proteomics: technologies and applications · ruedi h. aebersold, institute for systems biology...

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Program

* Session Chair • Number in ( ) equals speaker abstract number • Current as of February 20, 2003.

Tuesday, March 25

3 - 7pm Registration Shavano Peak Foyer

6:15 - 7:15pm Welcome Shavano Peak Foyer

7:15 - 7:30pm Orientation Shavano Peak

7:30 - 8:30pm KEYNOTE ADDRESSES Shavano Peak

*Ruedi H. Aebersold, Institute for Systems Biology Tony Pawson, Mount Sinai Hospital (002)Modular Protein Interactions and Signaling Networks

Summary of Meeting

The completion of the genomic sequences of a number of prokaryotic and eukaryotic species has catalyzed newresearch approaches to study the structure, function, and control of biological processes. They are characterized bythe systematic and, in many cases, quantitative analysis of all the molecules of a particular type expressed by a cellor tissue. The systematic analysis of proteins has been termed "proteomics." In an initial phase, most of the proteomicsefforts were focused on large-scale protein identification. More recently, the objectives and technologies of proteomicshave diversified and expanded. Current proteomics research attempts to systematically and, where applicable,quantitatively determine the many properties of proteins that determine their biological function. These include proteinabundance, state of modification, specific activity, interaction with other biomolecules, half-life, subcellular location,structure, and more. Significant current challenges include the development of suitable technologies to determine theseproperties on a proteome-wide scale, the interpretation of the large amounts of data obtained, the development ofcomputational approaches to infer biological function, and the integration of different types of data into a coherentmodel describing a biological process. The goal of the meeting is to bring together leading experts from the differentareas of proteomics research to discuss emerging technologies and their applications to biological and clinicalresearch.

Proteomics: Technologies and ApplicationsRuedi Aebersold and Stanley Fields

Supported by

Keystone Symposia

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Program


7 - 8pm Social Hour Torreys Peak

8 - 10pm POSTER SESSION 1 Torreys Peak

Identification and Quantification of Proteins/Management of Proteomics Data

Wednesday, March 26

7 - 8am Breakfast Torreys Peak

8 - 11:30am PLENARY SESSION Shavano Peak

Identification and Quantification of Proteins on a Proteome-Wide Scale*Pavel A. Pevzner, University of California, San Diego Ruedi H. Aebersold, Institute for Systems Biology (004)Toward a High Throughput Quantitative Proteomics PlatformMarvin Vestal, Applied Biosystems (005)Trends in Mass Spectrometry Instrumentation for Proteomics

Coffee Break Shavano Peak FoyerJohn R. Yates, Scripps Research Institute Proteomics in Model Systems

Short Oral Abstract Presentations of Selected Posters

11:30am - 1pm Poster Setup Torreys Peak

1 - 10pm Poster Viewing Torreys Peak

4 - 4:30pm SPECIAL LECTURE Shavano PeakGregory Milman, National Institutes of Health (008)Over $1 Billion in Biodefense Research Opportunities

4:30 - 5pm Coffee & Snacks Available Shavano Peak Foyer

5 - 7pm PLENARY SESSION Shavano Peak

Analysis, Interpretation and Management of Proteomics Data*John R. Yates, Scripps Research Institute Benno Schwikowski, Institute for Systems Biology (010)Computational Tools and Challenges for Integrating Proteomic DataPavel A. Pevzner, University of California, San Diego (011)New Algorithmic Approaches for the Analysis of Peptide Mass Spectral DataBruno M. Domon, Celera Genomics Group (012)Generation and Analysis of Data for Target Discovery in an Industrial Setting

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Program

* Session Chair • Number in ( ) equals speaker abstract number • Current as of February 20 2003

Thursday, March 27



In vivo Approaches/Analysis of Protein Activities



Protein Interactions and Protein Linkage Maps*Mark Johnston, Washington University Medical School Joachim Frank, SUNY-Albany (014)Single-Particle Reconstruction of Molecular Machines Using Cryo-Electron MicroscopyStanley Fields, University of Washington (015)Genetic and Genomic Approaches to Protein Interaction

Coffee Break Shavano Peak FoyerMarc Vidal, Dana Farber Cancer Institute (016)Toward a Proteome Atlas for C. elegansBertrand Séraphin, Centre National de la Recherche Scientifique (017)Large Scale Protein Linkage Maps via Purification of Protein Complexes and Mass Spectrometric Analysis




4 - 4:30pm SPECIAL LECTURE Shavano PeakGregory Milman, National Institutes of Health Whom to Contact at NIH and When?



In vivo Analysis*Stanley Fields, University of Washington Stephen W. Michnick, Université de Montréal (020)Mapping and Visualization of Biochemical Networks in Living CellsMark Johnston, Washington University (021)Finding Regulatory Sequences and their Protein Partners in Yeast by Comparative and Functional GenomicsAndrew Fraser, Wellcome Trust Sanger Institute (022)Functional Analysis of the C. elegans Genome by RNA-Mediated Interference

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Program


Friday, March 28



Structural Proteomics/Control of Protein Abundance and Activity/Computational Methods



Systematic Analysis of Protein Activities and Protein Arrays*Susan S. Taylor, University of California-San Diego Michael Snyder, Yale University (024)Global Analysis of the Yeast ProteomeDolores J. Cahill, Max Planck Institute of Molecular Genetics (025)Protein Arrays: Generation and Recent Applications

Coffee Break Shavano Peak FoyerGavin MacBeath, Harvard University (026)Protein Microarrays: From Functional Genomics to Protein ProfilingLarry Gold, SomaLogic Inc. (027)Aptamer Arrays and Proteomics






Structural Proteomics*Michael Snyder, Yale University David Baker, University of Washington Prediction and Design of Protein Structures and Protein-Protein Interactions Andrej Sali, University of California, San Francisco (030)Modeling the Structure of Proteins and Macromolecular AssembliesSusan S. Taylor, University of California-San Diego (031)Dynamic Integration of Signaling by PKA

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Program


Saturday, March 29


8 - 11pm Entertainment Torreys Peak



Control and Detection of Protein Abundance and Activity*Dolores J. Cahill, Max Planck Institute for Molecular Genetics David R. Morris, University of Washington (033)Translating the Transcriptome:High Throughput Analysis of mRNA TranslationBrian T. Chait, Rockefeller University Probes of Protein Interaction and Phosphorylation

Coffee Break Shavano Peak FoyerBenjamin F. Cravatt III, The Scripps Research Institute (035)Activity-Based Protein Profiling:Chemical Approaches for Functional ProteomicsElizabeth J. Grayhack, University of Rochester School of Medicine (036)Functional Proteomics




Methods to Explore Function*Brian T. Chait, Rockefeller University Tom W. Muir, Rockefeller University (038)From Protein Semisynthesis to Chemical Genetics:The Chemical Biology of Protein Splicing Edward M. Marcotte, University of Texas, Austin (039)Global Analysis of Protein Pathways, Systems and Interactions Peer Bork, European Molecular Biology Laboratory (040)Comparative Analysis of Protein Interaction Networks

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Speaker Abstracts

002 • Tuesday, March 25 • Keynote Address • Pawson

Modular Protein Interactions and Signaling Networks

Tony Pawson, Samuel Lunenfeld Research Institute, Mt. Sinai Hospital

Signaling from cell surface receptors involves a regulated series of protein-protein and protein-phospholipid interactions, mediated by modularinteraction domains. Such interactions play a broad role, not only in conventional signaling, but also in processes such as cytoskeletalorganization, vesicle/protein trafficking, cell polarity, and chromatin remodeling. Through their ability to recognize post-translationalmodifications, interaction domains control the dynamic response of cells to external and internal signals. The reiterated use of a limited set ofinteraction domains may have facilitated the evolution of complex cellular function, by the joining of domains in novel combinations. I willprovide evidence for these ideas, and show that cellular behaviour can be re-wired by juxtaposing domains in new ways. The use of proteomictechniques such as mass spectrometry to analyze signaling networks in yeast and mammalian cells will be discussed, and a novel strategy torapidly analyze protein function in the mouse embryo will be described.

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Speaker Abstracts

004 • Wednesday, March 26 • Identification and Quantification of Proteins on a Proteome-Wide Scale • Aebersold

Toward a High Throughput Quantitative Proteomics Platform

Ruedi H. Aebersold, Andrew Keller, Alexey Neshvizhskii, Xiao-Jun Li, Patrick Pedrioli, Sharon Chen, Eric Deutsch, Tim GriffinInstitute for Systems Biology, Seattle, WA 98103

The availability of the complete human genomic sequence has catalyzed the development of new technologies for the systematic andquantitative measurement of genomic and proteomic profiles to define comprehensive molecular signatures of tissues, cells and body fluids inhealth and disease. Such signatures are expected to impact a wide range of biological and clinical research questions, such as the systematicstudy of biological processes and the discovery of molecular clinical markers for detection, diagnosis and assessment of treatment outcome.The generation of such signatures via proteomics technology requires that many (ideally all) proteins in a sample can be identified andaccurately quantified. Since each study typically requires the analysis of multiple samples the generation of a high throughput, automatedplatform for quantitative proteomics is essential.

We have recently developed a new approach to quantitative proteomics. It is based on a new class of chemical reagents termed isotope codedaffinity tags (ICAT™ reagents), automated peptide tandem mass spectrometry and a suite of bioinformatics tools for the analysis,interpretation and validation of the results. In this presentation we will discuss recent conceptual and technical advances of the ICAT™ reagentmethod toward the generation of a robust, high throughput quantitative proteomics technology. The platform incorporates new methods forstable isotope labeling of proteins, new methods for high throughput, automated data collection by ESI and MALDI tandem mass spectrometryand a suite of software tools that provide probability-based scores for the determined protein identities and quantities and organize the data inrelational databases. Specific applications of the platform illustrating its current performance will also be discussed.

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Speaker Abstracts

005 • Wednesday, March 26 • Identification and Quantification of Proteins on a Proteome-Wide Scale • Vestal

Trends in Mass Spectrometry Instrumentation for Proteomics

Marvin VestalApplied Biosystems, Framingham, MA, USA 01701

Since 1988 two powerful ionization techniques, electrospray and MALDI, have emerged for efficiently ionizing proteins, peptides, and otherlarge nonvolatile molecules of biological importance. These ionization techniques have been combined with a variety of mass analyzersincluding several types of ion traps and time-of-flight systems for proteomic applications. Particularly important are systems employingtandem MS to first determine the accurate mass of an intact molecular ion, and then fragment that ion and employ a second MS to measure themasses of the fragments. These MS-MS spectra can be used for protein identification via database searching, or interpreted de novo todetermine sequence and/or post-translational modifications. Recent developments in MS-MS systems for proteomics are reviewed, and resultsare presented that illustrate the strengths and weaknesses of some of the alternatives. Anticipated trends for the future are briefly discussed.

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Speaker Abstracts

006 • Wednesday, March 26 • Identification and Quantification of Proteins on a Proteome-Wide Scale • Townsend

Strategies for Increasing Proteome Coverage

R. Reid Townsend, Oxford GlycoSciences Ltd., Abingdon, UK

The comprehensive identification of proteins from tissues, cells and biological fluids is an outstanding goal of proteomics. The natural rangeof protein concentrations in biological samples (at least six orders of magnitude) is a major impediment to increasing the proteome ‘coverage’of a genome. Often, much of the mass spectrometric analysis time is used to obtain spectra of peptides from the higher abundance proteins.Further, since peptides from lower abundance proteins are present in lower amounts, poorer quality spectra are often acquired. Proteinidentification criteria and database ‘search space’ can have considerable impact on the number of proteins incorrectly identified. To overcomesome of these limitations, depletion and enrichment methods were applied to cellular and biological fluid samples prior to MS-basedproteomic analysis. The selective removal of a set of high abundance proteins from plasma, serum, cerebrospinal fluid, and bronchioalveolarlavages enabled visualization and identification of minor components from two-dimensional gels. Cell-type enrichment was achieved fromfreshly-resected tissues and resulted in the identification of specific proteins and markers of histopathology in breast tumors. By combiningsubcellular fractionation and stable isotopic Cys- labeling, minor proteins in membranes and organelles were identified. As protoemicstechnology continues to improve, depletion and enrichment methods will remain key to identifying larger numbers of proteins from tissues,cells and biological fluids.

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Speaker Abstracts

008 • Wednesday, March 26 • Special Lecture • Milman

Over $1 Billion in Biodefense Research Opportunities

Gregory Milman, National Institutes of Health

The President’s NIH budget for FY2004 (http://www.hhs.gov/budget/04budget/fy2004bib.pdf#page=33) contains a modest 2%increase over FY2003, from $27.3 to $27.9 billion. For FY 2004, the budget proposes a total of $1.6 billion for NIH biodefense efforts.These funds will enable the National Institute of Allergy and Infectious Diseases (NIAID) and other NIH institutes to expand ongoingprojects and establish new initiatives as part of a comprehensive and sustained biodefense research program. The NIAIDbiodefense research agenda focuses on studies of microbial biology and host responses to microbes; the development of newvaccines, therapies, and diagnostic tools; and the development of research resources such as appropriate laboratory facilities. Weanticipate that the large investment in biodefense research will have many positive “spin-offs,” similar to the manner in whichHIV/AIDS research has advanced the understanding and treatment of many other diseases. NIAID research on organisms withbioterrorism potential will almost certainly lead to an enhanced understanding of other more common and naturally occurringinfectious diseases that afflict people here and abroad. In particular, the advancement of knowledge should have enormous positiveimpact on our ability to diagnose, treat and prevent major diseases such as malaria, tuberculosis, HIV/AIDS, and a spectrum ofemerging and re-emerging diseases such as West Nile fever, dengue, influenza, and multi-drug resistant microbes.

