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Genomes to Life Contractor-Grantee Workshop I
Arlington, Virginia, February 9-12, 2003

A6
Bioinformatics and Computing in the Genomes to Life Center for Molecular and Cellular Systems

D. A. Payne[1] (debbie.payne@pnl.gov), E. S. Mendoza[1], G. A. Anderson[1], D. K. Gracio*[1], W. R. Cannon[1], T. P. Straatsma[1], H. J. Sofia[1], D. A. Dixon*[1], M. Shah[2], D. Xu[2], D. Schmoyer[2], S. Passovets[2], I. Vokler[2], J. Razumovskaya[2], T. Fridman[2], V. Olman[2], A. Gorin[2], E. Uberbacher[2], F. Larimer[2], and Y. Xu[2]

*Presenters

[1]Pacific Northwest National Laboratory; and [2]Oak Ridge National Laboratory

Scientists will generate large amounts of experimental and computational data at the ORNL/PNNL Genomes to Life (GTL) Center for Molecular and Cellular Systems. Data will be generated at several collaborating facilities and will need to be shared among the collaborators and, ultimately, with the wider research community. The processing, analysis, management, and storage of this data will require a flexible, robust, and scalable information system. As the GTL project ramps up, many of the data and sample tracking and analysis functions will need to be automated and integrated to keep up with the high-throughput processes. Since the start of the project, our bioinformatics work has been focusing on three areas: 1) laboratory information management system (LIMS) in support of the Center’s data management and storage, 2) mass spectrometry proteomics analysis, and 3) bioinformatic analysis tools.

LIMS System

We have purchased a commercially available and proven LIMS system, Nautilus™ (from Thermo Lab Systems) to serve as the backbone for integrating data management and analysis. Nautilus, once configured, will provide comprehensive sample tracking from planning through experimentation, data analysis, reporting, and final archival or disposal. Nautilus will be interfaced with laboratory instruments and data analysis tools and services to enable automation and standardization of data processing. Data will be archived through integration with the Environmental Molecular Sciences Laboratory Northwest File System archive.

A key to the success of this project will be the ability for users to have ubiquitous, seamless access to LIMS data at both ORNL and PNNL. To accomplish this data sharing, a schema will be defined for components and workflow that are common to both facilities, and software will be written to access data from both instances of the LIMS system. Current activities include defining the overall system, defining the data management schema for the respective facilities at ORNL and PNNL, gathering requirements, and identifying common data structures.

Mass Spectrometry Proteomic Data Analysis

Before the GTL program started, PNNL developed the Proteomics Research Information System and Management (PRISM) system that stores, tracks pedigree of, and provides automated analyses of proteomic data. PRISM will be used both at PNNL and at ORNL for mass spectrometry data analysis. It is composed of distributed software components that operate cooperatively on several commercially available computer systems that communicate over standard network connections. PRISM collects data files directly from all mass spectrometers in the laboratory and manages storage and tracking of these data files as well as automates the processing into both intermediate results and final products.

PRISM will be installed at ORNL to provide a common proteomic data analysis capability. Additionally, a mass spectrometry data analysis pipeline for automated processing of large-scale mass spectrometry data of proteins and protein complexes has been designed and is in the early stages of implementation. The pipeline is designed to process data generated using both bottom-up and top-down approaches and to combine information derived from both approaches for identifying proteins and protein complexes. The pipeline builds a data interpretation capability based on three existing mass spectrometry data analysis software: SEQUEST, MASCOT, and COMET. These tools have been evaluated with systematic comparison using experimental data. Through these analyses, computational techniques have been developed for assessing the reliability of these identification tools. For example, in the case of SEQUEST, a neural network and a statistics-based method has been developed for such reliability assessment. Such a capability can significantly remove the need of human involvement in large-scale MS data interpretation. New methods for de novo sequencing that can complement database search-based methods for protein identification are also under development.

Bioinformatic Analysis Tools

In the area of bioinformatics, our project is focused in many areas: computational inferencing of protein complexes, including membrane-associated complexes, dynamic simulation of protein-protein interaction, and functional mechanism studies of protein complexes; characterizations of amino acids and peptide transport pathways; and identification of operons and regulons. Interactive analysis and visualization tools are being developed to support these goals.

This research is supported by the Office of Biological and Environmental Research of the U.S. Department of Energy. Pacific Northwest National Laboratory is operated for the U.S. Department of Energy by Battelle Memorial Institute through Contract No. DE-AC06-76RLO 1830.



