Facilities
Laboratories and research facilities available to PNNL staff engaged in environmental biomarkers research include various wet and dry laboratory facilities, combinations of equipment and approaches to biosignature discovery, assessment, validation, and implementation.
Laboratory capabilities include: protein labeling, gel imaging, spot detection image analysis, spot excision, sample prep, MALDI spotting, and MALDI ToF. The laboratory will focus on toxicoproteomics, and use global protein expression technologies to help understand the intrinsic (e.g., genetic, metabolic) and extrinsic (e.g., environmental) factors associated with acute and chronic exposure to toxicants and their contributions to chronic health effects and disease.
The primary goal of the laboratory will be the discovery of biomarkers in organs, tissue, serum, and other body fluids that reflect toxic exposures and/or adverse effects associated with such exposures. The secondary goal will be to analyze, interpret, and integrate genomics, transcriptomics, proteomics, and metabolomics data to better understand biological mechanisms of action and predict individual health risks.
Complementary facilities located throughout PNNL/Battelle provide capabilities used to support the discovery, analysis, validation and implementation of environmental biomarkers. Click on the listed capabilities for more information:
Nuclear Magnetic Resonance
PNNL staff use nuclear magnetic resonance (NMR) and electronic paramagnetic resonance (EPR) instruments in the High-Field Magnetic Resonance Facility. Ongoing research utilizes high-resolution spectroscopy of biological objects where slow (1 to 100 Hz) magic angle spinning is being used to discover and study biomarkers (e.g., pulmonary phospholipidosis accumulation) in live animals in real time.
Mass Spectrometry
PNNL's High-Performance Mass Spectrometry Facility with its cutting-edge mass spectrometry methods focus on global proteomics research and the Accurate Mass and Time (AMT) Tag process, and is being used to identify protein biomarkers. Capabilities allow visualization and analyses of cell proteins in great detail. State-of-the-art instruments are available for challenging research in proteomics, cell signaling, cellular molecular machines, and high-molecular weight systems.
Proteomics (top down & bottoms up approaches). Two approaches to proteomics analysis are supported at PNNL. The top down approach or intact protein analysis uses 2-dimensional gel electrophoresis and utilizes large amounts of sample, has a dynamic range of 10^4 and femtomole sensitivity. This approach is supported by LC-MS/MS, MALDI MS/MS and LC-FTMS, HPLC, MALD TOF/TOF. The bottoms up or peptide analysis (proteolyzed proteins) approach uses LC-FTICR and AMT tag approach, has ultra-high resolution, analyzes minute amounts of sample, has a dynamic range of 10^6 and a zetomole sensitivity. Methods are selected based upon desired outcomes.
Genomics
The lab utilizes Affymetrix arrays, Universal Fingerprint arrays, organism-specific microarrays, quantitative RT-PCR in genomic studies.
Proteomics (top down & bottom up approaches)
Two approaches to proteomics analysis are supported at PNNL. The top down approach or intact protein analysis uses 2-dimensional gel electrophoresis and utilizes large amounts of sample, has a dynamic range of 10^4 and femtomole sensitivity. This approach is supported by LC-MS/MS, MALDI MS/MS and LC-FTMS, HPLC, MALD TOF/TOF. The bottom up or peptide analysis (proteolyzed proteins) approach uses LC-FTICR and AMT tag approach, has ultra-high resolution, analyzes minute amounts of sample, has a dynamic range of 10^6 and a zetomole sensitivity. Methods are selected based upon desired outcomes.
Metabolomics
PNNL has developed methods to identify metabolome components and mass and time tags for highly sensitive, rapid metabolite detection and profiling.
Cell Signaling
Scientists are working to understand how cell signaling networks integrate with competing environmental stimuli to promote a range of cellular responses that underlie adaptive and pathologic behavior. Understanding the mechanisms will aid in identifying and detecting biomarkers of influence in these signaling pathways.
Biological Monitoring and Modeling
While analyses of various excised and intact tissues are performed routinely at the lab, more recently the focus has been on non-invasive biomonitoring (e.g., breath, urine and saliva). Modeling of biological structure and physiological function vary from the simple to the more complex (e.g., virtual respiratory tract). For deployment tools, sensor systems have been developed to address biomonitoring of both non-volatiles and volatiles.
Sensor Development
Integrated microfluidics and electrochemical sensor platforms, 3-D.
Computational, bioinformatics and Visualization Tools
PNNL is developing new approaches to handling the large amounts of data generated as part of the biosignature discovery process and the integration of that data into complex models. Researchers are in the midst of developing common databases and interfaces that will be linked into collaborative problem-solving environments. One such tool, OmniViz, has been developed to mine biology information. Another tool used includes ProMat, where the combination of the ELISA microarray platform with the ProMAT data analysis tool provides a powerful approach for the rapid, sensitive and high-throughput measurement of proteins in complex biological samples.
Inhalation Toxicology
In conjunction with Battelle Toxicology Northwest, unique capabilities in inhalation toxicology and pulmonary dosimetry exist on the PNNL campus. Generated aerosols are characterized for exposure concentration and particle size distribution and controlled using real-time monitoring, including optical light scattering instrumentation and particle mass monitors. Acute, subchronic, and chronic inhalation toxicity studies of new pharmaceutical and biopharmaceutical candidates are performed, and more recently toxic biological and chemical agents have been studied.
Microarray Technologies
PNNL has several efforts in microarray development. An example includes a protein ELISA (Enzyme-linked immunosorbent assay) microarray platform that is flexible, highly sensitive and capable of high-throughput protein measurements. The high sensitivity of this assay platform is due to the use of separate antibodies for capture and detection along with the integration of an amplification step for enhanced detection sensitivity. In order to efficiently evaluate the large data outputs from ELISA microarray experiments a bioinformatics program, Protein Microarray Analysis Tool (ProMAT) was developed. Another application of PNNL's microarray technology includes a salmonid-based DNA microarray for testing of exposure to contaminants in salmon populations.
Ecotoxicology
Notes: Toxin screening using reporter gene assays, phytoplankton analysis and phytoplankton species identification using MALDI-MS, endocrine disruption studies, micro-algae screening for maximum CO2 fixation, mesocosms.
Mesocosms
Mesocosms have been constructed at PNNL to study eelgrass propagation and growth, fisheries issues, contaminant flux and transport, and instrument development and testing. A hyporheic mesocosm will be constructed to conduct controlled laboratory-based biomarker studies and investigate the physical and biological dynamics of hyporheic zones as they relate to contaminants in surface waters from ground water sources.