Gregory Milman, Ph.D., is Director of the Office of Innovations and Special Programs in the National Institute of Allergy andInfectious diseases (NIAID). He is responsible for science initiatives, outreach activities, and the NIAID small business programs.He is well known for his Internet advice on grant preparation (http://www.niaid.nih.gov/ncn/impatica/). Dr. Milman serves on the NIHTrans-Agency Complementary and Alternative Medicine Coordinating Committee (http://nccam.nih.gov/), the NIH Committee forElectronic Research Administration (http://era.nih.gov/), and the Board of Directors of the Biotechnology Industry OrganizationCouncil of Biotechnology Centers (http://www.bio.org/aboutbio/cbc.html). Dr. Milman organized the NIH Bioengineering Consortium(http://www.becon1.nih.gov/becon.htm) and acted as its first Executive Secretary. In 2000, Dr. Milman served in the White HouseOffice of Science and Technology Policy (http://www.ostp.gov/). In addition to his NIH “day job,” Dr. Milman is a Visiting Professorat the University of Maryland.

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Speaker Abstracts

010 • Wednesday, March 26 • Analysis, Interpretation and Management of Proteomics Data • Schwikowski

Computational Tools and Challenges for Integrating Proteomic Data

Benno SchwikowskiInstitute for Systems Biology

One reason why proteomic data is so attractive for studying biological systems is that it represent a broad picture of biological processes at theprotein level. To unfold its full potential, this data needs to be interpreted together with other types of large-scale information, such as mRNAabundance measurements, protein-protein, and protein-DNA interaction data. As data volumes are enormous, the task of acquiring andintegrating proteomic data needs to be supported by a variety of computational tools.

In yeast these types of data are now becoming available on a large scale. This talk will go over the computational challenges in acquiring andintegrating proteomic data, and present a suite of open-source software tools under development at the ISB that support and automate thisprocess. In human, the available data is much more sparse, and the biological systems are much more complex. This requires strategies thatiterate between experiments and computational high-throughput analyses. This presentation will contain case studies in both yeast and humanthat illustrate these challenges.

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Speaker Abstracts

011 • Wednesday, March 26 • Analysis, Interpretation and Management of Proteomics Data • Pevzner

New Algorithmic Approaches for the Analysis of Peptide Mass Spectral

Pavel Pevzner Department of Computer Science and Engineering, University of California San Diego, CA

Although protein identification by matching tandem mass spectra against protein databases is a widespread tool in mass spectrometry, thequestion about reliability of such searches remains open. In particular, random database hits may lead to erroneous protein identification,particularly in the case of mutated or post-translationally modified peptides. This problem is especially important for high-throughput MS/MSprojects when the possibility of expert analysis is limited. Thus, algorithms that sort out reliable database hits from unreliable ones and identifymutated and modified peptides are sought. Most MS/MS database search algorithms rely on variations of the Shared Peaks Count approach thatscores pairs of spectra by the peaks (masses) they have in common. Although this approach proved to be useful, it has a high error rate inidentification of mutated and modified peptides. We describe new MS/MS database search tools, MS-CONVOLUTION and MS-ALIGNMENT,which implement the spectral convolution and spectral alignment approaches to peptide identification. We further analyze these approaches toidentification of modified peptides and demonstrate their advantages over the Shared Peaks Count. We also use the spectral alignment approachas a filter in a new database search algorithm.

This is joint work with Vlado Dancik, Zufar Muluykov and Chris Tang. The work is supported by the NIH “Computational mass-spectrometry”grant.

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Speaker Abstracts

012 • Wednesday, March 26 • Analysis, Interpretation and Management of Proteomics Data • Domon

Generation and Analysis of Data for Target Discovery in an Industrial Setting

Scott D. Patterson; Terence E. Ryan; Bruno M. Domon; Ian N. McCaffery; Proteomics Department, Celera Genomics Group, Rockville, MD,USA

The identification of potential targets for therapeutic intervention can be accomplished on a systematic basis by a variety of techniques thatinclude quantitative analysis of gene-specific mRNA levels and expressed proteins in normal and diseased cells. Differences in the expressionlevels of nucleic acid and protein gene products could suggest protein drug targets that are directly causative of disease, or reveal biochemicalpathways that could be modulated by therapeutic molecules. Any effort based on mRNA or protein expression level comparisons could beconfounded by a number of factors: (1) mRNA level in steady-state may not be correlated with actual encoded protein levels; (2) differentiallyexpressed protein levels might be a result of disease process, and not causative of the process, and therapeutic intervention based on such adifference will be unproductive and (3) the differential expression of mRNA or protein may be the result of biological variation unrelated to thedisease process under study. In order to address these possibly confounding factors, it is necessary to validate potential targets by establishingtheir firm association with disease, and their minimal distribution in non-diseased tissues of any type. This requirement suggests that emphasison true and reproducible quantitation of protein expression levels in a variety of samples will be an effective and highly efficient method ofgenerating drug targets with a high degree of utility. To achieve this aim, we have established an industrial-scale proteomics-based discoveryplatform consisting of cell biology, protein chemistry, and mass spectrometry technical groups. The analytical method used for quantitationemploys isotope labeling for differential analysis (ICAT, Applied Biosystems, Inc.). With this technique, tryptic peptides are generated fromlabeled proteins that have been specifically captured from various subcellular locations or protein families. The resulting peptides are identifiedand quantified by mass spectrometry. To evaluate this approach on a large-scale, we have applied it to a study of continuous cell lines derivedfrom human pancreatic adenocarcinomas. We have been able to establish processes for target discovery therapeutic antibody target identificationfor cell surface proteins. The results of these analyses will be presented together with the some of the issues that need to be addressed in such anundertaking.

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Speaker Abstracts

014 • Thursday, March 27 • Protein Interactions and Protein Linkage Maps • Frank

Single-Particle Reconstruction of Molecular Machines Using Cryo-Electron Microscopy

Joachim FrankHoward Hughes Medical Institute, Health Research, Inc. at the Wadsworth Center, and the Department of Biomedical Sciences, StateUniversity of New York at Albany, Empire State Plaza, Albany, NY 12201-0509

We call “molecular machines” the complexes formed by molecules intermittently in the cell for the purpose of catalyzing numerous reactionsof the cell metabolism. Well-studied examples are transcription, splicing, and translation. The term “machine” refers to the highly ordered,processive interactions of RNA, proteins and their ligands, which often go hand in hand with conformational changes of the interactingmolecules (Alberts, 1998).

Three-dimensional visualization of molecular machines in different processing states poses a difficult challenge since many of the varyingbinding constellation do not allow formation of crystals suitable for X-ray crystallography. On the other hand, they are as a rule too large tobe approached by NMR. Cryo-electron microscopy of single particles (see Frank, 1996), a relatively new technique, is able to overcome thisproblem, yielding three-dimensional density maps of the macromolecular assembly at close-to-native conditions. The success of the techniqueis exemplified by the ribosome. Molecular detail was revealed by cryo-EM well before the X-ray structure was solved. The currentresolution, 7.8Å, has been achieved by combining images of more than 100,000 particles in a single reconstruction (C.M.T. Spahn, R.Grassucci, K.H. Nierhaus, and J. Frank, in preparation). The use of antibiotics and GTP analogs allowed well-defined states to be analyzed asthree-dimensional “snapshots” of a dynamic process (Frank and Agrawal, 2000).

Current efforts to streamline and accelerate collection and processing of cryo-EM data should soon lead to a technology that allows routineachievement of ~10Å resolution. Another focus of efforts in cryo-EM of molecular machines is the development of time-resolved methods.

This work was supported by HHMI and NIH grants R37 GM29169 and R01 GM55440 (to J.F.).

B. Alberts (1998). The cell as a collection of protein machines: preparing the next generation of molecular biologists. Cell, 92: 291-294.

J. Frank and Rajendra K. Agrawal (2000). A ratched-like inter-subunit reorganization of the ribosome during translocation. Nature, 406: 318-322.

J. Frank (1996). Three-dimensional Electron Microscopy of Macromolecular Assemblies. Academic Press, San Diego.

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Speaker Abstracts

015 • Thursday, March 27 • Protein Interactions and Protein Linkage Maps • Fields

Genetic and Genomic Approaches to Protein Interaction

Stanley Fields, John Miller, Russell Lo, Victoria Brown-Kennerly, Chandra Tucker, Michael DeVit and Margaret Branson, Howard HughesMedical Institute and Departments of Genome Sciences and Medicine, University of Washington, Box 357730, Seattle, WA 98195

We have been interested in designing and implementing simple yeast-based assays to identify or characterize protein-protein and protein-ligandinteractions. In one approach, we have used the split-ubiquitin system of Johnsson and Varshavsky (Proc. Natl. Acad. Sci. USA 91:10340 (1994))as modified by Stagljar et al. (Proc. Natl. Acad. Sci. USA 95:5187 (1998)) to investigate interactions between integral membrane proteins of theyeast Saccharomyces cerevisiae. We generated an array of yeast transformants containing ~700 different membrane proteins fused to the N-terminal half of ubiquitin and screened it by mating to yeast bearing a membrane protein fused to the C-terminal half of ubiquitin. Interactionshave been detected for oligosaccharyltransferase components, ER-associated degradation (ERAD) constituents, and a variety of other proteinsas part of collaborative projects of the NCRR Yeast Resource Center. This assay has also been used with human Toll-like receptors (TLRs), whichdetect microbial antigens and activate an innate immune response. Initial experiments indicate that the assay can detect a ligand-dependentinteraction of TLRs as well as an interaction of TLRs with a downstream signaling component. In another approach, we are developing a geneticscreen to identify proteins that function in a common pathway or process. Interactions can be artificially forced via heterodimerizing leucinezippers, and the resulting yeast assayed for a gain-of-function phenotype. We have identified the minimal portions of Fos and Jun that will interactin yeast, and have shown that a Fos-GFP fusion can colocalize with Jun-protein fusions. In a third approach, we have initiated a systematicanalysis of the chemical sensitivities of the haploid deletion strains generated by the Saccharomyces genome deletion project. The loss of aprotective gene in a pathway affected by a drug can lead to a synthetic lethal effect in which the combination of a deletion and a normallysublethal dose of a chemical results in lethality. We have examined sensitivity to hydrogen peroxide and to several drugs.

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Speaker Abstracts

016 • Thursday, March 27 • Protein Interactions and Protein Linkage Maps • Vidal

Toward a Proteome Atlas for C. elegans

Marc VidalDana Farber Cancer Institute, Boston, MA 02115

The availability of complete genome sequences suggests new approaches for biological research to complement conventional genetics andbiochemistry. In this context, the goals of this laboratory are to generate a comprehensive protein-protein interaction, or interactome, map for C.elegans and develop new concepts to integrate this map with other functional maps such as expression profiles (transcriptome), global phenotypicanalysis (phenome), localization of expression projects (localizome), etc…. The resulting proteome atlas of integrated maps should be valuablefor a systems biology approach to the study of development.

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Speaker Abstracts

017 • Thursday, March 27 • Protein Interactions and Protein Linkage Maps • Seraphin

Large-Scale Protein Linkage Maps via Protein Complexes Purification and Mass Spectrometric Analysis

Bertrand SéraphinCentre de Génétique Moléculaire, CNRS, 91198 Gif sur Yvette, France

Many cellular processes are carried out by multiprotein complexes. Those include the ribosome, the proteasome, thespliceosome, RNA- and DNA-polymerases, etc.... To identify and analyse such assemblies, we have developed a genericprocedure that we named TAP for Tandem Affinity Purification. The TAP tag allows for the rapid purification of proteinsexpressed at their natural level under native conditions permitting thereby protein complex identification from arelatively small number of cells. The method does not require prior knowledge of the protein activity or function.Combined with mass spectrometry, the TAP strategy allows for the identification of proteins interacting with a giventarget protein providing insight into how the ensemble of expressed proteins (namely, proteome) is organised intofunctional units. The TAP method and mass spectrometry were used in a large-scale approach to characterisemultiprotein complexes in Saccharomyces cerevisiae allowing the systematic analysis of a physical protein network.Analysis of the assemblies analysed defined numerous distinct multiprotein complexes and suggested new functionsfor many proteins. Comparison of yeast and human complexes showed that conservation across species extends fromsingle proteins to their molecular environment. Application of the TAP method to various organisms will be discussed.

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Speaker Abstracts

020 • Thursday, March 27 • In vivo Analysis • Michnick

Mapping and Visualization of Biochemical Networks in Living Cells

Stephen W. Michnick U de Montreal, Biochimie, CP 6128, Succ. centre-ville, Montreal, Quebec, H3C 3J7, Canada, Fax: 514 343 2210

Cellular biochemical machineries, what we call pathways, consist of dynamically assembling and disassembling macromolecular complexes.While our models for the organization of biochemical machines are derived largely from in vitro experiments, do they reflect their organizationin intact, living cells? We have developed a general experimental strategy that addresses this question by allowing the quantitative probing ofmolecular interactions in intact, living cells. The experimental strategy is based on Protein fragment Complementation Assays (PCA), a methodwhereby protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as thedetector of a protein interaction. A biochemical machine or pathway is defined by grouping interacting proteins into those that are perturbed inthe same way by common factors (hormones, metabolites, enzyme inhibitors, etc). I will describe how we go from descriptive to quantitativerepresentations of biochemical networks at an individual to whole genome level and how our approach will lead ultimately to better descriptionsof the biochemical machineries that underlie living processes.

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Speaker Abstracts

021 • Thursday, March 27 • In vivo Analysis • Johnston

Finding Regulatory Sequences and their Protein Partners in Yeast by Comparative and Functional Genomics

Mark Johnston, Paul Cliften, Priya Sudarsanam, Su-wen Ho, and Barak Cohen. Department of Genetics, Washington University, St. Louis, MO63130

Functional sequences in genomes are more conserved through evolution than surrounding non-functional sequences, and can be recognized bythe ‘phylogenetic footprints’ they make in alignments of sequences of related species. Functional protein-coding sequences make relativelyobvious phylogenetic footprints; functional non-protein coding sequences, such as those regulating gene expression, are generally small andunder less evolutionary constraint, causing their footprints to fade quickly in evolution. Therefore, detection of regulatory sequence elementsrequires comparison of genome sequences of relatively closely-related species. To identify functional non-protein coding sequences in thegenome of bakers’ yeast, we determined partial genome sequences of five different Saccharomyces species and compared them to the S.cerevisiae genome sequence. Conserved sequences are not randomly distributed in the promoters: significantly more conserved sequence motifsappear in alignments of orthologous promoters than in shuffled versions of these alignments. The distribution of conserved residues across apromoter is heavily biased, with a significant peak of sequence identity between 125 and 250 bp upstream of the translation initiation codon,suggesting that the ‘average’ yeast promoter has its regulatory elements within 250 bp of coding sequences. Potential translational regulatorysequences are apparent in a subset of promoters that have highly conserved sequences immediately adjacent to their translational start codon.Searching the multiple sequence alignments of orthologous promoters revealed hundreds of conserved sequence motifs that appear in 2 or moregenes. Since only about 40 of these were previously identified, we have identified hundreds of novel potential functional sequences in the yeastgenome. We have developed and are employing high throughput genetic and biochemical methods for identifying the proteins that bind to theseconserved sequence motifs.

