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. 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.