Integrated Technologies

Research Emphasis

This center offers a suite of advanced sensor technologies designed to follow the dynamic properties of living cells, particularly as these relate to molecular transport and pathophysiology. Developed in-house are several electrochemical noninvasive microsensors relying on drift and noise reduction by using a modulation technique, termed self-referencing. Using differential signal analysis, the center can measure chemical gradients surrounding single cells and tissues. Sensor applications using voltammetry and amperometry are available for the detection of ions (e.g., potassium, hydrogen, and calcium) as well as electrically active molecules (e.g., oxygen, nitric oxide, and hydrogen peroxide). Recently the center has developed self-referencing sensors incorporating enzymes, allowing the detection of molecules such as glucose, lactate, and glutamate.

Current Research

Instrument development includes improvement of the existing electrochemical systems, all targeted to operate at the level of a single cell. Additionally, the center is working on inclusion of more complex electrochemical detection techniques; fusion of sensor modalities in electro-optical systems; faster signal processing techniques; transporter expression and biophysical analysis in expression systems and bilayers; and probe development for air-exposed surfaces.

The center also supports most conventional electrophysiological techniques working in conjunction with electrochemical detection. It also supports ratio imaging. Biological studies focus on the role of molecular transport in cell physiology and development with an emphasis on characterizing transport mechanisms and their role in basic biology and disease.

Resource Capabilities

Instruments

Resource staff design and manufacture amplifiers, micro-stepper motion controllers, and manipulators. There are seven experimental platforms of which five have compound microscopes, four inverted and one upright (Zeiss AxioScope). One Axiovert is equipped with an Attofluor Ratio imaging system. All platforms can be temperature-controlled. More than one detection system can be operated at a time. Culture facilities also are available for both mammalian and nonmammalian systems. Visiting investigators also have access to the extensive facilities of the Marine Biological Laboratory (MBL). This includes advanced imaging techniques such as multiphoton microscopy.

Software

Operating software: All experimental platforms run on PC Pentiums. The data collection and motion-controlling program (IonView written and maintained in-house) runs through Windows 98 and NT. All computers are linked to a local area network and the outside via the MBL server.

Informatics: As a service to the community, the center maintains a database of compounds targeting diverse aspects of cellular dynamics, from transporters to metabolism. Access is via the web site and free to all users.

1. Trimarchi, J. R., Liu, L., Smith, P. J. S., and Keefe, D. L., Apoptosis recruits two-pore domain potassium channels used for homeostatic volume regulation. American Journal of Physiology. Cell Physiology 282:C588–C594, 2002.
2. Boudko, D. Y., Moroz, L. L., Harvey, W. R., and Linser, P. J., Alkalinization by chloride/bicarbonate pathway in larval mosquito midgut. Proceedings of the National Academy of Sciences USA 98:15354–15359, 2001.
3. Jung, S.-K., Trimarchi, J. R., Sanger, R. H., and Smith, P. J. S., Development and application of a self-referencing glucose microsensor for the measurement of glucose consumption by pancreatic b-cells. Analytical Chemistry 73:3759–3767, 2001.

Research Emphasis

The primary focus of the Integrated Technology Resource for Biomedical Glycomics is the development of technologies to analyze and influence glycoprotein and glycolipid glycosylation found in animal cells. The resource has particular interest in developing tools and techniques to analyze glycosylation during animal cell differentiation and development, as well as during oncogenesis and tumor progression. The broad goal of these studies is to develop the means to describe the patterns of glycosylation changes during these differentiation events and determine if glycosylation inhibitors can influence differentiation pathways.

Current Research

The resource focuses on the early stages of embryonic mouse stem cell (ES) differentiation as the primary system of study for technology development. Glycoprotein and glycolipid glycosylation is being characterized during differentiation of ES cells into cells in the ectodermal primitive layer and during further differentiation into neuronal precursor cells. Glycoprotein analysis will identify N- and O-linked glycans and the proteins to which these glycans are attached using LC/MS-MS proteomic techniques coupled to serial lectin affinity chromatography schemes. In addition, sphingolipid and glycosphingolipid structures will be characterized to identify changes during differentiation. Changes in particular glycosyltransferases during differentiation will be determined by glycochip analysis, followed by quantification by kinetic PCR experiments. A bioinformatics component will integrate the glycosylation information obtained, as well as synergize with other glycodatabases being developed around the world.