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Speaker Abstracts

022 • Thursday, March 27 • In vivo Analysis • Fraser

Functional Analysis of the C. Elegans Genome by Rna-Mediated Interference

Andrew Fraser1, Ravi Kamath2, Yan Dong2, Peder Zipperlen2, Gino Poulin2, Monica Gotta2, Natalie le Bot2, Sergio Moreno and JulieAhringer2.

1The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK. 2Wellcome/CR UK Institute,Tennis Court Road, University of Cambridge, Cambridge CB2 1QR, UK.

Although there is a phenomenal quantity of sequence data available, biological function has still only been assigned to a small percentage ofpredicted genes in any metazoan. One systematic approach to analyse gene function is to examine the loss-of-function phenotypes of everypredicted gene in a genome — this is the approach that we have taken in C. elegans. We have generated a reagent that uses RNA-mediatedinterference (RNAi) to individually inhibit ~90% of all ~19,000 predicted genes in the C. elegans genome. Using this reagent, we examinedloss-of-function phenotypes for ~90% of all predicted genes and have found ~1700 genes to have detectable loss-of-function phenotypes.We find that genes with similar loss-of-function phenotypes tend to be clustered in specific regions of the genome – for example 36% of essentialgenes lie in 13% of the genome. These multi-megabase regions are also enriched for genes with similar transcriptional profiles, suggesting thatgenes with related functions may be clustered together to facilitate transcriptional coregulation.The library we have constructed is highly versatile and allows an essentially unlimited number of genome-wide RNAi screens. We are currentlycarry out multiple screens to identify genes involved in modulating ras signalling in C. elegans and will present these data.

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Speaker Abstracts

024 • Friday, March 28 • Systematic Analysis of Protein Activities and Protein Arrays • Snyder

Global Analysis of Protein Activities Using Protein Chips

Heng Zhu1, Metin Bilgin1, Jason Ptacek2, David Hall2, Antonio Casamayor1, Paul Bertone1, Nelson Lopez1, Ning Lan2, Ronald Jansen2, ScottBidlingmaier2, Geeta Devgan1, Perry Miller2, Mark Gerstein2, Michael Snyder1,2,1Department of Molecular, Cellular, and Developmental Biology, 2Department of Molecular Biophysics and Biochemistry, Yale University, NewHaven, Connecticut 06520

The genomes of a wide variety of organisms have now been sequenced; a major challenge ahead is to understand the function, regulation andmodification of the many encoded gene products. We have been carrying out proteomics approaches to the identification and analysis ofsignalling pathways in yeast. 121 of 122 protein kinases were cloned and purifed from yeast as GST fusions and analyzed for their ability tophosphorylate 60 different yeast substrates. More than 93% of the kinases exhibited activities that are 5 fold or higher, relative to controls,including 18 of 24 previously uncharacterized kinases. Many protein kinases had novel activities; for example 27 yeast kinases were found tophosphorylate Tyr. In addition, we have now cloned 6000 open reading frames and overexpressed their corresponding proteins. The proteinswere printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with a variety ofdifferent proteins, nucleic acids and phospholipids. As examples, we have probed yeast proteome chips with calmodulin and six differentphospholipids. These studies revealed many new calmodulin and phospholipid-interacting proteins; a common potential binding motif wasidentified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened fordiverse biochemical activities. They can also be used to screen protein-drug interactions and to detect posttranslational modifications.

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Speaker Abstracts

025 • Friday, March 28 • Systematic Analysis of Protein Activities and Protein Arrays • Cahill

Protein Arrays: Generation and Recent Applications

Dolores J. Cahill

Max-Planck-Institute of Molecular Genetics, Ihnestraße 73, D-14195 Berlin, Germany and PROT@GEN AG, Emil-Figge-Strasse 76 A, D-44227Dortmund, Germany and Dept. of Clinical Pharmacology, Royal College of Surgeons in Ireland, Dublin 2, Ireland.

In order to construct high-density, high content arrays, we have used cDNA libraries as a source of thousands of proteins and have developed asuccessful system for expression and purification of thousands of proteins in parallel. We have used these proteins to generate high density proteinarrays on glass slides and demonstrates their potential applications in the profiling of the antibody repertoire of auto-immune patients, and thedetermination of the specificity or cross-reactivity of antibodies. The optimisation of antibody arrays will also be described.

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Speaker Abstracts

026 • Friday, March 28 • Systematic Analysis of Protein Activities and Protein Arrays • MacBeath

Protein Microarrays: From Functional Genomics to Protein Profiling

Gavin MacBeath, Assistant ProfessorDepartment of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138

The system-wide study of protein function presents both a difficult and exciting challenge in this information-rich age of biology. We areapproaching this problem in two ways. First, we are developing and using protein microarray technology to elucidate molecular interactionswithin defined families of related proteins. In particular, we are focusing on families of protein domains – such as the PDZ domains – thatmediate interactions by binding linear peptides in a sequence-specific fashion. Second, we are adapting protein microarray technology toscreen small molecule libraries for compounds that disrupt or otherwise modulate these interactions in a highly specific fashion. Ultimately,we intend to study the cellular effects of these compounds at a systems level using focused profiling technologies. To this end, we are alsodeveloping methods to measure the abundance and post-translational modification state of multiple proteins in complex solutions (such ascellular lysates) using microarrays of antibodies. Our recent progress in these efforts will be reported.

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Speaker Abstracts

027 • Friday, March 28 • Systematic Analysis of Protein Activities and Protein Arrays • Gold

Aptamer Arrays and Proteomics

Larry Gold, Chairman of the Board and CSO, SomaLogic, Inc.

Photoaptamers, derived from the PhotoSELEXTM process which substitutes a brominated deoxyuridine (BrdU) for the thymidine (T)normally found in DNA, are high-affinity, high-specificity ssDNA compounds that can function as capture agents for proteins. Thephotocrosslinking of the photoaptamer’s BrdU to an electron rich amino acid side chain of the protein adds a second dimension of specificityto the protein binding event, comparable to the use of two antibodies in immunoassays.

Multiplexed photoaptamer-based arrays have been developed for candidate proteins, and allow for the simultaneous measurement of allproteins of interest without concern for crossreactivity of secondary binding agents. Since photoaptamers are covalently bound to their targetanalytes before signal detection, the arrays can be vigorously washed to remove background proteins, yielding superior signal-to-noise ratiosand lower limits of quantitation.

Multiplexed protein measurements will facilitate the quantitative analysis of numerous clinical questions such as the degree of tumor staging,potential drug responsiveness, or likely aggressiveness of a tumor. The greater analyte densities afforded by photoaptamer arrays are a keycomponent for this change of paradigm to more informative multiplexed analyses.

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Speaker Abstracts

030 • Friday, March 28 • Structural Proteomics • Sali

Modeling the Structure of Proteins and Macromolecular Assemblies

F. Alber, S. Dokudovskaya*, L. Veenhoff*, W. Zhang*, T. Suprapto*, J. Kipper*, B. Chait, * M. Rout*, A. Sali. University of California, SanFrancisco, CA. *The Rockefeller University, New York, NY.

The structures of most protein domains will eventually be characterized by structural genomics, which aims to determine most protein folds byexperiment, allowing the remaining protein sequences to be modeled with useful accuracy by computational methods. In the case of assemblies,however, the structure is usually obtained by a number of experimental methods of varying accuracy and resolution (eg, X-ray crystallographyof the subunits, low-resolution electron microscopy of the assembly, and chemical cross-linking). Therefore, there is a need for a computationalframework that can take into account all available information about the structure of an assembly and calculate at the appropriate resolution allmodels that are consistent with the given input. To this end, it is useful to express structure determination as an optimization problem. The threecomponents of this approach are (i) representation of an assembly; (ii) a scoring function consisting of individual spatial restraints; and (iii)optimization of the scoring function to obtain the models. This approach will be illustrated by the modeling of the yeast nuclear pore complex.

NIH CA89810 R33

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Speaker Abstracts

031 • Friday, March 28 • Structural Proteomics • Taylor

Dynamic Integration of Signaling by PKA.

S.S. Taylor, L. Burns-Hamura, G. Anand, P. Akamine, F. Ma, University of California, San Diego, Howard Hughes Medical Institute. La Jolla, CA 93093-0654.

Cyclic AMP is an ancient signaling molecule and in mammalian cells one of its primary targets is cAMP-dependent protein kinase (PKA). PKAis a prototype for the large protein kinase superfamily and the catalytic (C) subunit provides us with a template for the folding and dynamics ofthe protein kinase core. The very different properties of the two lobes, one highly dynamic and the other very stable, contribute to an extendedactive site, mostly preformed, for docking ATP and protein substrates. In addition, the extensive conserved networks that radiate outward fromthe site of phosphoryl transfer provide many opportunities for communication across long distances. The regulatory subunits harbor the cAMPbinding modules that regulate the activity of the enzyme. The dynamic properties of the R subunits, specifically how the cAMP binding modulestoggle between a cAMP bound state and the holoenzyme state has been probed by hydrogen/deuterium exchange coupled with massspectrometry. In addition to the R and C subunits, the enzyme is anchored to scaffold proteins referred to as A Kinase Anchoring Proteins(AKAPs). By probing the binding properties of a dual specific AKAP, DAKAP2, that binds to RI and RII, we have developed isoform-specificbinding peptides that mimic the AKAP PKA binding site. These probes have been used in vitro and in cells to establish the importance of thebinding of RI and/or RII subunits. In addition to probing the spatial organization of the type I and II holoenzymes in cells, we have usedrecombinant FRET-based activity reporters to probe the activity of PKA in living cells. These multiple strategies have allowed us to betterappreciate the dyanmic features of the this enzyme and its targeting in living cells while high resolution structure analysis has provided anunderstanding of the molecular basis for the isoform-specific targeting and activation of PKA.(This research has been funded by grants from the NIH and by the Howard Hughes Medical Institute.)

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Speaker Abstracts

033 • Saturday, March 29 • Control and Detection of Protein Abundance and Activity • Morris

TRANSLATING THE TRANSCRIPTOME: High Throughput Analysis of mRNA Translation

David R. Morris1,Vivian L. MacKay1, Xiaohong Li2, Kyle A. Serikawa1, Xie L. Xu2, G. Lynn Law1, Lue Ping Zhao2, and RogerBumgarner3, Departments of 1Biochemistry and 3Microbiology, University of Washington, Seattle, WA 98193 and 2Division of Public HealthSciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109

The transcriptome provides the database from which a cell assembles its collection of proteins. However, translation of individual mRNAspecies into their encoded proteins can be regulated, sometimes resulting in discordance between the level of an mRNA and the abundance ofits protein product. We have reported a technology, Translation State Array Analysis or TSAA, that simultaneously assesses the level of anmRNA and its association with the translational machinery. Detailed descriptions through TSAA of the translational behavior of 4931transcripts of Saccharomyces cerevisiae show that, indeed, there is a striking diversity across the constellation of mRNA molecules.Transcripts of different genes can vary from complete engagement with polysomes to total sequestration in mRNP particles. Of those mRNAsprimarily located in polysomes, the average ribosome spacing on the message can vary by more than an order of magnitude. Thus, thisspectrum of translational behavior provides an extraordinary dynamic range of regulation, exclusive of actual transcript levels. These diversetranslational properties do not show a strong correlation with global mRNA properties such as transcript abundance, codon bias, initiator AUGcontext or estimated lengths of the untranslated regions. For example, abundant mRNAs tend on average to be better translated than thepopulation as a whole, but some representatives of this class, such as the mRNA products of GCN4 and HAC1, are located only to a minorextent in polysomes. On the other hand, low abundance mRNAs often localize greater than 90% in polysomes, examples being the CDC28,CLB3 and ADE1 products. These results are consistent with each mRNA having its own set of unique structural properties that confer itsspecific translation activity. The information garnered from this analysis allows one to estimate relative rates of protein production across thetranscriptome, as well as providing new insights into this additional level of gene expression control.

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Speaker Abstracts

035 • Saturday, March 29 • Control and Detection of Protein Abundance and Activity • Cravatt III

Activity-Based Protein Profiling: Chemical Approaches for Functional Proteomics

Benjamin F. Cravatt, Departments of Chemistry and Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA92037, Fax: 858-784-2798, [email protected]

The field of proteomics aims to characterize dynamics in protein function on a global scale. However, several classes of enzymes are regulatedby posttranslational mechanisms, limiting the utility of conventional proteomics techniques for the characterization of these proteins. Ourresearch group has initiated a program aimed at generating chemical probes that interrogate the state of enzyme active sites in wholeproteomes, thereby facilitating the simultaneous activity-based profiling of many enzymes in samples of high complexity. Progress towards thegeneration and utilization of active site-directed chemical probes for the proteomic characterization of several enzyme classes will bedescribed. These enzyme classes fall into two general categories: 1) enzymes for which active site-directed affinity agents have been well-defined, and 2) enzymes for which active site-directed affinity agents have been lacking. The application of activity-based protein profiling tothe functional characterization of enzyme activities that vary in human cancer specimens will be highlighted, as will be the use of this strategyas a screen to discover potent and selective reversible enzyme inhibitors.

