Biomarkers Facility Core
Objectives
The goal of the Biomarker Facility Core (BFC) is to provide access to state-of-the-art facilities for proteomics, metabolomics, mass spectrometry and DNA damage, microarray, and mouse genotyping for Center members, in order to facilitate and enhance research on environmentally related disease. This will be accomplished by achieving the following objectives:
- To make available to Center members well-equipped laboratories for determination and/or quantitation of analytical, molecular, and genomic biomarkers.
- To assist Center members with experimental design, tissue collection, and assays for various biomarkers.
- To foster the application of biomarkers in pilot projects and new collaborative research projects.
- To provide education and training in methods related to biomarker discovery and/or analysis.
Core Director, Members and Affiliates
Facilities and Services
The Biomarker Facility Core operates several facilities including the following:
- Proteomics Facility
- Metabolomics Facility
- Mass Spectrometry Facility
- DNA Damage (Slot-blot) Facility
- Microarray Facility
- Mouse Genotyping Facility
Proteomics Facility (Director, Carol Parker, PhD)
The UNC-Duke Michael Hooker Proteomics Center, created in January 2002 to assist researchers at UNC and (since July 2005) at Duke, in identifying and characterizing proteins, is managed by Dr. Carol Parker and overseen by Dr. Xian Chen (both of whom hold faculty appointments in Biochemistry & Biophysics). In combination, Drs. Parker and Chen have over 40 years of mass spectrometry and protein research experience. Facility staff currently includes five post-doctoral research associates, two research technicians, and an IT specialist. In addition to fee-for-service customers, the UNC-Duke Michael Hooker Proteomics Center is supported by research groups including the UNC Lineberger Comprehensive Cancer Center, the Specialized Cooperative Centers Program in Reproduction Research (an NIH funded consortium for whom the facility is the national proteomics facility), the Center for Gastrointestinal Biology and Disease, and CEHS.
The Proteomics Facility has acquired several new instruments—the ABI 4000 Q-TRAP is now interfaced to an Eksigent capillary LC (for high-sensitivity phosphorylation site determination and quantitation), and the Bruker Reflex III MALDI/TOF was replaced with a Bruker Ultraflex MALDI/TOF/TOF for high-sensitivity MALDI analyses.
Protein identification from gels is done routinely on our MALDI-TOF/TOF (ABI 4700). An off-line LC-MALDI interface [a Dionex ProBot coupled to capillary LC (HP1100 capLC)] is used for ultra-sensitive analysis of modification sites. A Quadruple/Time of Flight (Q-STAR) is used mainly for nano-electrospray, but has also an oMALDI interface. This instrument provides high mass accuracy molecular weight determination, as well as targeted modification site analysis. A Micromass Q/TOF API US (also a Quadruple/Time of Flight instrument) coupled on-line to a Waters capillary LC system is used for characterization of posttranslational modifications, and for performing high-sensitivity LC-MS/MS experiments. We are also the only US university with a 12T Fourier Transform Ion Cyclotron Resonance (the Apex QqFTICR, from Bruker Daltonics). This instrument is capable of “top down” proteomics technologies, and is interfaced to a Dionex 3000 LC instrument for ultrahigh-resolution biomarker studies. During the past year, a major initiative has begun in FT-based metabolomics as a joint effort between the proteomics and metabolomics facility and several CEHS members.
We currently have a ProXpress imaging camera for visible and fluorescently-stained gels, as well as access to a GE Heatlhcare Typhoon imager which we use for differential protein expression analysis. ProGenesis 2D analysis software is available for analyzing the gels in order to determine differential protein expression. A 2DiD (Leap Technologies) robotic gel processing workstation and three ProGest robotic digestors are used for cutting gel plugs and extracting proteolytic peptides. The facility provides services at different levels ranging from consultation, sample preparation to the final analysis and data interpretation. In collaborative effort students, post-docs or technicians from other departments are trained on site or in courses to aid interdisciplinary integration of proteomic technology. More details are listed on the facility web site at http://proteomics.unc.edu/cehs.html.
For data acquisition, handling, and processing, custom and commercial software programs have been implemented for protein and genome database searching on a multi-processor server supported by the UNC Center for Bioinformatics. Our site-licensed Mascot database search engine can process peak lists generated by each instrument, providing a common platform for interpreting experiments. For sample processing, data storage, and archiving, the facility has acquired a custom-built Laboratory Information Managing Systems from Applied Biosystems, allowing the access to the raw and processed data over the internet.