A8
Mass Spectrometry in the Genomes to Life Center for Molecular and Cellular Systems

Gregory B. Hurst[1] (hurstgb@ornl.gov), Robert L. Hettich[1], Nathan C. Verberkmoes[1], Gary J. Van Berkel[1], Frank W. Larimer[1], Trish K. Lankford[1], Steven J. Kennel[1], Dale Pelletier[1], Jane Razumovskaya[1], Richard D. Smith[2], Mary Lipton[2], Michael Giddings[5], Ray Gesteland[4], Malin Young[3], and Carol Giometti[6]

[1]Oak Ridge National Laboratory; [2]Pacific Northwest National Laboratory; [3]Sandia National Laboratories; [4]University of Utah; [5]University of North Carolina; and [6]Argonne National Laboratory

Mass spectrometry is a significant contributor to the Center for Molecular and Cellular Systems due to its capability for high-throughput identification of proteins and, by extension, protein complexes. From the outset of the Genomes To Life (GTL) Program, therefore, mass spectrometry has an important role to play in the pursuit of Goal 1 of the GTL—the identification of the “machines of life.” The potential utility of mass spectrometry to GTL, however, extends far beyond current capabilities. In addition to incorporation of state-of-the- art mass spectrometry as a resource, we have also included a mass spectrometry research component as part of the Center for Molecular and Cellular Systems. The aim of this research component is to improve on existing mass spectrometry tools for protein complex characterization, as well as to produce new tools that will further the goals of the GTL program. Key to the success of this research component is close interaction with the protein expression, complex isolation,  computational and imaging components of the Center.

Currently, mass spectrometry is contributing heavily to the process of identifying target proteins that are likely to be members of complexes in Rhodopseudomonas palustris. These target proteins will be evaluated for expression as fusions with affinity labels to facilitate isolation of complexes. This identification process is based on mass spectrometric detection, in pelleted fractions, of proteins that one would normally expect to find in soluble fractions, indicating possible membrane association or membership in a large complex. From MS analysis of proteins from two different growth conditions of R. palustris, an initial list of target proteins has been assembled. The MS analysis strategy  at ORNL measures both intact molecular masses (“top-down”) and tandem mass spectra of tryptic digests of proteins (“bottom-up”). The “bottom-up” approach allows more comprehensive identification of proteins in a sample, while the “top-down” approach, which exploits the high-performance characteristics of Fourier transform mass spectrometry, provides information on post-translational modifications. The accurate mass tag (AMT) approach at PNNL is aimed at increasing throughput, sensitivity, and dynamic range for enhancing the detection of low-copy-number proteins and complexes.

We have also obtained initial mass spectrometry results from affinity purifications of fusions of R. palustris genes with GST and 6-HIS affinity tags, expressed in E. coli, verifying correct expression of the fusion proteins. Two strategies are being compared for this measurement. The first strategy is to elute affinity-captured proteins from the resin, separate by 1D SDS-PAGE, excise bands, digest, and analyze by reverse-phase nanoscale liquid chromatography on line with nano-electrospray/ tandem mass spectrometry. The second strategy is to eliminate the gel separation, and simply digest the entire mixture eluted from the affinity resin. The latter strategy will improve throughput considerably. “Top-down” measurements of affinity-captured fusion proteins are also underway. Current experiments directed toward expression of affinity-labeled proteins in R. palustris will provide our first opportunity for mass spectrometric identification of proteins that associate with these labeled targets-an important first step for Goal 1 of GTL.

Combined mass spectrometric and computational methods for characterizing crosslinked protein complexes are also under development. Crosslinking offers the opportunity to stabilize “fragile” complexes. Furthermore, it provides an alternative method to introduce an affinity tag into a protein complex, potentially increasing the throughput of analysis of complexes. Technical issues to be solved include increasing the robustness of crosslinking protocols, mass spectrometric detection of crosslinks, and computational methods for data interpretation. We have made progress in optimizing an affinity purification procedure based on peptides that have been crosslinked using a biotinylated reagent. Computer programs for interpretation of mass spectra of crosslinked samples have been initiated. Demonstration of integrating these various components on a model protein complex is underway.

Although not all funded by GTL, other mass spectrometric techniques relevant to the goals of the GTL are also under development. At ORNL, these include a method for characterizing surfaces of proteins and protein complexes via oxidative chemistry combined with mass spectometry, and sampling by electrospray mass spectrometry of proteins captured on surfaces displaying arrays of affinity-capture reagents surfaces. PNNL is developing hardware improvements for increasing the speed, sensitivity, and dynamic range of measurements, as well as informatic methods for incorporating chromatography elution information in protein identification techniques.