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Speaker Abstracts

036 • Saturday, March 29 • Control and Detection of Protein Abundance and Activity • Grayhack

Functional Proteomics

Elizabeth Grayhack, Andre Alexandrov, Jane Jackman, Feng Xing, Weifeng Gu, Yoshiko Kon, Martha Wilkinson, Michael White, Mark Dumont, and Eric Phizicky, University of Rochester, School of Medicine and Dentistry, Rochester, NY 14642

To facilitate biochemical analysis of the yeast proteome, a genomic array of S. cerevisiae GST-ORF fusions was produced in yeast by fusingeach ORF to GST. Purification and biochemical assay of pools of GST-ORF proteins allows analysis of the proteome for biochemicalfunction. Assignment of biochemical activity is rapid, versatile, and sensitive using this method. Both binding and catalytic assays have beenused to detect more than 35 proteins (and their cognate genes) with this method. The catalytic activities include a wide variety of differentchemical reactions (ligation, group transfer, redox, isomerization, phosphorylation, and hydrolysis). Proteins that act in a complex have alsobeen with this system. In collaboration with Mike Snyder, Erin O’Shea, and Jonathan Weissman, we are producing a new C-terminal fusionarray that should be more useful for membrane proteins, and will have a number of improved features.

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Speaker Abstracts

038 • Saturday, March 29 • Methods to Explore Function • Muir

From Protein Semisythesis to Chemical Genetics: The Chemical Biology of Protein Splicing

Tom W. Muir. The Rockefeller University, 1230 York Ave, New York City, NY 10021

Protein splicing is a posttranslational process in which an intervening sequence, an intein, is removed from a host protein, the extein. In proteintrans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. We have shown that thiolysis ofmutant intein-fusions leads to the generation of recombinant protein-thioester derivatives that can be chemically ligated to polypeptides (syntheticor recombinant) bearing an N-terminal Cys. This semisynthetic process, generally referred to as Expressed Protein Ligation (EPL), has been usedto incorporate unnatural amino acids, posttranslational modifications and isotopic probes site-specifically into proteins. EPL has been applied tonumerous systems over the last few years and has allowed a variety of biological questions to be addressed. Selected examples from our ownwork will be discussed. A second application of protein splicing that will be discussed is in the area of chemical genetics. We have recentlydeveloped a novel system that allows protein trans-splicing to occur only in the presence of the small molecule, rapamycin. This ‘conditionalprotein transsplicing’ (CPS) technique provides a means to trigger the post-translational synthesis of a target protein from two fragments. Inprinciple, CPS provides a level of temporal control over protein function that is difficult to achieve using standard genetic approaches. Recentprogress in this area will be described.

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Speaker Abstracts

039 • Saturday, March 29 • Methods to Explore Function • Marcotte

Global Analysis of Protein Pathways, Systems, and Interactions

Alex Adai, Shaliesh V. Date, Brie Fuqua, Peng Lu, Aleksey Nakorchevskiy, John Prince, Arun Ramani, Rong Wang, and Edward M. MarcotteInstitute for Cellular and Molecular Biology, Center for Computational Biology & Bioinformatics, University of Texas at Austin, 1 University Station, A4800, Austin, Texas, 78712-0159

About half of the roughly 40,000 genes encoded by the human genome, as in every other genome sequenced to date, are completelyuncharacterized and of unknown function. There is a broad need for methods to discover the functions of these thousands of uncharacterizedgenes and how the corresponding proteins participate in networks, pathways, and systems in the cell. It has now become clear that thegenomes themselves contain extensive information about the relationships between the proteins. To begin to reveal the functions of theseproteins, and how the proteins associate into systems and pathways, we have developed computational approaches for inferring the functionsof proteins that have never before been studied. These methods work by examining the contexts of genes, such as which organisms carry thegenes, the occurrence of gene fusion events, and the relative positions of genes on the chromosomes. Proteins whose genes occur in similarcontexts can be shown to work in similar cellular pathways. By using such methods, cellular networks consisting of thousands of proteinsfrom a single organism can be reconstructed. The networks contain many known cellular systems, as well as potentially novel pathways andsystems.

Supported by grants from the Welch Foundation, the National Science Foundation, the Texas Advanced Research Program, a Dreyfus NewFaculty Award, and a Packard Fellowship.

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Speaker Abstracts

040 • Saturday, March 29 • Methods to Explore Function • Bork

Comparative Analysis of Protein Interaction Networks

Peer BorkEMBL, Meyerhofstr.1, 69012 Heidelberg and MDC, [email protected]

Recent advances in proteomics and computational biology have lead to a flood of protein interaction data and resulting interaction networks(e.g. [1]). Here I first analyze the status and quality of parts lists (genes and proteins), then comparatively assess large-scale protein interactiondata [2] and finally try to identify biological meaningful units (e.g. pathway, cellular process) within interaction networks [3] that are derivedfrom the conservation of gene neighborhood, gene fusion or gene co-occurrence [4]

[1] Gavin AC et al. Nature. 2002 Jan 10;415(6868):141-7[2] von Mering C, Krause R, Snel B, Cornell M, Oliver SG, Fields S, Bork P. Nature. 2002 May 23;417(6887):399-403[3] Snel B., Bork P, Huynen MA. Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):5890-5[4] von Mering C, Huynen MA, Jaeggi D, Schmidt S, Bork P, Snel, B. Nucl.Ac. Res. 2003, in press

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Poster AbstractsWednesday, March 26:Identification and Quantification of Proteins/Management of Proteomics Data

Analysis of redox sensitive proteins by tandem massspectrometry

Nancy Andon1, Robert Cumming2, David R. Schubert2, JohnR.Yates III, and Paul A.Haynes1 1The Torrey Mesa ResearchInstitute of Syngenta, San Diego, CA, USA, 92121; 2The SalkInstitute for Biological Studies, San Diego, CA, USA, 92121

The toxicity of chemically reactive oxygen species isthought to make a significant contribution to the death of nervecells in stroke, Parkinson’s disease and Alzheimer’s disease.

We have developed an oxidative stress model in whichmurine hippocampal glutamate sensitive and resistant cell linesare exposed to glutamate, and then compared using a modified 2-D system, which allows us to specifically identify proteins whosedisulfide binding properties change with oxidative stress. We havealso used this technique to test a severe oxidative stress model, inwhich the glutamate resistant and sensitive cell lines wereexposed to either diamide or H2O2 .

A number of changes in protein interactions wereidentified between the stressed and control cell lines. Of 117unique proteins identified, the vast majority are heat shockproteins, which are known to be involved in oxidative stress.Several other proteins, including elongation factor-1, calreticulin,oxoglutarate dehydrogenase, and GAPDH, all of which are knownto be involved in either oxidative stress or apoptosis were alsoidentified, along with a number of other proteins implicated inthese pathways, and, most interestingly, a number of proteins ofas yet unknown function.

Longitudinal Study of Rheumatoid Arthritis (RA) byMass Spectrometry: Serum Proteins and Metabolites

H. Lin, W. Wang, S. Roy, T. A. Shaler, H. Zhou, L. Hill, G. Frenzel,J. Thompson, M. Anderle, P. Kumar, and C. H. Becker, SurroMed,Inc., 2375 Garcia Ave., Mountain View, California 94043 USA

SurroMed has enrolled > 160 subjects in a longitudinal registry tocollect serum, urine, and clinical outcome measures at multipletimepoints over the course of 3 years. An interim analysis of thebaseline timepoint was performed on 20 subjects with chronic RAand 20 healthy subjects that were matched for age, gender, and co-morbid conditions.RA is a chronic inflammatory disease. There is an unmet clinicalneed to identify biomarkers that are predictive of progression ofdisease as well as response to therapeutics. The interim analysis is apromising first step towards realizing that goal as it successfullyrevealed expected and novel molecules. With < 1 mL of serum, peptides and metabolites were quantitativelyprofiled on high-resolution time-of-flight instruments using on-lineliquid chromatography and electrospray ionization (LC-ESI-MS) fornonvolatile molecules and GC-MS for volatile molecules. Usingone-dimensional chromatography, 2500 molecular ions werequantified per sample yielding approximately 70 differences at p <0.001 (~ 3 expected by chance). The analysis is currently beingextended using two-dimensional chromatography. Molecular identification is made using tandem mass spectrometrywith ion-trap and triple-quadrupole instruments along with computeranalysis of the fragmentation spectra.

Identification of the surface interacting protein of decoyreceptor 3

Yung-Chi Chang, Shie-Liang Hsieh, Institute and Department ofMicrobiology and Immunology, National Yang-Ming University,Taipei, Taiwan

Decoy receptor 3 (DcR3) is a soluble receptor belonging to the tumornecrosis factor receptor (TNFR) superfamily and is readily detected incertain cancer patients. Recently, we further demonstrated that DcR3-treated dendritic cells (DCs) skew T cells response to Th2 phenotype.Moreover, the presence of DcR3 also downregulated thedifferentiation and function of CD14+ monocytes into macrophages.We then want to address which surface molecule can exert suchfunction. The monocytic cell line, THP-1 was lysed in lysis buffer andthen enriching the glycoprotein fraction of cell lysate using ConAcolumn. Finally the eluant of ConA column was further purified byDcR3.Fc affinity column and subjected to MALDI-TOF and LCMS/MS analysis. The potential molecule was about 67 kDa and highglycosylated, and the biological function of molecule should be furtherverified.

This work was mainly supported by grants NSC 89-2320-B-010-124,NSC 90-2320-B010-109 from the National Science Council, Taiwan.

Withdrawn

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Poster Abstracts Wednesday, March 26:Identification and Quantification of Proteins/Management of Proteomics Data

Systematic, quantitative proteomic analysis of theSaccharomyces cerevisiae cell cycle

Mark R. Flory, Hookeun Lee, Kyle Serikawa, Roger Bumgartner, DavidMorris, David Goodlett, Ruedi AebersoldInstitute for Systems Biology, Seattle, WA 98103

Fundamental to a complete understanding of the structure, functionand control of biological systems is the ability to quantitatively analyzeproteins, the catalysts and effectors of nearly all biological activities. To thisend, we are using quantitative mass spectrometry for the systematicmeasurement of cell cycle-regulated protein expression levels inSaccharomyces cerevisiae following release from mating factorsynchronization. Protein samples harvested from both timepoint andreference (asynchronous) cultures were reduced and labeled with isotopicallyheavy or light ICAT (isotope-coded affinity tag) reagent prior to trypsindigestion. ICAT-labeled peptide mixtures separated by off-line liquidchromatography were analyzed by reverse-phase tandem mass spectrometry.Using a second-generation acid-cleavable ICAT reagent we achieve aconsistent sampling depth over 1500 proteins.

The proteomic profiles were integrated with translation andtranscription expression profiles from the same yeast culture to create acomposite, simultaneous description of three modes of expression. The datawere analyzed using cluster analysis tools such as Cytoscape. Thecombination of three types of data representing gene expression at differentlevels extends traditional profiling approaches that measure only transcriptlevels. This dataset is expected to catalyze the development of informaticstools for the integration and analysis of large, diverse datasets and to increasethe understanding of the eukaryotic cell cycle.

Novel Reagents for Quantitative Proteomics.

M.H. Gelb, P. Bottari, Y. Li, D. Wang, F. TurecekDepts. of Chemistry and Biochemistry, Univ. of Washington, Seattle,WA 98195

R. Aebersold, A. Tao, H. ZhangInstitute for Systems Biology, Seattle, WA

We have synthesized a variety of novel reagents for the quantitativeanalysis of enzymes in complex mixtures (cell lysates). Thetechnique is based on biotinylated substrates combined with heavyisotope-containing product internal standars. Mass spectrometry isused for analysis after biotin conjugates are captured by solid phasestreptavidin. It will be shown that the technique is applicable to theanalysis of virtually any enzyme and is fully automable forapplication in clinical or research laboratories. Furthermore, severalenzymes can be quantified in a signal analysis. A related technique isthe development of Isotope Coded Affinity Tags (ICAT). Newgeneration ICAT reagents have been designed and are being testedfor quantitative analysis of specific protein functionalities.

A Protein Chemist’s Guide To The Gel-Free ProteomeGalaxy

Kris Gevaert, An Staes, Marc Goethals, Jozef Van Damme, Lennart Martens,Hans Demol, Bart Ghesquière, Sara De Groot, Magda Puype, Grégoire R.Thomas & Joël Vandekerckhove, Department of Medical Protein Research,Ghent University, B-9000 Ghent, Belgium.

We have developed a highly versatile procedure for the isolation of subsetsof peptides out of complex mixtures such as proteolytic digests of total cellor tissue extracts without the use of affinity tags. Because of its homologyto diagonal electrophoresis and diagonal chromatography, we have calledthis novel procedure combined fractional diagonal chromatography orCOFRADIC® (Gevaert et al., Mol. Cell. Proteomics, in press). Following aproteolytic digest, peptide mixtures are fractionated by reverse phase (RP)HPLC and in each fraction, representative peptides (i.e. peptides containingsufficiently rare amino acids) are altered such that their chromatographicbehaviour changes. Upon a secondary RP-HPLC separation, these alteredpeptides elute differently and are specifically collected for further sequenceanalysis.

COFRADIC® is a highly versatile technique since it allows to isolate anypeptide containing a group that can be specifically altered. We here showthat peptides containing methionine and/or cysteine as well asphosphorylated peptides and peptides spanning the amino terminus can bespecifically isolated and used to identify their parent proteins. Furthermore,the complete procedure of peptide isolation, LC-MS/MS analysis andpeptide/protein identification is hugely automated. The use of COFRADIC®

for proteome studies will be addressed by the results of various proteomestudies in which classes of proteins that frequently escape analysis in gel-based studies, are now clearly identified. These include highly hydrophobicproteins, low abundant proteins, extremely large proteins and highlycharged ones.

