ANALYTICAL PROTEIN PROFILING

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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.