Metabolomics Facility (Director Thomas O’Connell, PhD)
The Metabolomics Facility began operation in the Fall of 2005 to provide a collaborative resource for high throughput metabolomics studies. The Metabolomics Facility focuses on NMR based methods to provide unbiased analyses of any of a wide variety of biofluids, cells and tissues. The major instrumentation in the Metabolomics Facility includes a 400 MHz multinuclear NMR spectrometer housed in 400 sq ft of space in room 4 of the Glaxo building. This instrument has a 100 sample automation robot to provide high throughput analyses of large sample sets. In addition to the standard inverse detection coil for 1H NMR analysis, a Protasis microcoil NMR probe will be installed in December 2006. This probe allows for sample volumes as low as 5 microliters to be analyzed. After concentration, samples with less than 10 nanomoles can be analyzed in approximately 20 minutes. The UNC Metabolomics Facility is one of only two academic metabolomics laboratories in the country to have such capacity. In October, a 600 MHz NMR instrument was purchased to accommodate the increase in number of projects and the need for higher field analyses. This instrument has three probes, the standard inverse detection probe, a direct detection probe, optimized for the detection of 13C for metabolic flux analyses and a magic angle spinning probe for the analysis of whole cell and tissue samples. This instrument has a 50-sample automation robot.
The Facility was initiated with funds from the North Carolina Biotechnology Center with matching funds from industry and the UNC School of Medicine and the UNC Graduate School. The Facility is equipped with lyophilizers, centrifuges and speed-vacs for processing biofluid and tissue samples. In addition the Facility can perform perfused cell studies using an NMR-compatible bioreactor (patented by Dr. MacDonald) and cell encapsulation technology. Surface coil probe construction for in vivo analyses can be carried out in Dr. MacDonald’s laboratory using an S-parameter network analyzer.
The Facility has acquired state of the art software for data acquisition and processing along with suites of both commercial and home written software for data reduction and pattern recognition analysis. In addition, it has a collaborative development agreement with Chenomx Inc. (Alberta, Canada) to develop and refine their NMR metabolite database software. This software provides a unique capability to efficiently and quantitatively profile specific metabolites by NMR. The statistical analyses of the facility are enhanced by a collaboration with Dr. Oleg Favorov, Assoc. Professor, UNC Department of Biomedical Engineering. Mr. Clarence Thomas, Informatics Core Directory of the General Clinical Research Center provides assistance with database management.
The newly available FTICR mass spectrometer in the Proteomics Facility is being used to develop FT-based metabolomics. The FTICR-MS is located in the same building as the metabolomics facility. Because of limited availability the center is currently evaluation several mass spectrometric platforms that will in future complement the NMR base metabolomic studies. It is planed for 2007 to submit an joint instrumentation grant to NIH and the NCBC to obtain funding for an MS platform that will be dedicated for metabolomic analyses.
Mass Spectrometry Facility (Director Gunnar Boysen, PhD)
The Mass Spectrometry Facility provides qualitative and quantitative analyses to CEHS members. The analyses and services provided enhance epidemiology and toxicology research among UNC scientists investigating various molecular mechanism of environmentally related disease processes. The facility also provides expertise for quantitative measurement of DNA and protein adducts as markers of exposure, oxidative stress and endogenous damage. Such biomarkers are important for evaluating gene-environment interactions.
The UNC Biomarker Mass Spectrometry Facility operates two ThermoFinnigan TSQ 7000 triple quad mass analyzers: one is mainly operated in GC mode and the other is set up for micro LC-MS and MS/MS analyses; and a ThermoFinnigan LCQDeca ion trap mass analyzer equipped with an LC. Further, two new Quantum Ultra triple quad mass analyzers (Thermo) have been purchased and installed. One is equipped with an regular UPLC (Waters) and the other with a nano UPLC (Waters) liquid chromatograph allowing ultra high pressure liquid chromatography at flow rates between 200 nL and 2 ml per minute. Lastly, the facility houses a Thermo TSQ-Quantum Classic triple quad mass analyzer equipped with an Agilent 1100 capillary LC system (including autosampler and diode array detection) as part of Dr. Swenberg’s Lab equipment. In addition the facility has several HPLC units available with multi-channel ECD, fluorescence, UV, and diode array detection to aid characterization of standard and method development.
During most of 2006 the Mass Spectrometry Facility was in a temporary location due to renovation and asbestos removal. It will be relocated early 2007. The renovated Facility will consist out of an analysis laboratory, a sample preparation laboratory, technical appliance room and an office. The suite will have uninterruptible power, central vacuum and nitrogen systems for continuously supply of power, vacuum and ultra-pure nitrogen. The n vacuum system will reduce noise and improve air quality in the laboratories.