Parallel Peptide Tandem Mass Spectrometry on a Time-of-flight MassAnalyzer

Eugene C. Yi, Ning Zhang, Marcello Marelli, John Aitchison, Benno Schwikowski andDavid R. Goodlett

Mass spectrometry (MS) is currently the most important discovery based tool in the fieldof Proteomics (1). It is used extensively for indirect descriptive analysis of proteins byanalyzing peptides produced by proteolytic action on proteins. Two methods arecommonly used to identify proteins through database search of MS data acquired onpeptides: 1) mass fingerprinting and 2) tandem MS (MS/MS). In our laboratorymicrocapillary HPLC (µLC) MS/MS in an ion trap (IT) mass spectrometer is thepreferred method. However, we have recently shown that standard data-dependent (DD)ion selection during µLC/MS/MS when applied to very complex samples reproduciblyidentifies the same proteins (~ same for peptides) in replicates 25% of the time (2). Thereasons for poor reproducibility are complex, but include: 1) high sample complexity, 2)low MS duty cycle with serial ion selection and 3) poor chromatographic peak capacity.To increase protein coverage during µLC we have begun to explore parallel collisioninduced dissociation (CID) of peptides in-source on an ESI-TOF-MS and in collision cellon an ESI-QTOF-MS. Parallel MS/MS of peptides rather than the current standard ofserial MS/MS has been previously demonstrated using the high mass accuracy/resolutionof Fourier transform ion cyclotron resonance MS (FTICRMS), referred to as multiplexMS (3), to de-convolute the mixed fragment ion mass spectra. We refer to the parallelMS/MS method carried out on lower mass accuracy/resolution TOF mass analyzers asshotgun MS/MS to distinguish it from the FTICRMS method. A combination of massfingerprinting, MS/MS and chromatographic synchronization of parent to fragment ionsis used to identify proteins from mixed fragment ion mass spectra of peptides.Additionally, the method may be used to quantify relative changes in protein expressionby incorporating stable isotope labeling. When differential stable isotope labeling isincorporated, then sequence tags may be generated to aid cases of degeneracy at theprotein level that arise from the lower mass accuracy of a TOF mass analyzer. In additionto proof of principle using standards, we will present some initial data using shotgun CIDto characterize membrane proteins isolated from yeast peroxisomes and discussbioinformatic approaches to identification of parent proteins from tandem MS dataproduced by fragmenting peptides in parallel rather than in series.1. Aebersold, R. and Goodlett, D.R., Chem Rev, 2001, 101, 269.2. Eugene C. Yi et al. Electrophoresis 2002, 23, 3205.3. Masselon, C Anal Chem 2000, 72, 1918.

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Proteins induced by organophosphate exposure in OP-susceptible and -resistant strains of the southern cattle tick

Felix D. Guerrero and Andrew Y. Li, USDA-ARS Livestock InsectsResearch Laboratory, Kerrville, TX, USA, 78028

Organophosphate (OP) resistance has developed in Mexicanpopulations of the southern cattle tick, Boophilus microplus.Reintroduction of this tick and its associated pathogens into theUnited States would cause severe economic losses to the cattleindustry. We have begun a comparative study of the proteome ofOP-susceptible and -resistant strains of B. microplus. As an initialstep, we have exposed larvae from an OP-susceptible strain, Munoz,and an OP-resistant strain, San Roman, to 0.03 and 0.4% coumaphos,respectively, which are sublethal doses, well below the LD50 foreach strain. Additionally, untreated larvae from each strain have alsobeen collected. Soluble proteins were extracted from each sampleand analyzed by 2-dimensional IEF-PAGE to identify OP-inducibleproteins and strain-specific protein expression.

Biomarkers of Inflammation from SELDI Analysis ofRat Serum after Acute Lipopolysaccharide (LPS) Treatment.

JE Hartis, A Xu, ME Bruno, BA Wetmore, JA Taylor, BA Merrick.NIEHS, PO Box 12233, RTP, NC 27709; Ciphergen, Freemont, CA94555

Bacterial LPS can induce septic shock by release of cytokines andvasoactive peptides. SELDI-MS (surface enhanced laser desorptionionization) analysis of serum was conducted for biomarker discoveryof circulating bioactive peptides during acute inflammatory responseto LPS in a rat model. Male rats at 12 rats per group were treated byi.p. with saline or 5 mg/kg, LPS for 2 and 6 hr. Serum TNFα levelswere high at 2 hr after LPS but returned to control after 6 hr. Organhistopathology was limited to vascular endothelial changes. Massspectra (MS) were measured from surface retentates of whole serum ormultidimensional fractionation (MDF) of serum by strong anionexchange and step-wise pH elution. SELDI analysis of whole serumshowed 4 ion peak changes compared to 36 peak changes at p<0.01 inMDF serum at 2 hr. At 6 hr after LPS, 20 peak changes were found inMDF serum compared to 2 peak changes with whole serum. About twofold more ions were increased as were repressed in whole serumcompared to MDF serum. Comparison of LPS treated rats at 2 and 6hr revealed 17 ion changes, none of which were found in whole serum.Prominent ions in each fraction are being isolated for further MSanalysis to obtain the identity of the underlying proteins. We concludeSELDI analysis of MDF serum fractions is a more sensitive means ofdetecting LPS responsive biopeptides than from whole serum alone.

Identification of Ubiquitinated ER Proteins

Amy L. Hitchcock, Kathryn Auld, Seth Frietze, Steven P. Gygi, and PamelaA. SilverDepartments of Cancer Biology, Biological Chemistry and MolecularPharmacology, and Cell Biology; The Dana Farber Cancer Institute andHarvard Medical School, Boston, MA USA 02115

The highly conserved Cdc48/p97-Npl4-Ufd1 protein complex is requiredfor the proteasome-dependent degradation and processing of ubiquitinatedendoplasmic reticulum (ER) proteins. As such, Saccharomyces cerevisiae cellscontaining the conditional npl4-1 mutation display a striking accumulationof ER-associated ubiquitinated proteins. However, it is unknown whetherall ubiquitinated ER proteins require Npl4p function for proteasomaldegradation, or if there might be an Npl4p-specific pathway fordegradation of a subset of ubiquitinated ER proteins. In the current study,a combined biochemical/proteomic approach has been undertaken toidentify (1) all ubiquitinated ER proteins in yeast, and (2) the ubiquitinatedER substrates of Npl4p-dependent proteasomal degradation. Using a yeaststrain expressing 6xHis-tagged ubiquitin, we have purified ubiquitin-conjugates from wild type and npl4-1 ER membrane fractions byimmobilized Ni++ affinity chromatography. Proteins present in the boundfraction are identified by direct mixture analysis of tryptic peptides usingnano-scale microcapillary LC-MS/MS. Currently we have identified >100putative ubiquitinated ER proteins from wild type membranes. Theseproteins are qualitatively compared to those isolated from npl4-1membranes, as well as to a negative control strain. To quantitativelyidentify ubiquitinated proteins that are specifically stabilized in npl4-1 ERmembranes, we will directly compare wild type and npl4-1 ubiquitinconjugates by combining the above strategy with isotope-coded affinitytags (ICAT). Ultimately, this approach could be utilized to identify, forexample, the substrates of specific E2 or E3 enzymes, or proteinsdifferentially ubiquitinated in response to extracellular stimuli.

Quantitative Fungal Proteome Analysis FollowingSeparation by Multidimensional Chromatography

P. T Jedrzejewski, G. Ganshaw, A. GaertnerGenencor International, Inc., 925 Page Mill Road, Palo Alto, CA 94304,USA

The filamentous fungus Trichoderma reesei is an importantbiological host for the production of industrially relevant enzymes, suchas biomass degrading cellulases, and enzymes for food and beverageprocessing. In order to optimize enzyme production for this fungalhost, and to design the appropriate enzyme mix for its respectiveapplication, a number of approaches can be utilized, for example,genes can be knocked out or added and fermentation conditions canbe manipulated. Optimization of the fermentation conditions may beimproved by gaining insight at the proteome level (proteinidentification, protein metabolism).

We have applied multidimensional chromatography for theseparation and purification of the exoproteome of a cellulase producingstrain of the T. reesei. Following multidimensional chromatography(ion-exchange chromatography followed by various formats ofreversed-phase chromatography) on the protein level, samples weredigested and analyzed by nanoLC-MS/MS. Protein identification wasperformed using database search software.

We successfully identified over 35 proteins from this fungalexoproteome. This compares favorably with other proteomic methods(e.g. 2D-PAGE) in terms of throughput as well as number of proteinsidentified. We show the results of quantitative characterization for anumber of identified proteins across various fermentation conditions.

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PROTEOMIC STRATEGY FOR IDENTIFICATION OF GPI-ANCHORED PROTEINS IN H. SAPIENS AND A. THALIANA PLASMAMEMBRANES.

Felix Elortza1, Allan Stensballe1, Leonard Foster1, Thomas Nühse2, Scott Peck2,and Ole N. Jensen1.

1Department of Biochemistry and Molecular Biology, University of SouthernDenmark, Campusvej 55, DK 5230 Odense M, Denmark 2Sainsbury Laboratory, Norwich Research Park, Colney, Norwich NR4 7UH, UK.

In eukaryotic cells, a subset of proteins are attached to the external leaflet of theplasma membrane by a glycosylphosphatidylinositol (GPI) anchor. There issubstantial evidence suggesting that these GPI-anchored proteins are clustered insphingolipid-sterol microdomains or lipid rafts. Rafts play a crucial role in manycellular processes including membrane traffic, cell signalling and human diseases.We have combined detergent-based phase separation methods and enzyme treatmentto enrich and isolate GPI-proteins from lipid rafts from human cells and GPI-proteinsfrom plasma membrane fractions isolated from Arabidopsis thaliana. The isolatedGPI-protein fractions were precipitated with acetone, separated by SDS-PAGE andvisualized by silver staining. Protein bands were excised for in-gel digestion and theresulting peptides were subsequently analyzed by nanoscale liquid chromatographycoupled online to electrospray Q-TOF tandem mass spectrometry (nLC-MS/MS).Tandem mass spectra were interpreted automatically using the MASCOT sequencedatabase search engine. With this combination of methods we have identified morethan 50 GPI anchored proteins in H. sapiens and A. thaliana, validating thisproteomic approach for analysis of GPI anchored membrane proteins and generatingthe largest experimental data set on GPI-proteins to date. The technique is expectedto be generally applicable and highly useful for studying membrane proteins, forexample for determination of molecular markers on the cell surface for diagnostic ortherapeutic purposes.

This research was supported by a post-doctoral fellowship to F.E. from the Basquegovernment and by resources provided by the Danish Biotechnology InstrumentCenter.

Selective Chemical Profiling of PPAR Agonists Utilizing ProteomicsTechnology

Linda Kochanski, Anita Y. Lee, Patrick Griffin*, Margaret E. McCann††, Ching H.Chang†, Ellen Rohde and William A. Hanlon. Departments of Molecular ProfilingProteomics Research, Basic Chemistry† and Pharmacology††. Merck ResearchLaboratories, Rahway, NJ 07065. ExSAR, Monmouth Junction, NJ 08852*

Peroxisome Proliferator Activated Receptors (PPARs) are nuclear transcription factorsthat regulate genes involved in lipid and glucose homeostasis. Three PPAR isotypes exist.PPARδ is ubiquitously expressed. PPARα, which is highly expressed in hepatocytes,results in peroxisomal proliferation and β-oxidation of fatty acids in rodent liver whenactivated. PPARγ, which is predominately expressed in adipose tissue, is responsible foradipocyte differentiation, fat storage and improved insulin signaling of matureadipocytes. PPARγ and α receptors may function as targets for the treatment ofdyslipidemia and insulin resistance. It has been found that activation of these PPARreceptor isotypes causes different effects in the liver of rodents. Sustained induction ofperoxisome proliferation and the peroxisomal β-oxidation system in liver of rodentschronically treated with peroxisome proliferators leads to development of hepatocellularcarcinomas. To better understand the toxicity associated with PPAR agonists wecompared proteomic profiles of liver tissue from mice treated for up to 7 days with aselective α agonist (Compound 1) and two γ selective agonists, rosiglitazone (Compound2) and a Merck TZD (Compound 3), both of which are from different chemical classes.Utilizing high-resolution two dimensional difference gel electrophoresis (2D-DIGE),Decyder spot detection and matching software and µLC-MS/MS we detected proteinexpression profile changes and identified proteins showing altered expression. Sixtyeight proteins, induced by the PPARα agonist, had altered protein expression levels.Forty of these proteins were unique to the PPARα agonist. Treatment with the MerckTZD and rosiglitazone induced altered expression in 24 and 26 total proteinsrespectively. Thirteen of the proteins with altered expression were common between allthree agonists. Proteins with altered expression were identified as enzymes involved inperoxisome proliferation and β-oxidation of fatty acids. Various isoforms of theseenzymes, which were differentially expressed in response to drug treatment, wereidentified as well. The identification of these proteins demonstrates that proteinexpression maps produced using 2D electrophoresis can be used to profile compounds ofdifferent chemical classes.

Use of discovery science-approach to elucidate biological effects induced bymobile phone radiation: A pilot study

1Dariusz Leszczynski, 1Reetta Kuokka, 1Sakari Joenväärä, 1Jukka Reivinen, 1Teemu Kallonen,2Kari Jokela, 2Ari-Pekka Sihvonen, 2Tim Toivo, 3Niels Kuster and 3Juergen Schuderer; 1Bio-NIR Research Group & 2NIR Laboratory, STUK-Radiation and Nuclear Safety Authority,Helsinki, Finland, 3ITIS, Zurich, Switzerland; [email protected]

Elucidation of the biological or health effects of the electromagnetic fields (EMF), e.g. thoseemitted by electrical power lines or mobile phones, has been done for decades but the reliableanswers are still missing. Extensive epidemiological studies are commonly expected to give theanswers. However, finding and scientific validation of any potential health hazard (whether it iscancer or non-cancer effect) might not be possible using epidemiological approach alone becauseof the “low sensitivity” of this method. Recently, it has been suggested that high-throughputscreening techniques (HTST) will be crucial in providing important information for thoroughdetermination of all possible bio-effects of the EMF (Leszczynski, Lancet 358, 2001, 1733).The “discovery science” postulates use of combination of data obtained by transcriptomics (globalgene expression) and proteomics (global protein expression) to enumerate the behavior ofbiological system (Aebersold et al., Nature Biotechnology 18, 2000, 359). This approach seemsto be particularly suited for elucidation EMF health hazard issue because it might reveal a varietyof unpredictable biological effects. Also, it might provide valuable information about the potentiallong term chronic exposure effects that are at this time completely unknown.There are only a handful of published studies and conference abstracts where HTST were used toelucidate biological responses to EMF. We have used HTST to examine gene and protein changesthat occur in two human endothelial cell lines: fast proliferating EA.hy926 and its slowproliferating variant EA.hy926v1. Cells were exposed for the 1-hour exposure to 900 or 1800MHz GSM signal at an average specific absorption rate (SAR) of ∼2.0 - 2.4 W/kg and attemperature of 37±0.1-0.3oC. We found that few tens of genes (out of 3600 examined) alter theirexpression level. This was accompanied by changes in the activity of few hundreds of proteins(based on the protein phosphorylation status) and changes in the expression level of several tensof proteins (out of ∼1300 examined). Combination of the HTST data on gene and proteinexpression and protein activity has revealed several signal transduction pathways that might beaffected by GSM signal exposure. E.g. activation of hsp27/p38MAPK stress response pathwayaltered stability of stress fibers and caused endothelial cell shrinkage. This, if occurring in vivo inbrain capillaries might affect stability of blood-brain barrier (Leszczynski et al. Differentiation 70,2002, 120).In conclusion, although the use of discovery science approach employing HTST will not providedirect evidence of health hazard or its absence, it will be essential in unraveling of all biologicaleffects induced by EMF exposure. Further elucidation of the physiological significance of thesebiological effects for the health and well-being, in short- and long-term exposure conditions, willallow determination whether any health hazard might be associated with the use of EMF-emittingdevices at the present safety levels.Acknowledgements: Pia Kontturi and Hanna Tammio for help in performing biologicalexperiments. Funding support: the Finnish Technology Development Center – Tekes (LaVitaproject), the 5th Framework Programme of the European Union (REFLEX project), the Academyof Finland (post-doc fellowship), the VerUm Foundation, Munich, Germany, and STUK, Finland.