Resource Capabilities

Instruments

Hewlett-Packard GC-MS instruments, DIONEX HPAEC system, Applied Biosystems Voyager DE MALDI-MS mass spectrometer, Metrohm-Peak HPAEC system, Thermo Finnegan LCQ and LTQ Advantage mass spectrometers.

Software

Innovative software is currently under development.

Service and Training

Services offered include initial characterization of glycoprotein and glycosphingolipid glycans, including estimation of sample purity, glycosyl composition analysis, glycosyl linkage analysis, and molecular weight determinations using gas-liquid chromatography/mass spectrometry; electrospray ionization, fast-atom bombardment, matrix-assisted laser desorption/ionization, time-of-flight, and tandem mass spectrometry. Proteomic analysis of N-glycosylated glycopeptides also is available. More sophisticated, customized, or nonroutine service can be individually designed and performed as a collaborative investigation. Several workshops emphasizing methodology for glycoconjugate analysis are offered each year, and participants come from both industry and academia.

Research Emphasis

The Laboratory of Neuro Imaging Resource (LONIR) develops novel strategies to investigate brain structure and function in their full multidimensional complexity. There is a rapidly growing need for brain models comprehensive enough to represent brain structure and function as they change across time in large populations, in different disease states, across imaging modalities, across age and gender, and even across species. International networks of collaborators are provided with a diverse array of tools to create, analyze, visualize, and interact with models of the brain. A major focus of these collaborations is to develop 4-dimensional brain models that track and analyze complex patterns of dynamically changing brain structure in development and disease, expanding investigations of brain structure-function relationships to four dimensions.

Current Research

The development of modeling approaches focuses on new strategies for surface and volume parameterization that provide an advanced analysis of surface and volumetric brain models, tracking their change across time. Additional research cores focus on anatomical fundamentals, analyzing anatomical and cytoarchitectural attributes across multiple scales and across time. Another core focuses on visualization and animation, for the dissemination of brain models that visualize complex variations in brain structure and function across time. Specialized approaches are under development for handling cortical data. Ongoing national and international collaborations are analyzing normal and aberrant growth processes, brain development, tumor growth, Alzheimer's disease and related degenerative disease processes, schizophrenia, and brain structure in normal and diseased twins.

BIRN

Resource Capabilities

Computer Resources

The LONIR relies on a SGI Onyx2 Reality Monster with 32 processors and 4 InfiniteReality2 pipes to drive graphics-intensive applications and interactive multidimensional visualization of structural models and datasets. For computer-intensive operations, the laboratory relies on a 64-processor SGI Origin 3800, a 32-processor SGI 1200 cluster, a 32-processor SGI Origin 3900, and a 24-processor SUN Sunfire 6800. In addition, a 6-processor SGI Onyx2 with a single InfiniteReality2E pipe serves a backup graphics mainframe.

The resource has made a decisive move towards highly available systems design to ensure maximum uptime. To that extent, the laboratory uses a fault-tolerant storage area network (SAN). The SAN hardware infrastructure is composed of the supercomputers above, RAID5 storage, a 360-terabyte StorageTek robotic tape silo, and three 400-megabyte-per-second Brocade fiber channel switches. Two dual 360-MHz MIPS-R12000 processor SGI Origin 200 servers mediate all data transactions and provide networking services.

Numerous desktop workstations are networked to the SAN via switched 100baseT ethernet. 4 HP laser printers, 1 Tektronix Phaser 560 color printer, 1 HP DesignJet 3500CP poster printer, and a publication-quality Fujix Pictography 3000 color printer facilitate document preparation.