A dedicated server for file storage and back-up has been set-up. Newly generated data are backed up from instrument PCs daily and stored for 2 years. In addition, space is reserved to store critical data on a separate hard drive for up to 5 years. After that, the data is moved to long tern storage by burning on DVDs. The server can be accessed from office PCs with read-only permission in order to prevent possible deletion or overwriting of data and intrusion from the Internet. This permits researchers to analyze data from office PCs, without interrupting ongoing instrument operations.
Please visit the CEHS Facility Core Service Request System (at http://cehs.sph.unc.edu/srs) for more information.
DNA Damage (Slot-blot) Facility (Director Jun Nakamura, PhD)
The DNA Damage Facility operates a Kodak Imager, plate reader and equipment for slotblot assays. It provides slotblot assays for aldehydic DNA lesions such as abasic sites, as well as real time real time assay to monitor single strand breaks in DNA by indirectly measuring PARP-1 activation through depletion of intracellular NAD(P)H.
From 12/16/2005 to 12/15/2006, Slot-Blot (DNA damage) Facility provides DNA/protein extraction, abasic site assay, and DNA damage response analysis using DT40 cell model system to members of the CEHS center at UNC as well as Lovelace Respiratory Research Institute, NM (NIEHS Center). During this year our Facility set-up and utilize a powerful model system consisting of a series of isogenic chicken B-lymphocyte DT40 cell lines lacking various cell cycle checkpoint and DNA repair genes to determine the vital proteins necessary for cells to respond to environmental mutagens. The DT40 cell system has been widely used by genetic researchers for more than a decade. Since DNA damage response systems in DT40 cells appear to be quite similar to that in human cells, the DT40 model system has a great advantage over yeast models for the functional investigation of the DNA damage response by genetic approaches. Therefore, our DNA damage response analysis using DT40 cell system is an excellent tool to identify essential genes to counteract against environmental carcinogens and the assays are ready to be run for members of the CEHS.
Microarray Facility (Director Ivan Rusyn, PhD)
The Microarray Facility provides investigators with low cost access to high quality commercial microarrays that have been customized in the areas of toxicology and cancer research. The facility consolidates expensive equipment required for gene expression experiments and well-trained personnel required for microarray research. It collaborates closely with the UNC Center for Bioinformatics and provides a seamless interface to the UNC Microarray Database (UNC MD - https://genome.unc.edu).
Recent progress in sequencing human and mouse genomes, as well as advancements made in sequencing of other model organisms, allows the analysis of gene expression on a nearly genome-wide scale. While microarray technology is widely used, the field is notorious for multiple array formats and competing technologies that can overwhelm the investigators new to this technology. Our group has intimate knowledge of all widely-used array formats and can offer its expertise to other investigators. The Consortium's effort to standardize gene expression profiling experiments has shown that great accuracy and reproducibility in microarray experiments across different laboratories can be achieved through the use of SOPs, standardized equipment, and high quality commercial arrays.
The Microarray Facility is located in the Michael Hooker Research Center and includes an Agilent array scanner interfaced to a dedicated computer for running the latest version of the feature extraction software and depositing the results into UNC MD, a hybridization oven and rotator customized for several array formats, a high speed centrifuge with interchangeable rotors, and other small items required for processing microarrays. The RNA isolation and quality check can be performed at the core as well, and the use of Agilent BioAnalyzer is required for QA&QC purposes and minimizing the waste of reagents and microarrays.
The Facility also provides scientific expertise in small-scale quantitative gene expression techniques like multi-probe RNase Protection Assays (RPA) and Quantitative Real Time PCR (QRT-PCR). These techniques allow the analysis and/or verifyification of expression changes for up to several hundred genes as a follow-up to microarray analysis or in unrelated experiments. The Core is equipped with electrophoresis equipment required for RPA, a phosporimager to acquire data and appropriate image analysis tools for data extraction.
Mouse Genotyping Facility (Director David Threadgrill, PhD)
The Mouse Genotyping Facility is being develop to support small-to-medium size genotyping projects including that needed for management of colonies of research animals carrying various engineered mutations used for CEHS-related research and crosses to develop congenic lines or to localize genetic factors controlling inter-individual response to environmental exposures. The Facility has been developing a standardized platform in collaboration with BioTrove in order to offer a highly affordable system using the OpenArray platform based upon a miniturized version of TaqMan (20nl reaction volumes). Almost 200 assays have been developed and are currently being assembled the standardized genotyping platform. Concurrently, the core is developing tissue samples preparation SOPs , whose processing is based upon a Qiagen BioRobot 3000 to prepare DNA. Laslty, a server will be used to upload genotypes where they can be remotely accessed by the individual users. The first standardized array will be available in March, 2007, shortly after delivery of the new OpenArray NT Imager.