Application proteomic approach toward mapping trans-regulatory elements of myosin heavy chain

Qishan Lin, Proteomics Core Facility, CCFG, State University of New York atAlbany, *Myomatrix Therapeutic, LLC. Rensselaer, NY12144

Rapid advances in genomic sequencing, bioinformatics, and analyticalinstrumentation have created the field of proteomics, which at present is basedlargely on two-dimensional electrophoresis separation of complex proteinmixture and identification of individual protein using mass spectrometry. Theseanalyses provide a wealth of data, which upon further evaluation lead to manyquestions regarding the structure and function of the proteins. The challenge ofanswering these questions creates a need for high-specificity approaches thatmay be used in the analysis of bio-molecular recognition events and interactingpartners, and thereby places great demands on new methodology development.It has been shown that myosin heavy chain (MHC) plays an important role inthe fetal gene program (One of the characteristic changes that occur in a failingheart is a change in gene expression wherein genes that were turned off shortlyafter birth are reactivated in the disease process. This reactivation of fetal genesis known as the induction of the fetal gene program). Thus, developing a high-throughput screening assay to understand the gene switch on/off machinery willaid us in our search for compounds that would reverse this change in geneexpression and potentially reverse the progression of heart failure. In this study,nuclear fractionation, DNA promoter affinity chromatography, two-dimensional electrophoresis, multiple dimension chromatography, and massspectrometry were integrated toward mapping the trans-regulatory elements ofmyosin heavy chain in rat neonatal cardiomyocytes. To demonstrate proof ofprinciple of the working platform, transcription factors including GATA 4,P300, and MEF2 were identified.

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Identification of androgen-regulated genes in prostate cancercells using isotope-coded affinity tags and mass spectrometry

Katie L. Meehan and Marianne D. Sadar. Department of Cancer Endocrinology, BC CancerAgency, Vancouver, BC, Canada, V5Z 4E6

Prostate cancer is the most common cancer diagnosed and the second leading causeof death from cancer for Canadian men. The molecular and biochemical mechanismsinvolved in prostate carcinogenesis are unknown. However, increasing reportssuggest that androgens are involved in development and progression of prostatecancer. The objective of this study was to apply a new quantitative technology for theproteomic analysis of the mitogenic effect of androgens on prostate cancer cells.Nuclear, cytoplasmic and membrane protein fractions were isolated from androgenstimulated (10 nM of the synthetic androgen, R1881) and unstimulated LNCaP cells.Protein fractions were subjected to stringent denaturation, labelled with heavy andlight ICAT reagents respectively then combined and proteolysed. Resulting peptideswere first separated using a high-resolution cation exchange column and then furtherpurified using an avidin affinity column. ICAT-labelled peptides were analysed byLC/MS/MS. Application of cell fractionation, ICAT labelling and LC/MS/MSenabled the concurrent quantification and identification of 2502 proteins in complexprotein mixtures obtained from stimulated and un-stimulated LNCaP cells.Differential expression of proteins associated with stimulated LNCaP cells wererevealed and consistent with previously identified proteins in prostate cancer cells.These proteins included members of the cyclin family, calreticulin, c-myc, Kruppel-like factor, transforming growth factor-b, disintegrin-like metalloprotease,selenoprotein P and laminin. However, of considerable interest was the identificationand quantitative differences in expression of a large number of proteins that have notbeen previously reported to be expressed in prostate cells. Some of these proteinsincluded polyadenylate-binding protein 2, valosin, phosphoglycerate kinase 1,triosephosphate isomerase, tektin 3, sema domain, MUL-protein and PTPN13-likeprotein. In addition, many uncharacterised proteins were also detected and displayedaberrant expression in stimulated LNCaP cells compared to unstimulated. Thiscomprehensive proteome analysis has generated a differential protein expressioncatalogue containing thousands of proteins differentially expressed in prostate cancercells in response to androgens. Results from these studies will enhance ourunderstanding of the molecular pathways involved in the proliferation of prostatecancer and give rise to potential therapeutic targets and novel biomarkers.

Functional characterization of transgenic Arabidopsislines with ectopic expression of thetranscription factor gene MYB13

Schlesier, B., Bäumlein, H. & Mock, H.-P. Institute of Plant Genetics and Crop Plant Research, Corrensstrasse 3,D-06466 Gatersleben

Members of the large family of MYB transcription factors in plants areinvolved in the control of a number of cellular functions such asdevelopmental processes, hormone responses as well asphenylpropanoid metabolism. The completion of the Arabidopsisgenome sequencing enables new approaches to define the functionalsignificance of individual transcription factors in plants. The paperdescribes a proteome analysis of transgenic lines with ectopicexpression of AtMYB13 in comparison to control plants. Plants werecultivated in a vessel under controlled environmental conditions tominimize biological variation. Protein extracts of roots and leaveswere separated by 2-D gel electrophoresis. Image analysis revealeddistinct changes in protein patterns for the transgenic lines. Proteinsdifferentially expressed were identified using mass spectrometrictechniques. Results from proteome analysis are compared with dataobtained by transcript profiling.

Funding by the Deutsche Forschungsgemeinschaft to H.-P. Mock isgratefully acknowledged.

Energy coupling between the PspF AAA+ protein andRNA-Polymerase

J Schumacher; P Bordes; X Zhang and M Buck Department ofBiological Sciences, Imperial College of Science, Technology andMedicine, London SW7 2AZ, UK

Regulation of bacterial transcription by σ54 dependent enhancerbinding proteins critically involves their tri-nucleotide hydrolysis torestructure the σ54 RNA polymerase and the promoter DNA to whichit is bound, resulting in open promoter complex formation andtranscription. We show by biochemical studies and cryo-electronmicroscopy that the activated enhancer binding protein PspF fromEscherichia coli is organised in hexameric rings, demonstrating thatPspF belongs to the AAA+ superfamily. AAA+ proteins are molecularmotors that couple the energy derived from ATP hydrolysis torestructure their substrate. We have identified residues within PspF thatare directly involved interacting and energy coupling between PspFand the closed promoter complex. These residues are mapped on ahomology based PspF model to illustrate the energy couplingmechanism.

Quantitative analysis of protein complexes using solid-phase isotope tags

W. Andy Tao, Jeffrey Ranish, Ruedi AebersoldInstitute for Systems Biology, Seattle, WA 98103

The identification and accurate quantification of proteins at highthroughput are essential components of proteomic strategies forstudying cellular functions and processes. Techniques largely based onstable isotope labeling and tandem mass spectrometry are increasinglybeing applied in quantitative proteomic studies. A solid-phase approach for the accurate quantification of individualproteins within complex mixtures is described. The method uses aclass of new solid-phase chemical reagents to introduce a stableisotope tag on every peptide via its N-terminal for quantitative analysisby mass spectrometry. Using this approach, peptides derived fromimmunopurified STE12 protein complexes isolated from yeast cells indifferent states were analyzed for protein identification and changes inrelative abundance. The method is redundant since multiple peptides from each protein aretagged, allowing better accuracy in quantification. In addition, thesolid-phase based approach can be easily adapted for the automationand high throughput experiments, and for quantitative analysis ofprotein post-translational modifications.This work was supported by grants from NCI (R33, CA 93302),NHBLI Proteomics Initiative (N01-HV-28179), and from OxfordGlycoScience. W.A.T. is a Damon Runyon Fellow supported by theDamon Runyon Cancer Research Foundation (DRG 1740-02).

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Proteomic analysis of food-induced gastrointestinalhormones using SELDI proteinchip technology

*Roel Vonk, *Han Roelofsen, *He Tao, *Renate Hagedoorn, †GuidoRychen, ‡Jean-Michel Antoine.*Dept.of Pediatrics, University of Groningen, Hanzeplein 1 9713 GZGroningen,The Netherlands. †Lab. Animal Science of INRA, Nancy,France. ‡Danone Vitapole, Paris, France.

The physiological functions of gastrointestinal hormones such asincretins (insulinotropic hormones e.g. GIP and GLP-1) are more andmore recognized for their role in glucose intolerance and energy intake.In this respect interactions with other peptide hormones (resistin, leptinetc.), originating from different organs are not clear. A major drawback inthis field is the complexity of the measurement of peptide hormones. Toovercome this problem we applied SELDI proteinchip technology(Ciphergen) (see also www.medical proteomics.com). Methods: pigsprovided with permanent canula’s in the portal vein and artery were usedas a model (Rerat et al. Br. J. Nutr. 1992; 68:111-138). The pigs receivedwell characterized test meals. Blood samples of both vessels were takenbefore and after ingestion at time points up to 240 minutes. Sera werediluted 1:1 in 9 M urea/2% CHAPS containing protease inhibitor(Complete, Roche) and were diluted further in binding buffer. Weakcation exchange (WCX) and hydrophobic (H4) chips were used foranalysis. Results: comparison of the obtained peptide profiles revealedat least 12 peptides in the range of 1 to 10 kDa that showed time-dependent change in expression. A SWISSPROT database search withthe found masses gave several hits for gastrointestinal hormones.Further identification of the peptides is necessary to validate thismethod. Conclusion: these data indicate that SELDI proteinchiptechnology is a promising tool for measurement and discovery of awhole range of food-induced peptide hormones.

Intact Mass Measurements Show That Current BioinformaticsPrograms Do Not Reliably Predict The Transit Peptide Cleavage Sites Of IntegralMembrane Proteins.

Stephen M. Gómeza, Karl Y. Bilb, John N. Nishioc, Kym F. Faulla and Julian P.Whiteleggea,1a Pasarow Mass Spectrometry Laboratory - Departments of Psychiatry & BiobehavioralSciences and Chemisty & Biochemisty, and the Neuropsychiatric Institute. University ofCalifornia, Los Angeles, California 90095, U.S.A.b Biosphere 2, Columbia University, Oracle, Arizona 85623, U.S.A. c College of Natural Sciences, California State University, Chico, CA 95929, U.S.A.1 To whom correspondence should be addressed. E-mail [email protected]; tel. 310-794-5156; fax 310-206-2161

Electrospray–ionization mass spectrometry coupled with reverse-phase liquidchromatography was used to identify 35 nuclear-encoded thylakoid-associated proteinsfrom the chloroplasts of Arabidopsis thaliana. Identifications were made based upon intactmass measurements, cyanogen bromide mass tags and/or MS/MS sequencing. With themature amino-terminus of each protein unequivocally assigned we constructed a data setto challenge several Web-based programs for predicting organelle targeting and/or theproteolytic processing site of the transit peptide. ChloroP and TargetP were found to bereliable at predicting chloroplast targeting, but were only able to reliably predict the transitpeptide cleavage site for proteins targeted to the stroma. SignalP (eukaryote settings) wasaccurate at predicting the transit peptide cleavage site for proteins targeted to the lumen.SignalP (Gram-negative bacteria settings) was reliable at predicting peptide cleavage ofthyakoid proteins inserted into the membrane via the “spontaneous” pathway, while theprocessing sites of thylakoid-integral proteins inserted by the SRP-dependent pathwaywere not well predicted by any of the programs tested. These results suggest that there is asecond thylakoid processing protease that recognizes the transit peptide of proteins insertedvia the “spontaneous” mechanism and that this mechanism may be related to the secretorymechanism of Gram-negative bacteria. None of the bioinformatics tools we tested wereable to predict the transit peptide cleavage site from SRP-dependent thylakoid-integralproteins, however improvements in these tools could be made by more judicious selectionof training data sets, including separate data sets for vascular plants and green algae and/orseparate data sets for each sub-organellar compartment.

Support from DOE (DE-FG03-01ER15251 to KFF/JPW) is gratefully acknowledged.

Quantitative Mass Spectrometry uncovers an androgen co-regulatedprotein network in prostate cancer cells.