1. Toga, A. W. and Thompson, P. M., New approaches in brain morphometry. American Journal of Geriatric Psychiatry 10:13–23, 2002
2. Thompson, P. M., Cannon, T. D., and Toga, A. W., Mapping genetic influences on human brain structure. Annals of Medicine 34:523–526, 2002.
3. Narr, K. L., Cannon, T. D., Woods, R. P., Thompson, P. M., et al., Genetic contributions to altered callosal morphology in schizophrenia. Journal of Neuroscience 22:3720–3729, 2002.

Research Emphasis

The Neuroimaging Analysis Center (NAC) develops image processing and analysis techniques for basic and clinical neurosciences. The NAC research approach emphasizes both specific core technologies and collaborative application projects.

The core activity of the NAC is the development of algorithms and techniques for postprocessing of imaging data. New segmentation techniques aid identification of brain structures and disease. Registration methods are used for relating image data to specific patient anatomy or one set of images to another. Visualization tools allow the display of complex anatomical and quantitative information. High-performance computing hardware and associated software techniques further accelerate algorithms and methods. Digital anatomy atlases are developed for the support of both interactive and algorithmic computational tools. Although the emphasis of the NAC is on the dissemination of concepts and techniques, specific elements of the core software technologies have been made available to outside researchers or the community at large.

The NAC’s core technologies serve the following major collaborative projects: Alzheimer’s disease and the aging brain, morphometric measures in schizophrenia and schizotypal disorder, quantitative analysis of multiple sclerosis, and interactive image-based planning and guidance in neurosurgery. One or more NAC researchers has been designated as responsible for each of the core technologies and the collaborative projects.

BIRN

Resource Capabilities

Hardware

The research work of the NAC is supported by a large, state-of-the-art network of more than 70 UNIX workstations, a central application and file server, and several enterprise-class computers from Sun Microsystems connected by Gigabit Ethernet. Storage needs are met by a high-reliability network-attached storage device from Procom Technology that holds 2.5 terabytes of data. The dissemination component of the NAC is achieved through a web-centric approach. The Internet is used to disseminate results and other documentation, for the distribution of software, and for data exchange with outside collaborators.

Software

The core technologies of NAC consist of custom, internally developed software algorithms, libraries, and applications that are available to support collaborating projects. When possible, this software is built on open software standards.

1. McCarley, R. W., Salisbury, D. F., et al., Association between smaller left posterior superior temporal gyrus volume on magnetic resonance imaging and smaller left temporal P300 amplitude in first-episode schizophrenia. Archives of General Psychiatry 59:321–331, 2002.
2. Warfield, S. K., Talos, F., Tei, A., Bharatha, A., Nabavi, A., Ferrant, M., et al., Real-time registration of volumetric brain MRI by biomechanical simulation of deformation during image guided neurosurgery. Computer Visual Science 5:3–11, 2002.
3. Wei, X., Warfield, S. K., et al., Quantitative analysis of MRI signal abnormalities of brain white matter with high reproducibility and accuracy. Journal of Magnetic Resonance Imaging 15:203–209, 2002.
4. Westin, C.-F., Maier, S. E., et al., Processing and visualization for diffusion tensor MRI. Medical Image Analysis 6:93–108, 2002.

Research Emphasis

This resource focuses on assisting biomedical researchers through the development and application of advanced proteomics technologies designed for the characterization of biomedically important systems, including pathogenic microbes, viruses, animal models, human tissues, fluids, cultured cell lines, and clinically obtained samples. Unique capabilities include nanoscale sample handling combined with high-throughput, high-pressure, high-resolution capillary liquid chromatography (LC) coupled to electro-spray ionization interfaced high-sensitivity, wide-dynamic-range Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers (MS).

Current Research

Development and application of new MS instrumentation and online and offline LC separation methods for the characterization of highly complex proteomic systems, including peptide/protein identification and quantitation methods; characterization methods implement bottom-up and top-down approaches to facilitate identification of protein posttranslational modifications. The development and application of single-chain antibody technology for the characterization and quantitation of posttranslational modifications and for the enrichment of specific protein-protein complexes. Development of new software tools to facilitate the quantitation, validation, and interpretation of proteomic data.

