1. Metabolomics and Proteomics Core Facilities are composed of several major operations that involve a variety of expertise for metabolomic and proteomic analysis. Each laboratory provides complementary expertise that globally serves to enhance the research capabilities of the Life Science research community on campus and in the region. The Bindley Bioscience Center provides research administrative oversight to coordinate the functional linkages between these various operations and to ensure the continued improvement of the technical capabilities. OVERVIEW RESOURCES RESEARCH CONTRIBUTION HIGHLIGHTLIST OF SERVICES QUALITY CONTROL AND ASSURANCES CONTACT INFORMATION Metabolomics and Proteomics Core Centers High throughput protein/metabolite profiling with LC-MS n or GCxGC- MS Various off-line HPLC separations and molecule isolation Identification and characterization of molecules Automated/manual sample preparation and Solid Phase Extraction (SPE) Automated/manual computer search and data analyses Methods development and consultation Bioinformatic consultation Complete training in metabolomics/proteomics technologies Proteomics and Metabolomics Facilities share roughly 3,000 ft 2 space of the BIND, HORT, and HANS buildings at Purdue University. Major equipment includes: Waters Micromass Q-TOF micro coupled to HPLC (equipped with regular and nano electrospray ionization source; capable of MS/MS analyses). The instrument is a high resolution time-of- flight (TOF) mass spectrometer that enables automated exact mass measurements. The instrument also features a quadrupole mass filter and collision cell for MS/MS analyses. LECO Pegasus 4D GCxGC-TOF (capable of MS analyses) enables to detect hundreds-to-thousands more compounds than previously seen using conventional GC techniques. Powerful, easy-to-use Windows ® -based ChromaTOF ® software simplifies component identification providing a significant increase in efficiency and productivity. Agilent MSD/TOF coupled to HPLC (equipped with regular and nano electrospray ionization source; capable of MS analyses). The MSD TOF offers outstanding mass resolution and accuracy. Waters Micromass LCT Premier (TOF) coupled to HPLC (equipped with regular and nano electrospray ionization source; capable of MS analyses). The instrument is equipped with W- Optics, novel method for enhancing resolution without increasing footprint. Agilent nanoLC-ion trap XCT plus can perform multiple MS/MS scan with rapid scanning speed and very high sensitivity. The XCT plus is coupled to the Chip Cube system for low sample volume analyses. AB QSTAR Pulsar (Q/TOF) coupled to nanoUPLC (Waters) combines ESI ionization with the hybrid quadrupole TOF analyzer for multi charge, high resolution analysis. AB 4800 MALDI TOF/TOF provides high sensitivity and mass accuracy. In addition, since there is usually only a singly charged ion formed, it is a good choice for the analysis of heterogeneous samples. Furthermore, the static nature of the sample allows for multiple evaluations on a single sample. Cellular response to stimuli is reflected by changes in concentration of metabolites and/or protein expression, post-translation modifications or post-translational processing of proteins. The identification of these changes is essential for understanding biological processes. In proteomics, large number of proteins from one or more samples is analyzed simultaneously and at least one or more quantitative and qualitative analyses are made on the sample components. Sample complexity generally varies from a few thousand to tens of thousands of proteins. It is recognized that complex samples require extensive separation before any quantitative and qualitative analyses can be used. The metabolome, on the other hand, is unique from the proteome in that it is not directly encoded by the genome. This makes the task for characterizing the metabolome more difficult because there is not a unique correspondence between the number of genes and metabolites present. Metabolomics and Proteomics Core Facilities use state-of-the-art technologies, develop new methods, and provide resources for both qualitative and quantitative analysis. Examples of ongoing projects include: Determination of Energy Related Metabolites by GC/MS and LC/MS (with Dr. Ho from Laboratory of Renewable Resources Engineering at Purdue University). To improve precision in absolute quantification, we have introduced a new post-biosynthetic stable isotope encoding concept called Group Specific Internal Standard Technology (GSIST). In GSIST, Metabolite standards and experimental samples are derivatized with two different labeling agents that are chemically identical but isotopically distinct (12C vs 13C). After mixing these derivatized metabolites, each molecule from control or standard sample serves as an internal standard for determining the concentration of the chemically identical component in experimental sample. This Method was used in studies focused on improvement of the conversion of lignocellulosic biomass to ethanol by recombinant saccharomyces yeast. Use of proteomics and metabolomics techniques for Biomarker Identification in Vertebrate and Invertebrate Species Exposed to Various Environmental Stressors (with Dr. Sepulveda from Forestry and Natural Resources at Purdue University). A 2DGE and GCxGC/MS analyses are utilized to evaluate proteomic and metabolomic changes unique to specific environmental stressors. Significant variations were observed in great blue heron (Ardea herodias) eggs exposed to varying polychlorinated biphenyl (PCB) concentrations, different populations of a freshwater amphipod, Diporeia, residing in Lake Michigan as well as Diporeia exposed to atrazine and its metabolite desethylatrazine (DEA). Oxidative Stress in Drosophila (with Dr. Pittendrigh from Department of Entomology at Purdue University). Novel proteomics and metabolomics approaches were developed for the quantification and identification of differentially expressed proteins and metabolites involved in energy metabolism of Drosophila exposed to oxidative stressors such as peroxide and methamphetamine. Lipidomic Profiling of Cells Infected by Dengue Virus (with Dr. Kuhn from Biological Sciences at Purdue University). Improved HPLC-MS approach was used for the identification of phospholipid biomarkers of Dengue virus infection. Of those, 23 phospholipids were up or down regulated in 80% of the experimental samples. Future work will expand to cholesterol and sphingolipid evaluations. Determination of Ganoderic Acids in Mushrooms as Potential Therapeutic Agents for Cancer (with Dr. Sliva from Indiana University). GSIST approach was used to identify active componds in Ganoderma mushroom extract These compounds are under investigation as potential cancer therapeutic agents. A protocol was also developed to evaluate ganoderic acids in rat plasma collected over 24 hours after ingestion and their adsorption profile was determined while simultaneously monitoring for related metabolites. The inclusion of mass spectral data from higher-throughput methods creates a bottleneck with laborious data management and analysis phases that are increasingly error prone due to the complexity of the information. To overcome this issue innovative and efficient bioinformatics infrastructure has been created at the Bindley for data management and data mining. The Purdue Discovery Pipeline (PDP) automatically performs online experimental data quality control. Data that meet quality standards are transferred into a common data format and archived in the central database. The user can build a data mining workflow from a pool of available informatics tools. Jiri Adamec, Ph.D. Purdue University Bindley Bioscience Center, BIND 112 1203 W. State Street West Lafayette, IN 47907 Email: jadamec@purdue.edu Phone: 765-496-6148 Fax: 765-496-1518