Michael E. Wright, Ph.D, Senior Research Fellow, Dr. Ruedi Aebersold Laboratory,Institute for Systems Biology, Seattle, WA 98103

Comprehensive analysis of complex protein mixtures from biological samples hasrecently been accomplished by coupling microcapillary liquid chromatography (µLC) totandem mass spectrometry (MS/MS) and database searching (Opiteck, Lewis et al. 1997;Link, Eng et al. 1999; Tong, Link et al. 1999). More importantly our laboratory hasrecently developed a new class of reagents termed isotope-coded affinity tags (ICAT) thatselectively reacts and isotopically labels cysteine residues in proteins (Gygi, Rist et al.1999). Combining the ICAT technology with µLC-MS/MS and database searching hasallowed us to simultaneously sequence identify and accurately quantify individualproteins within complex mixtures (Gygi, Rist et al. 1999). In this report we demonstratehow the ICAT methodology can be used to quantify protein abundance changes inmammalian cells. We have successfully identified and quantified greater than 1,000proteins from the microsomal fraction of LNCaP prostate cancer cells. Androgenincreased the levels of a number of proteins involved in fatty acid biosynthesis, includingfatty acid synthase (FAS), a known androgen-regulated target in prostate epithelia(Swinnen, Esquenet et al. 1997). Androgen also increased the levels of secreted andmembrane-bound proteases, which included prostate-specific antigen (PSA), TMPRSS2,and kallikrein 2 (hK2). Several families of previously characterized membrane receptorsand proteins involved in adhesion, secretion and vesicular trafficking were alsoandrogen-regulated. We will integrate the results of this proteomic analysis with ourcDNA microarray analyses and present a working model of androgen-regulated genesand proteins in prostate cancer cells.

Gygi, S. P., B. Rist, et al. (1999). “Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.” Nat Biotechnol 17(10): 994-9.

Link, A. J., J. Eng, et al. (1999). “Direct analysis of protein complexes using massspectrometry.” Nat Biotechnol 17(7): 676-82.

Opiteck, G. J., K. C. Lewis, et al. (1997). “Comprehensive on-line LC/LC/MS ofproteins.” Anal Chem 69(8): 1518-24.

Swinnen, J. V., M. Esquenet, et al. (1997). “Androgens stimulate fatty acid synthase inthe human prostate cancer cell line LNCaP.” Cancer Res 57(6): 1086-90.

Tong, W., A. Link, et al. (1999). “Identification of proteins in complexes by solid-phasemicroextraction/multistep elution/capillary electrophoresis/tandem massspectrometry.” Anal Chem 71(13): 2270-8.

Proteomic analysis of skeletal muscle calcium homeostasis

Michael Zeece, Steve Jones, Ron Cerny, Joe Schultz, Nathan Palmer, & BorisKornileav. University of Nebraska, Lincoln NE, USA, 68583-0919

Calcium homeostasis is a critical process in muscle because this cationrepresents the trigger for several physiological functions including metabolism,contraction and protein turnover. There is also evidence that calcium level islinked to apoptosis and programmed cell death. Abnormal regulation of calciumlevel is widely held as the principle cause of pathological conditions such asmalignant hyperthermia (MH) and central core disease (CCD) in humans.Malignant hyperthermia is also manifested in some animals and often results intheir sudden death.

Significant progress has been made in the past decade regarding ourunderstanding of calcium regulation and in the identification of potential causesfor its abnormalities. The discovery of a single mutation in ryanodine receptor(RyR1) gene stirred hopes that definitive testing would enable early detection ofthese potentially fatal diseases. However, a more complicated picture hasemerged from recent research. For example, numerous (>20) mutations havebeen identified in RyR1 gene that correlate with MH in humans. It has also beenshown that calcium homeostasis results from the coordinated interaction ofseveral sarcoplasmic reticulum (SR)-associated proteins including: RyR1,dihydropyridine receptor, calsequestrin, tradin, junctin and calmodulin. Theseproteins form a functional complex that is responsible for calcium release.Several key proteins undergo reversible phosphorylation that modulates calciumflux. Thus there may be multiple mutations and/or modifications that collectivelycontribute to elevated calcium level and its adverse consequences.

Our group has taken a proteomic approach to investigate alterations in thiscomplex protein system that are linked to abnormal calcium level. Two-dimensional electrophoresis in combination with mass spectrometry methods arebeing used to separate, identify and characterize a targeted proteome ofconstituent SR proteins. This group of membrane proteins represents significantchallenges to widely used proteomic separation methods. Thus we havedeveloped appropriate 2-D techniques to separate these hydrophobic proteins.We are also developing non-gel based approaches to investigate potentialpolymorphisms in the 565 kDa RyR1 protein obtained from porcine skeletalmuscle. The results of this work including separations of SR-associated proteinsand their identification will be presented.

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Quantitative analysis of microsomal proteins from IL-12induced lymphoblasts

Jan-Jonas Filén1,2, Tuula Nyman1, Juha Korhonen1 and Riitta Lahesmaa11 Turku Centre for Biotechnology, University of Turku and Åbo AkademiUniversity, P.O.Box 123, 20521 Turku, Finland, 2 National Graduate School inInformational and Structural Biology

T helper cells (Th) are divided into Th1 and Th2 subsets based upon theircytokine profiles and function. Naïve Th cells differentiate into Th1 and Th2subsets depending on the antigens, costimulatory molecules, and cytokines theymeet. Cytokines IL-12 and IL-4 play a major role in the regulation of thedevelopment and differentiation of the Th cell subsets. IL-12 enhances thegeneration of Th1 lymphocytes and inhibits the production of Th2 subset. Viceversa IL-4 enhances the generation of Th2 subset and inhibits the production ofTh1 lymphocytes. The selective activation of Th1 and Th2 lymphocytes playsan important role in the pathogenesis of allergy, asthma and autoimmunediseases.

In this study peripheral blood mononuclear cells obtained from healthy blooddonors (Finnish Red Cross Blood Transfusion Service) were activated withphytohemagglutinin (PHA). After 48 hours IL-2 was added to cultures and Th1cell differentiation was induced by addition of IL-12. After 48 hours cells werelysed and microsomal fraction was prepared. The microsomal fraction of IL-12induced Th cells was studied using isotope-coded affinity tag (ICAT) reagentlabelling and 2-dimensional chromatography followed by nanoLC-ESI-MS/MSanalysis. The data was analysed with ProICAT software. As a preliminary resultseveral proteins from microsomal fraction were identified and quantified.

Unbiased quantitative proteomics of lipid raftsreveals high specificity for signaling factors

Leonard J. Foster, Carmen L. de Hoog, Matthias Mann*

Center for Experimental Bioinformatics (CEBI), Institute forBiochemistry and Molecular Biology, University of SouthernDenmark, Campusvej 55, DK-5230 Odense M, Denmark

Membrane lipids were once thought to be homogenously distributed inthe two-dimensional surface of a membrane but the lipid raft theorysuggests that cholesterol and sphingolipids partition away from othermembrane lipids. Lipid raft theory further implicates these cholesterol-rich domains in many processes such as signaling and vesicle traffic.However, direct characterization of rafts has been difficult due to theirsmall size and largely biochemical definition. In the first large-scaleproteomic analysis of rafts we use quantitative high-resolution massspectrometry to specifically detect proteins depleted from rafts bycholesterol-disrupting drugs, resulting in a set of 241 authentic lipidraft components from over 1000 identified proteins. We detect a largeproportion of signaling molecules, highly enriched versus totalmembranes and detergent-resistant fractions, which thus farbiochemically defined rafts. Our results provide the first large-scaleand unbiased evidence for the connection of rafts with signaling andplace limits on the fraction of plasma membrane composed by rafts.

Proteome analysis of lipid rafts subtracted from activatedprimary CD4+ T lymphocytes

Juha T. Korhonen, Tuula A. Nyman, Jan-Jonas R. Filen, Riitta LahesmaaTurku Centre for Biotechnology, University of Turku and Abo AkademiUniversity, Turku, Finland, P.O. POX 123.

The selective activation of functionally distinct human CD4+ T helperlymphocytes, Th1 and Th2 cells, plays an important role in the pathogenesis ofasthma and many inflammatory diseases. T cell receptor mediated (TCR)signaling differs in these T helper cell subsets, but the molecular basis for thisis currently unclear. However, it was found recently, that the composition ofmembrane microdomains, also referred to as lipid rafts, is distinct in Th1 andTh2 cells, leading to functional differences in TCR signaling in these T cellsubsets. We have analyzed the proteome of lipid rafts subtracted from activatedhuman primary CD4+ T lymphocytes in order to identify known and novelsignaling molecules relevant for TCR signaling in these cells. Membranesresistant to 1% Brij 58 detergent were isolated using sucrose density gradientultracentrifugation. Proteins co-purified with these lipid rafts were digestedwith trypsin, and tryptic peptide mixture was fractioned by cation exchangechromatography. Peptides were further separated and analyzed using nanoLC-ESI-MS/MS. Proteins were identified by comparison of tandem mass spectra toprotein and DNA sequence databases using Mascot and ProID softwares. As apreliminary result many of the proteins known to be associated with lipid raftsin different cell lines were also identified from these primary CD4+ Tlymphocytes. We were also able to identify some novel proteins co-purifyingwith lipid rafts and validation of these results is going on. In conclusion, themethodology described here is powerful in identifying novel proteinspotentially important in cell signaling.

Plasma Membrane (PM) Expression Profiles in Restingand Activated Murine Splenocytes

Matthew Peirce, Robin Wait, Shajna Begum, Andrew Cope andJeremy Saklatvala, Kennedy Institute of Rheumatology Division,Imperial College London, 1, Aspenlea Road, Hammersmith, LondonW6 8LH, United Kingdom.

PM proteins of mammalian cells are frequently refractory to analysisby two-dimensional electrophoresis. Using surface labelling withbiotin followed by affinity capture, PM proteins were isolated (purity>50%) and separated by solution phase isoelectric focussing and SDS-PAGE then identified by HPLC MS/MS. Using these methods weidentified 75 PM proteins from a murine T cell hybridoma and murinesplenocytes. These included one hypothetical protein and threeproteins not previously reported in these cells. The expression of 12PM proteins appeared sensitive to activation with phorbol ester andionomycin. Activation-induced differences in the expression ofMHCII, Glucocorticoid Induced TNF Receptor-related gene (GITR),CD69, IgM and IgD were independently verified by Western blot orFACS analyses. This approach may be applicable to the PMs ofdiverse mammalian cell types.

This work was funded by the Arthritis Research Campaign, UK, theMRC and the Wellcome Trust.

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Low molecular weight proteomics in Arabidopsisthaliana

Jon Griffin, Prof. Johnathan Napier and Prof. Peter Shewry CropPerformance and Improvement, Long Ashton Research Station,Department of Agricultural Sciences, University of Bristol, UK,BS41 9AF

The number of predicted genes in the genome of Arabidopsisthaliana is 25,498 and analysis of these sequences revealed that 18%were low molecular weight (<20 kDa) proteins. Most currentproteomic studies have been biased to proteins greater than 15 kDa.Using an adapted proteomic approach we aim to identify lowmolecular mass proteins and peptides that have either not beenpreviously characterised or studied, or have not been identified by theuse of bioinformatic algorithms when the Arabidopsis genome wasanalysed. Two different separation approaches have been undertaken.The first based on 2-Dimensional Electrophoresis using highpercentage Tris-Tricine gels and the second using 2-DimensionalChromatography with size exclusion and reverse phasechromatography. Identification of proteins was using MALDI-TOFMS and ESI-MS/MS following trypsin digestion. It is anticipated thatsome of the components identified will have biological activities (e.g.anti-microbial) or physical properties (e.g. surface activity), whichwill allow their exploitation. This poster details our findings so far.

PhD studentship supported by the [email protected]

High-resolution and High-throughput GlobalProteome Profiling by Three-dimensional LC-MS/MSAnalysis

Jing Wei, Wen Yu, Jun Sun, Arianna Jones, Martin Keller, Jay ShortDiversa Corp., San Diego, CA 92121

The complexity and dynamic range of the proteome is resolvedby a novel approach involving on-line three-dimensionalchromatography separation, mass spectrometry separationand detection, as well as novel bioinformatics softwaredevelopment. Comprehensive view of the yeast proteome,including semi-quantitative results, was obtained. Yeast cells atlog-phase were homogenized and fractioned as solubleproteins, urea solubilized proteins, and SDS solubilizedproteins. More than 2500 unique proteins were identified fromthese fractions with an average coverage of 11 peptides perprotein by the 3D LC-MS/MS. Proteins of all classes werepresented, including many lower abundance proteins. Majorityof the primary metabolism pathways were fully identified. Post-translational modifications including N-acetrylation andmethylation were systematically surveyed. The proteinabundance estimated by the “coverage index” correlated wellwith the published copy numbers of the corresponding genes.The higher sequence coverage and better separation willprovide a powerful tool for detailed mapping of the proteome.

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Poster AbstractsThursday, March 27: In vivo Approaches/Analysis of Protein Activities

Global analysis of changes in protein-phosphorylationupon TNF-αα signaling

Stephan Bek, Anja Pfenninger, Thomas Wendrich, Jochen Kruip.Aventis Pharma Germany, Industriepark Höchst, 65926 Frankfurt amMain.

Protein phosphorylation represents a major control step of proteinfunction, which cannot be evaluated with conventional genomic tools. Itplays central roles in signal transduction, metabolism, apoptosis and celldevelopment. Among the most important signal transduction pathways,tightly regulated by phosphorylation, are the NFκB pathways. Theymediate inflammatory reactions in response to pathogenic mechanismthat are involved in various diseases like osteoarthritis, rheumatoidarthritis, arteriosclerosis, ischemia, diabetes 2, and others. Kinases,phosphatases, and substrates involved in these pathways are thereforefavoured targets for therapeutical strategies. Consequently tools tocarefully analyse global changes in protein phosphorylation areessential for pharmaceutical drug research. Currently no generalstrategy is able to address these dynamic phoshorylation changes In order to obtain a quite sensitive and comprehensive overview aboutthe phospho-proteome of a cell that has received inflammatory signals,we have established the 2D-separation of metabolically labelled cell-lysates. The acquired phosphorylation maps have been linked to overallprotein pattern, and differentially phosphorylated proteins were identifiedby MALDI peptide mass fingerprint. In doing so, we were able to identifya significant number of proteins, that were differentially phosphorylatedupon TNF-α signalling. Whereas some of them are already knownplayers in inflammatory pathways, others are not known up to know tobe regulated by phosphorylation. In summary, this method allows the analysis of 32P-labelled proteins indefinied biological/experimental backgrounds.