Resource Capabilities

Instruments

Five FT-ICR mass spectrometers have been developed or modified, including an instrument with world's highest magnetic field (based around an 11.5 tesla superconducting magnet) high-performance FT-ICR, as well as 7, 9.4, and 12 tesla FT-ICR instruments. A Micromass Ultima QTOF, a QSTAR Hybrid LC/MS/MS System, a Thermoquest MAT TSQ 7000, a PE Sciex API 3000, and six Thermoquest LCQ quadrupole ion trap mass spectrometers complete the instrumentation suite. These instruments are equipped with automated high-pressure capillary LC systems and in-house electrospray or nanospray ionization interfaces for ultra-high sensitivity.

Software

In-house developed ICR-2LS is publicly accessible at the web site.

Special Features

Automated, high-throughput, ultra-sensitive, wide-dynamic-range proteome characterization of microbial, mammalian, and viral systems is afforded by the instrumentation suite at the Pacific Northwest National Laboratory.

1. Shen, Y., Jacobs, J. M., Camp II, D. G., et al., High efficiency SCXLC/RPLC/MS/MS for high dynamic range characterization of the human plasma proteome. Analytical Chemistry In press.
2. Blonder, J., Goshe, M. B., Xiao, W., et al., Global analysis of the membrane subproteome of Pseudomonas aeruginosa using liquid chromatography-tandem mass spectrometry. Journal of Proteome Resesearch In press.
3. Shen, Y., Tolic, N., Masselon, C., et al., Ultrasensitive nanoscale proteomics using high-efficiency on-line micro-SPE-nanoLC-nanoESI MS and MS/MS. Analytical Chemistry November 25, 2003.
4. Jacobs, J. M., Mottaz, H. M., Yu, L.-R., et al., Multidimensional proteome analysis of human mammary epithelial cells Journal of Proteome Research November 5, 2003.
5. Lipton, M. S., Pasa-Tolic, L., Anderson, G. A., et al., Global analysis of the Deinococcus radiodurans proteome by using accurate mass tags Proceeding of the National Academy of Sciences USA 99:11049-11054, 2002.
6. Smith, R. D., Anderson, G. A., Lipton, M. S., et al., An accurate mass tag strategy for quantitative and high-throughput proteome measurements Proteomics 2:513-523, 2002.

Research Emphasis

The central goal of the Resource Center for Biomedical Complex Carbohydrates is to increase understanding of the molecular basis of the involvement of carbohydrates in protein-carbohydrate interactions and to develop more powerful technologies necessary to achieve this goal. Complex carbohydrates play an important role in many biomedical processes, including inflammatory response, hormone action, malignancy, viral and bacterial infections, and cell differentiation. As new technologies are developed, application of these processes is pursued through collaborative and service projects.

Current Research

Two broad goals of current research are developing integrated technologies to study oligosaccharide-protein interactions, and developing and applying new methodologies for the structural analyses of glycoconjugates. This includes developing molecular biological methods for the identification and expression of carbohydrate-binding domains of proteins; developing physical methods to elucidate the structural and energetic basis of carbohydrate recognition; testing these methodologies in two lectin systems (the galectin-1 and the Xenopus laevis oocyte lectin systems); optimizing and developing mass spectrometric methods for the structural analysis of saccharides; and using FT-IR to obtain rapid and accurate determination of mono- and oligosaccharide sugar composition. Physical methods development includes advancing mass spectrometric techniques to aid in characterization of structural properties of multimeric lectins and identification of ligand-binding sites in these lectins, computational modeling methods to more accurately predict bound geometries for novel ligands and provide a rational basis for ligand design, biosynthetic and synthetic schemes to produce a library of potential ligands, and NMR structural methods specifically targeted to characterization of the binding sites of carbohydrate-recognizing proteins and the bound state of carbohydrate ligands.