This research sponsored by Office of Biological and Environmental Research, U.S. Department of Energy. Oak Ridge National Laboratory (ORNL) is managed by UT-Battelle, LLC, for the U. S. Department of Energy under Contract No. DE-AC05-00OR22725.



A10
Genomes to Life Center for Molecular and Cellular Systems: A Research Program for Identification and Characterization of Protein Complexes

Joshua N. Adkins[1], Deanna Auberry[1], Baowei Chen[1], James R. Coleman[1], Priscilla A. Garza[1], Jane M. Weaver Feldhaus[1], Michael J. Feldhaus[1], Yuri A. Gorby[1], Eric A. Hill[1], Brian S. Hooker[1], Chian-Tso Lin[1], Mary S. Lipton[1], L. Meng Markillie[1], M. Uljana Mayer[1], Keith D. Miller[1], Sewite Negash[1], Margaret F. Romine[1], Liang Shi[1], Robert W. Siegel[1], Richard D. Smith[1], David L. Springer[1], Thomas C. Squier[1], H. Steven Wiley[1] (steven.wiley@pnl.gov), Linda J. Foote[2], Trish K. Lankford[2], Frank W. Larimer[2], T-Y. S. Lu[2], Dale Pelletier[2], Stephen J. Kennel[2], and Yisong Wang[2]

[1]Pacific Northwest National Laboratory; and [2]Oak Ridge National Laboratory

Summary: We have developed methodologies for isolating and identifying multiprotein complexes in Shewanella oneidensis MR-1 (PNNL) and Rhodopseudomonas palustris (ORNL), whose metabolisms are important in both understanding microbial energy production and environmental remediation. We are comparing complementary methods involving the isolation and identification of transient and stable protein complexes, with a current focus on validating the physiological relevance of isolated protein complexes.

Cloning, Expression, and Purification: To date, 23 S. oneidensis genes have been cloned into the GATEWAY™ expression vector pDEST™ containing a His6-tag for purification. Initial screening tests indicate that ~73% of cloned genes were expressed. Among those expressed proteins, 8 were purified to homogeneity using a Ni-NTA column under nondenaturing conditions. The yields of purified proteins obtained from 1 L of culture varied from 5 to 29 mg. We have also constructed new GATEWAY™-compatible vectors that will permit the expression of His6-tagged proteins in both S. oneidensis and R. palustris and the subsequent isolation of preformed complexes from microbes. Using four modified pDEST vectors, 7 R. palustris, genes have been cloned and expressed in E. coli. We are testing both N and C-terminal 6-his and GST tags for efficiency of expression and purification . Western blots of proteins and MS spectra of tryptic digests (see MS poster) of the GST-tagged nitrite reductase verify the expression and purification of polyproteins at high yield. The modified vector containing the GroEL gene has been inserted into R. palustris and it appears to be retained and convey drug resistance to the bacteria. Pull down experiments are in progress to isolate complexes from this target organism.

Affinity Reagent Generation: Purified proteins from S. oneidensis are currently being screened against a cell surface display of single-chain fragment variable (scFv) antibodies on the yeast Saccharomyces cerevisiae developed at PNNL, allowing rapid generation of affinity reagents that will permit the capture of protein complexes formed in vivo. We expect that these affinity reagents will cross-react with homologous protein complexes in different microbes, permitting the rapid isolation of protein complexes in a generalized manner.

Tagging and Cross-Linking Approaches for Complex Isolation:  In addition to the His6-tag, additional epitope tags are being assessed for their utility in enhancing the specificity of complex isolation under milder isolation conditions that will retain low-affinity binding partners in protein complexes. To date, we have demonstrated the utility of the CCXXCC epitope sequence for protein purification. Likewise, commercially available light-activated cross-linking reagents have been used to stabilize protein complexes in cellular homogenates from Shewanella, permitting the affinity purification of protein complexes under more stringent conditions that remove nonspecifically associated proteins. Under these conditions a limited range of cross-linked products are observed that are readily characterized by mass spectrometry.

