Transcript Metabolomics and Proteomics Center

Metabolomics and Proteomics Core Centers
RESEARCH CONTRIBUTION HIGHLIGHT
OVERVIEW
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.
RESOURCES
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Proteomics and Metabolomics Facilities share roughly 3,000
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-offlight (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 WOptics, 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.
LIST OF SERVICES
•High throughput protein/metabolite profiling with LC-MSn or GCxGCMS
•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
QUALITY CONTROL AND ASSURANCES
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.
CONTACT INFORMATION
Jiri Adamec, Ph.D.
Purdue University
Bindley Bioscience Center, BIND 112
1203 W. State Street
West Lafayette, IN 47907
Email:
Phone:
Fax:
[email protected]
765-496-6148
765-496-1518