Service and Training

Services offered include initial characterization of complex carbohydrates, including estimation of sample purity, glycosyl composition analysis, glycosyl linkage analysis, and molecular weight determinations using gas-liquid chromatography/mass spectrometry; electrospray ionization, fast-atom-bombardment, matrix-assisted laser desorption/ionization, time-of-flight, and tandem mass spectrometry; NMR analysis using 500-, 600-, and 800-MHz ¹H- and ¹³C NMR spectroscopy. More sophisticated, customized, or nonroutine services can be individually designed and/or set up as a more extensive, collaborative investigation. Several workshops emphasizing methodology for glycoconjugate analysis are offered yearly.

1. Arranz-Plaza, E., Tracy, A. S., Siriwardena, A., Pierce, J. M., and Boons, G. J., High avidity, low affinity multivalent interactions and the block to polyspermy. Journal of the American Chemical Society 124:13035–13046, 2002.
2. King, D., Bergmann, C., Orlando, R., Benen, J.A.E., Kester, H.C.M., and Visser, J., The use of amide exchange-mass spectrometry to study conformational changes within the endopoly-galacturonase II/homogalacturonan/ polygalacturonase-inhibiting protein system. Biochemistry 41:10225–10233, 2002.
3. Clarke, C., Woods, R. J., Gluska, J., Cooper, A., Nutley, M. A., and Boons, G. J., Involvement of water in carbohydrate-protein binding. Journal of the American Chemical Society 123:12238–12247, 2001.
4. Jain, N. U., Venot, A., Umemoto, K., Leffler, H., and Prestegard, J. H., Distance mapping of protein-binding sites using spin labeled oligosaccharide ligands. Protein Science 10:2393–2400, 2001.

Research Emphasis

The complete genome sequence of the yeast Saccharomyces cerevisiae has identified approximately 6,000 genes, almost a third of which have not been assigned a function. The concept of this center is to provide a central resource to focus complementary technologies on the analysis of yeast protein complexes using the methods of mass spectrometry, the two-hybrid system, microscopy, and protein structure prediction. Mass spectrometry is used to identify the proteins present in complex mixtures from cells and the two-hybrid assay finds interacting partners for specific proteins. Microscopy is used to analyze interactions within living cells by fluorescent resonance energy transfer. Protein structure prediction predicts the structure of proteins for which no structural information exists. These technologies can be applied to identify new functions for previously characterized yeast proteins, to implicate novel proteins in various biological processes, and to uncover links between different processes. The center collaborates with yeast biologists working in such research areas as the development of cell polarity, transcriptional control, signaling pathways, response to DNA damage, and protein degradation.

Research Capabilities

Instruments

Finnigan MAT LCQ electrospray ion trap mass spectrometers, MALDI source for ion trap mass spectrometer, HPLC and autosampler for mass spectrometer, ABI Integral multi-dimensional HPLC system, Applied Biosystems MALDI-Q-Star mass spectrometer, Biomek 2000, Deltavision optical sectioning microscope.

Software

SEQUEST software for database searching using MS/MS spectra of peptides; Autoquest software for protein complex identification using SEQUEST; SEQUEST summary.

1. Cheeseman, I., Anderson, S., Jwa, M., Green, E., Kang, J.-S., Yates, R., Chan, C., Drubin, D., and Barnes, G., Phospho-regulation of kinetochore-microtubule attachments by the Aurora kinase lpl1p. Cell 111:163–172, 2002.
2. Drees, B. L., Sundin, B., et al., A protein interaction map for cell polarity development. Journal of Cell Biology 154:549–571, 2002.
3. Tong, A. H., Drees, B., Nardelli, G., Bader, G. D., Brannetti, B., et al., A combined experimental and computational strategy to define protein interaction networks for peptide recognition modules. Science 295:321–324, 2002.
4. Boddy, M. N., Gaillard, P. H., McDonald, W. H., Shanahan, P., and Yates, J. R. 3rd, Mus81-Eme1 are essential components of a Holliday junction resolvase. Cell 107:537–548, 2001.