Complex Isolation and Identification: Critical to the development of robust methods to rapidly isolate protein complexes is the assessment of standard protocols to isolate and identify different classes of protein complexes. We have therefore developed parallel methods focusing on the isolation and identification of membrane and soluble protein complexes that are known to form either stable or transient protein-protein interactions. Initial measurements have focused on the identification of stable and soluble protein complexes (e.g., RNA polymerase A), which has permitted the validation of protein isolation and cross-linking methods and the development of conditions that minimize nonspecific protein associations. However, because dynamic changes in protein complexes are expected to provide important insights into the metabolic regulatory strategies used by these organisms to adapt to environmental changes, we have extended these methods to assess transient protein interactions associated with signal transduction proteins (phosphotyrosine phosphatase A ) and stress-regulated proteins (e.g., methionine sulfoxide reductases A and B). In the latter cases, these proteins are known to interact and reduce oxidized substrates on a time scale of minutes. The development of immunoprecipitation methods that permit the isolation of transient complexes involving these proteins suggests that generalizable strategies to rapidly isolate protein complexes can be used to identify the formation of transient protein complexes. Surprisingly, the catalytic activity of methionine sulfoxide reductases from Shewanella has additional catalytic activities relative to those found in either E. coli or vertebrates, consistent with Shewanella’s known ability to thrive under harsh environmental conditions. We expect that identifying binding partners between this critical antioxidant protein will, furthermore, provide important information regarding oxidatively sensitive proteins and associated regulatory strategies that these organisms implement to survive.

Of the 7 R. palustris proteins expressed in the modified pDEST vector, we are concentrating on the GroEL chaparonin protein to validate complex formation. The tagged protein expressed in E. coli can be used to complex with GroES from R. palustris to document complex formation and pull-down efficiency. R. palustris has two different genes for GroEL type proteins and we will test if each is expressed and if they form co-complexes or if they are used separately for different functions. Dissimilatory nitrite reductases are capable of generating a membrane potential, as well as providing an electron sink for maintenance of balanced photosynthetic growth in the presence of highly reduced C-sources. In addition, there is a report that cells engaged in denitrification have an altered chemotactic response. Other systems being expressed include subunits of the uptake hydrogenase and components of sulfite oxidation, i.e., sulfite dehydrogenase, and sulfite oxidase.

This research is supported by the Office of Biological and Environmental Research of the U.S. Department of Energy. Pacific Northwest National Laboratory is operated for the U.S. Department of Energy by Battelle Memorial Institute through Contract No. DE-AC06-76RLO 1830. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U. S. Department of Energy under Contract No. DE-AC05-00OR22725.



A12
New Approaches for High-Throughput Identification and Characterization of Protein Complexes

Michelle Buchanan[1] (buchananmv@ornl.gov), Frank Larimer[1], Steven Wiley[2], Steven Kennel[1], Thomas Squier[2], Michael Ramsey[1], Karin Rodland[2], Gregory Hurst[1], Richard Smith[2], Ying Xu[1], David Dixon[2], Mitchel Doktycz[1], Steve Colson[2], Carol Giometti[3], Raymond Gesteland[4], Malin Young[5], and Michael Giddings[6]

[1]Oak Ridge National Laboratory; [2]Pacific Northwest National Laboratory; [3]Argonne National Laboratory; [4]University of Utah; [5]Sandia National Laboratories; and [6]University of North Carolina

The Center for Molecular and Cellular Systems (CMCS) is a recently established project that focuses specifically on Goal 1 of the GTL program. Its aim is to identify and characterize the complete set of protein complexes within a cell to provide a mechanistic basis of biochemical functions. Achieving this Goal would provide the ability to understand cells and their components in sufficient detail to allow the creation of network maps of cells that could be used in building models to predict, test and understand the responses of a biological system to its environment. Further, Goal 1 forms the foundation necessary to accomplish all of the other objectives of the GTL program, which are focused on gene regulatory networks and molecular level characterization of interactions in microbial communities.

A stated goal of the GTL program is to identify greater than 80% of the protein complexes in an organism per year within the first five years of the program. Ultimately, the GTL program will require the analysis of thousands of protein complexes from hundreds of microbes each year. The central task of the CMCS (Core Project) is to integrate biological, analytical, and computational tools to allow identification and characterization of protein complexes in a robust, high-throughput manner. The Core includes systems for growth of microbial cells under well-characterized conditions, isolation of protein complexes from cells, and their analysis by mass spectrometry (MS), followed by verification and characterization by imaging techniques. Several approaches for the isolation of the complexes are currently being examined and compared, including affinity tags (e.g., GST and 6-HIS affinity tags) and single chain antibodies. Computational tools are being integrated into this process to track samples, interpret the data, and to archive and disseminate data. Automated, parallel sample handling processes will be incorporated to maximize throughput and minimize amount of sample required.

