Detector (FD) and a high sensitivity Gilson fluorescence detector; 3) 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. In addition to the HPLC systems, the ACL has a Spectromax Plus is a UV/VIS variable wavelength microtiter plate reader with Softmax Pro software used for performing colorimetric spectrophotometry and immunoassays and two Agilent Gas Chromatographs (GC) systems for the detection of drugs and their metabolites. The GCs 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. 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. Other, more complicated analysis, are charged from an estimate of the time required. Currently, we are offering BioPlex analysis by having users purchase their own kits and then providing them with a workstation with all of the materials needed for analysis. We are working on a user fee to cover the cost of materials and the time necessary for calibrations and validations for the platform. With the addition of our core manager this year we should be able to increase the efficiency of our charge structure. 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. For non-NIH sponsored projects (MSM and outside institutions and corporations), labor costs are assessed. In addition, since the ACL receives no operating budget from either the NIH or MSM, an additional fee is assessed to all projects to help cover equipment maintenance and other laboratory overhead. Costs are periodically adjusted to reflect vendor price changes.