The CMCS is initially focused on the identification and characterization of protein complexes in two microbial systems, Shewanella oneidensis and Rhodopseudomonas palustrus. The aim is to obtain a knowledge base that can provide insight into the relationship between the complement of protein complexes in these microbes and their biological function. Early activities within the Core have focused on setting up isolation, purification and analysis techniques and obtaining data on specific complexes in these two microbes. For R. palustris, we have performed baseline growth studies in two important metabolic states, anaerobic photohetero-trophic and dark aerobic heterotrophic. Wild-type cultivations at up to 2-L have generated samples for proteome analysis and for isolation of protein complexes. Data has been obtained from affinity purification of fusion proteins between several R. palustris genes and GST and 6-HIS affinity tags have been expressed in E. coli. We have verified correct expression of the fusion proteins and affinity-labeled proteins in R. palustrus. Various forms of chaperonin60, nitrite reductase, hydrogenase subunits, sulfite dehydrogenase, and thiosulfite oxidase are currently being examined. Work with Shewanella has focused on an initial set of tagged proteins expressed in E. coli; 20 proteins are in progress, among them, phosphotyrosine phosphatase, methionine sulfoxide reductase and RNA polymerase–alpha subunit have been purified and carried forward to use as bait with Shewanella extracts, with MS-MS analysis proceeding.

The Core of the CMCS will generate large amounts of experimental data at different sites and these data will need to be shared among the collaborators and, ultimately, with the wider research community. The management and storage of this data requires a flexible, robust and scalable information system. After a comprehensive analysis and evaluation of the CMCS’s process and data flow information need, we selected a Laboratory Information Systems (LIMS) that will serve as the backbone for integrating data management and analysis. Concurrent with evaluation of LIMS systems, we have also examined the processes within the Core that can be readily automated and incorporated into parallel processes (e.g., 96 well plate format), such as cell lysis, complex isolation, and final purification prior to MS analysis.

As initial data are generated within the Core, we are also evaluating the technologies to identify bottlenecks and needs for technology improvement. Current technologies for the identification and characterization of protein complexes will not be sufficient to meet the long-term goals of the GTL program. Therefore, a number of research tasks have been devised to address specific requirements of the Core, including new approaches for high throughput complex processing. For example, as part of the efforts to improve sample processing, we are evaluating microfluidic devices for microbial cell lysis and protein/peptide separation. We are also examining novel approaches for optimizing molecular characterization by MS, such as improving sensitivity and dynamic range. Combined MS and computational methods for characterizing crosslinked protein complexes area also under development. Crosslinking offers the opportunity to stabilize “fragile” complexes, and is an alternative to introducing an affinity tag into the complex, potentially increasing analysis throughput. Initial investigations have included optimization of an affinity purification procedure based on crosslinked biotinylated peptides, and the identification of putative cross-links in model protein complexes. In addition, imaging techniques are being developed to validate the presence of complexes in cells and to provide physical characterization of the complexes. Finally, bioinformatics tools for data tracking, acquisition, interpretation, and dissemination, along with computational tools for modeling and simulation of protein complexes are being developed.

This research sponsored by Office of Biological and Environmental Research, U.S. Department of Energy. Oak Ridge National Laboratory (ORNL) is managed by UT-Battelle, LLC, for the U. S. Department of Energy under Contract No. DE-AC05-00OR22725.



A14
Automation of Protein Complex Analyses in Rhodopseudomonas palustris and Shewanella oneidensis

P. R. Hoyt[1] (hoytpr@ornl.gov), C. J. Bruckner-Lea[2], S. J. Kennel[1], P. K. Lankford[1], M. S. Lipton[2], R. S. Foote[1], J. M. Ramsey[1], K. D. Rodland[2], and M. J. Doktycz[1]

[1]Oak Ridge National Laboratory; and [2]Pacific Northwest National Laboratory

High-throughput analyses afforded by mass spectroscopy require sample preparation processes that can keep pace. Standardization and automation of protein “pulldowns”, and related reagents are being developed. The processes are designed to provide a straightforward material flow in high-throughput format for the pulldown of protein complexes from the Rhodopseudomonas palustris and Shewanella oneidensis genomes. Existing techniques are well developed; however, some processes in clone library, antibody, and protein complex production have never been automated and few established protocols are available. In order to provide the highest level of biological significance and protein interaction coverage, the protein complex pulldowns from the different organisms will use different strategies. Subsequently, automation is designed to use flexible, compatible processes of varied scale during the program such that advances in technology can be evolved into innovative high-throughput techniques for sample preparation. The result will be a unique and robust system for protein expression and complex pulldown in bacterial systems.

The process for production of native tagged proteins for complex pulldown experiments uses conventional fluidics scale of 96-well format and liquid handling robotics. It is subdivided into the molecular preparation of a complete genomic library of expression clones for in vivo expression of R. palustris genes, followed by the production of proteins and “pull-downs” of protein complexes for analyses by mass spectrometry. The gene library and protein production scheme involves a suite of high-throughput molecular biology techniques based on the Gateway™ technology cloning strategy supplied by Invitrogen Corporation. This process requires two rounds of recombination between purified DNAs to produce protein expression vectors suitable for pull-down experiments in RP. At this time, all PCR setup, PCR purification, plasmid isolation, and redistribution steps, have been fully automated and integrated into an information management system for sample tracking. Recombination reactions should be fully automated in the near future using existing instrumentation. High-throughput automation of the electroporation steps, as well as colony picking can be automated using commercially available products, which are currently being evaluated. This leaves only the plating of bacteria on selective media to rely on manual processes.

Because detergents are not compatible with mass spectroscopic analyses, manual disruption processes were required. We were able to adapt a high-throughput, closed container non-detergent bead-milling technology (used originally for high-throughput isolation of RNA from animal tissues), to disrupt the R. paulutris cell walls. This process results in comparable protein profiles generated using other physical disruption techniques. Bead milling has been found to be most compatible with downstream MS analyses. Additionally, it reduces cross-contamination, and provides an extraordinary level of automation to the production process.

An heterologous-tagged protein pulldown system, for S. oneidensis using single-chain antibodies (Ab) to specific expressed proteins is also under automation development. This process uses a microfluidics platform combined with functionalized microbeads for the purification of protein complexes. A renewable microcolumn system with optical detection has been assembled and automated procedures developed. The renewable microcolumn consists of small volumes (microliters) of microbeads that are automatically packed, perfused with cell lysates, and wash solutions, and proteins eluted using a solution that is suitable for mass spectrometry analysis. After each purification, the small volume of microbeads is automatically flushed from the microcolumn and a new microcolumn is automatically packed. The microbeads are functionalized for the capture of a specific protein, for example by derivatization with an antibody for the protein of interest. Optical monitoring of the microcolumn during processing provides information about the amount of material on the column during each binding and washing step. The current automated procedure can process a cell lysate volume ranging from 10 microliters to 1 millilter, and the purified proteins are eluted into 150 microliters of a low salt buffer solution. Automated procedures are currently being tested for the capture of Shewanella proteins tagged with yellow fluorescent protein (YFP), along with the proteins that associate with the YPF-tagged protein. As new reagents for protein capture such as single chain antibodies for Shewanella proteins of interest are developed, they will be linked to microbeads and renewable column protocols will be developed for automated purification of the protein complexes for mass spectrometry. In the next stage of this work, the eluted protein complexes will be analyzed by mass spectrometry and the automated protocols will be optimized.

For the ultimate in throughput and sensitivity, a lab-on-a-chip complex isolation and identification program is also under development. Many of the individual steps involved in sample processing and analysis, including cell lysis, protein/peptide separations and enzyme digestions, have been implemented in microfluidic devices that can be interfaced with mass spectrometry for on-line analysis. (We have previously demonstrated electrically induced lysis of mammalian cells in microfluidic devices and will apply this technique to bacterial protoplasts). The integration of these functions with a pull-down step would provide high-throughput analyses of protein complexes in extremely small numbers of cells.

In summary, protein complex analysis by mass spectroscopy will require a high-throughput reagent production scheme. Because the complexes isolated are different for the different organisms, different schemes for complex isolation have been implemented. At scales ranging from macro to micro we are automating the production of reagents and samples to produce these different complexes, and the processes are being optimized to feed into mass spectroscopic analyses. The automation development is concomitant with establishment of sample tracking and information management processes so that integration of these systems will be seamless.

This research sponsored by Office of Biological and Environmental Research, U.S. Department of Energy. Oak Ridge National Laboratory (ORNL) is managed by UT-Battelle, LLC, for the U. S. Department of Energy under Contract No. DE-AC05-00OR22725.