Protein Function Prediction Using Protein Interactions

Minghua Deng, Ting Chen and Fengzhu Sun, Molecular andComputational Biology Program, Department of Biological Sciences,University of Southern California, Los Angeles, CA 90089-1113

Assigning functions to novel proteins is one of the most importantproblems in the post-genomic era. We develop a novel approach thatapplies the theory of Markov random fields (MRF) to infer a protein’sfunctions using protein-protein interaction data and the functionalannotations of its interaction protein partners. For each function ofinterest and a protein, we predict the probability that the protein has thatfunction using Bayesian approaches. Unlike in other available approachesfor protein functions where a protein has or does not have a function ofinterest, we give a probability for having the function.We apply our method to predict cellular functions for yeast proteinsdefined in the Yeast Proteome Database (YPD), using the protein-proteininteraction data from the Munich Information Center for ProteinSequences (MIPS, http://mips.gsf.de). We show that the MRF basedapproach outperforms other available methods for function predictionbased on protein interaction data.

This research is partially supported by the NSF ITR EIA-0112934 andNIH NIGMS 1-R01-RR16522-01.

Analysis of Mycobacterium tuberculosis cell wall glycoproteinsusing a Novel Two-Dimesional Gel Electrophoresis System.

Karen M. Dobos1, John T. Belisle1, Regina Rooney2, Bradley Scott2, and ReneNunez2 1Mycobacteria Research Laboratories, Dept. of Microbiology,Immunology, and Pathology, Colorado State University, Ft. Collins, CO, 80523;2Invitrogen Corporation, Carlsbad, CA, 92008

The evaluation and characterization of the proteome of M. tuberculosis is centralto the development of diagnostic antigens, drug targets, new vaccines, anddefinition of pathogenic mechanisms; and thus crucial to the control anderadication of tuberculosis. Moreover, the proteome holds secrets that are notreadily obtained or proven by direct analysis of the genome, including post-translational modifications. Previously, we have described the glycosylation ofsecreted M. tuberculosis proteins through rigorous biochemical and moleculartechnologies. In contrast, the description of similar proteins that define the cellularenvelope of M. tuberculosis is lacking. The study of these proteins is hindered bytheir poor solubility and recovery from mycobacteria lysates (restricting theiravailability for study) and the lack of technologies to study these macromolecules.We have employed traditional biochemical methods to purified cell wallpreparations of M. tuberculosis to extract and isolate these proteins. This classicalbio-separation methodology has been partnered with a novel vertical 2D-GEsystem (IPGZoom Runner) to afford high throughput analysis of this complexmixture of proteins. Thus far, we have been able to resolve over seventy uniqueprotein spots from the mycobacterial cell wall, including four products of 19, 25, 26,and 38 kDa previously defined as mycobacterial lipoproteins; three of which maybe glycosylated, and thus are likely to be prominent structural features of thetubercle bacillus. Concanavalin-A probing of these 2D-GE resolved productsrevealed the presence of four additional putative glycoproteins present in thisfraction, with observed molecular masses of 99, 80, and two products of 58 kDa.Elucidation of these products and their role in the physiology of M. tuberculosis areunderway.This work was funded in part by NIH,NIAID Contract NO1 A1-75320 and GrantRO1 A1-44042.

A Mass Spectrometric Approach to Identify Novel ActiveProtein Kinases

Pasan Fernando*, Wen Ding+, John F. Kelly+ and Lynn A. Megeney*. *OttawaHealth Research Institute and the +National Research Council, Ottawa,Ontario, Canada.

Protein phosphorylation is a primary mechanism for the regulation of cellularprocesses. As such, the characterization of protein kinases remains central tothe understanding of phospho-signaling related events. However, the ability toscreen active protein kinases on a large scale remains a daunting challenge.Here, we describe an adaptation of the previously established in-gel kinasemethod for screening the activities of protein kinases from murine tissue andcultured cells. Protein lysates from mice harbouring the activated MAP kinasekinase-6 (MKK6) gene were analyzed by IEF and 2D PAGE in gels crosslinkedwith myelin basic protein (MBP). Following electrophoresis, gels weredenatured in a solution of guanidine-hydrochloride and then renatured in asolution of 2-mercaptoethanol. Incubation of the gels in a kinase buffercontaining [γ-32P]ATP followed by autoradiography allowed the visualizationof specific protein spots, ie. phosphorylated MBP by a putative protein kinasethat co-migrated to the same location during IEF/PAGE. Nanoflow-LC/electrospray ionization mass spectrometry allowed for sensitive detection ofnumerous protein kinases. Importantly, the kinase profile generated with thisapproach allowed us to map a network of interactions within the cellularcontext of active MKK6. With additional refinements and alterations in samplepreparation, we are able to demonstrate the utility of this method as a platformfor the comparison of proteomes based on differences in kinase activities.

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Poster Abstracts Thursday, March 27: In vivo Approaches/Analysis of Protein Activities

Characterization of the Phospho-Proteome of HumanBreast Cancer Cells

Brie K. Fuqua and Dr. Edward M. MarcotteDepartment of Chemistry and Biochemistry, The University of Texas atAustin, Austin, Texas USA 78712

Phytoestrogens are natural plant compounds that have astructure and function similar to that of the mammalian female sexhormone 17β-estradiol. Studies have correlated a reduced risk of breastcancer development with diets rich in phytoestrogens, such as soy-baseddiets. However, the mechanism of phytoestrogen action on cells is notfully understood.

Various cellular signaling cascades are triggered by exposureto estrogen and phytoestrogens. Classically, studies have concentrated oncharacterizing long-term transcriptional effects. However, rapid changesalso occur via phosphorylation of serine, threonine, and tyrosine residueson key signaling molecules. We are using proteomic methods to identifyproteins that have been modified by phosphorylation in response tophytoestrogen exposure.

The total fraction of phosphorylated proteins, or phospho-proteome, is isolated from cancerous breast epithelial cell lysates withaffinity chromatography following treatment with phytoestrogens. Afterfurther separation with HPLC, phosphorylated proteins are analyzed byESI ion trap mass spectrometry. Global identification of proteins whosephosphorylation status is altered in response to estrogenic exposure willprovide insight into the signaling processes shaping cancer cell growthand metastasis.

Acknowledgements: Project supported by the University Co-OperativeSociety 2002-2003 Undergraduate Research Fellowship

Identification of in vivo phosphorylation changes in topoisomerase IIααand ββ in HL-60 cells that are differentially sensitive to topoisomerase II-poisons

K. Chikamori, D. R. Grabowski, M.T. Kinter, R. H. Aebersold, A. H. Andersen, I. D. Hickson,R. M. Bukowski, R. Ganapathi and M. K. Ganapathi. Cleveland Clinic Foundation,Cleveland, OH 44124, U.S.A., Systems Biology Institute, Seattle, WA 98195, U.S.A.,University of Aarhus, Aarhus DK-8200, Denmark and Cancer Research UK OxfordCancer Centre, Oxford OX39DS, U.K.

Topoisomerase (topo) alters DNA topology for the processing of genetic material. Type IItopo consists of two isoforms, topo IIα and topo IIβ. Phosphorylation of topo II regulatesits activity and sensitivity of cells to drugs that poison the enzyme. Resistance to topo II-poisons is associated with hypophosphorylation of topo IIα and hyperphosphorylation of

topo IIβ. Decreasing intracellular Ca2+-transients can mimic this resistant phenotype. Tolocate specific phosphorylation site(s) on topo IIα and topo IIβ that are altered in HL-60cells resistant to topo-poisons we compared CNBr and tryptic phosphopeptide maps ofthese isozymes in cells: a) sensitive to topo II-poisons (HL-60/S), b) resistant to topo II-poisons (HL-60/R), and c) treated with the intracellular calcium chelator, BAPTA-AM (HL-60/S/B). CNBr or tryptic phosphopeptides generated from topo IIα or topo IIβimmunoprecipitated from extracts of cells labeled with [32P]Pi were analyzed by 1D-gelelectrophoresis or 2D-phosphopeptide mapping, respectively. CNBr phosphopeptidemaps of topo IIα revealed hypophosphorylation of a 12 kDa peptide in HL-60/R and HL-60/S/B cells, as compared to HL-60/S cells. This peptide was identified by N-terminalEdman sequencing to correspond to the 10.4 kDa CNBr peptide 34 (aa1041-1131),localized to the catalytic domain of topo IIα. Tryptic digestion of peptide 34 generated twophosphopeptides which migrated similarly to the two hypophosphorylated trypticpeptides observed in HL-60/R and HL-60/S/B cells. Comparison of CNBrphosphopeptide maps of topo IIβ in HL-60/S and HL-60/R cells revealedhyperphosphorylation of a 65 kDa peptide in HL-60/R compared to HL-60/S cells. Thispeptide was one of two major phosphopeptides of MW 65 and 53 kDa detected in HL-60 cells. Edman sequencing revealed that both peptides have identical N terminalsequences corresponding to the penultimate CNBr peptide starting at aa1248 . Since thelast peptide consists of only two amino acids the difference in the size (∼12 kDa) is likelydue to differential post translational modification(s). Tryptic phosphopeptide maps alsoidentified hyperphosphorylation of three peptides in HL-60/R compared to HL-60/S cells.Studies to identify, by mass spectrometry, the specific site(s) in the catalytic domain oftopo IIα and in the C-terminus of topo IIβ that are differentially phosphorylated in sensitivecells and in cells displaying a resistant phenotype are currently underway. In summary,these results suggest that resistance of HL-60 cells to topo II-poisons is accompanied byalterations in site specific phosphorylation of topo II α and topo IIβ, albeit by reciprocalmechanisms. (USPHS CA-749349 and DK56917)

Development of a system for the study of protein-proteininteractions in planta, and application of the method tocharacterization of a TATA-box binding protein complex in Oryzasativa

Paul A. Haynes, Jingping Zhong, Shiping Zhang, Xinping Yang, NancyAndon, Donna Eckert, John R. Yates III, Xun Wang, and Paul Budworth,The Torrey Mesa Research Institute of Syngenta, San Diego, CA, USA,92121

We have developed a simple and rapid method for in vivoprotein complex purification from plants using a tomato Biotin peptide asan affinity tag. The Biotin tag was fused in-frame to the carboxylterminus of TATA-box binding protein and the fusion cassette wastransformed into rice callus. The expressed chimeric protein waspurified from rice suspension cell cultures, along with the associatedprotein complex components, using immobilized streptavidin. Theproteins present in the purified complex were visualized by gelelectrophoresis and 86 unique proteins were identified by tandem massspectrometry. The bait protein and a number of proteins known to beassociated with TBP proteins were identified, as well as many otherproteins involved in pre-mRNA processing, chromatin remodeling, andtranslation. The identification of these novel protein-protein associationsprovides important new insights into the mechanisms of mRNAtranscription and pre-mRNA processing, and demonstrates that thissystem can potentially be used for the identification of all proteinsinteracting with a given target protein under physiological conditions inrice. This system is also applicable to other tissues and organisms, andcould be applied in a high-throughput fashion to enable systematicanalysis of protein-protein interactions on a proteome-wide scale.

Identification of protein tyrosine kinases and tyrosinephosphorylated proteins overexpressed in transitional cellcarcinoma

Shie-Liang Hsieh, Department of Microbiology and Immunology,National Yang-Ming University, Taipei, Taiwan

Cancer is the leading factor of annual death in Taiwan, and is the mostprevalent disease leading to death in the developed countries. Sincethe up-regulated tyrosine kinase activity found in cancer cells isusually resulted from the altered structure of tyrosine kinase due togene mutation and deletion, therefore techniques based on thedetection of mRNA (such as DNA microarray, RT-PCR etc) can notdetect the upregulated PTK activity under this situation, though PTKgenes amplification and the increase of mRNA also occur in certaintumor cells. Therefore we use proteomic approach to study the alteredkinase activity and the up-regulated tyrosine phosphorylated proteinsin TCC. Several tyrosine phosphrylated proteins were identified viathis approach, and their roles in TCC is under intensive investigationat this moment.

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Proteomic analysis for oxidative stress-induced proteinmodifications

Narae Hwang, Ja Won Seo, Seung-Hee Yim, Eun Joo Song, Min ChungKim, Ji Young Chung, and Kong-Joo LeeFrom the Center for Cell Signaling Research, Division of MolecularLife Sciences and College of Pharmacy, Ewha Womans University,Seoul, 120-750, Korea

Exposure of cells to oxidative stresses including hydrogen peroxide,heat shock and growth factors, induces the modifications of variousproteins. We have identified the target protein of oxidative stresses,GAPDH known to play various cellular roles including apoptosis,proliferation and regulation of transcription factors such as AP-1. Toexamine the physiological role of GADPH in oxidative stress, we haveidentified the protein modifications and interacting proteins using 2D-gel electrophoresis and MALDI-TOF MS and ESI-Q-TOF MS. Proteinmodifications including cystein oxidation, phosphorylation and lysine-acetylation were detected and binding proteins having various cellularfunctions such as RNA binding, chaperone, and redox signal relatedproteins were identified. Redox dependent protein modificationsregulate the protein functions. These results suggest that the variousmodifications and the change of binding partners in oxidative stresspropagate differential signaling pathways. [Supported by MOST 21C Frontier Functional Human Genome Project(FG-4-14) by KISTEP, by KOSEF through the Center for Cell SignalingResearch (CCSR) at Ewha Womans University and by IMT2000 projectfor IT-BT. Students (N Hwang, JW Seo, MC Kim, JY Chung and EJ Song)and Research Professor (SH Yim) were financially supported by BrainKorea 21 program]

Proteome and phosphoproteome Analysis in BovineAdrenal Medulla using MALDI-MS and LC/MS/MS

Pegah R. Jalili and Chhabil Dass, Department of Chemistry, TheUniversity of Memphis, TN 38152, USA

In this study, LC/MS-based proteomic approach is used to identifyproteins and phosphoproteins in bovine adrenal medulla. This gland is amajor source of hormones, opioids, neurotransmitters, and severalimportant proteins. These biomolecules play a variety of roles in thefunctioning of hypothalamic-pituitary-adrenal axis (HPA), including painsuppression, influence on the immune system, maintenance of the vascularresponse, protection of the body against stress and in patho-physiologicalprocesses that lead to Alzheimer and Parkinson diseases. Phosphoproteinsare involved in the control of cell growth, metabolism, differentiation, and inthe function of many proteins, hormones, neurotransmitters and enzymes.

After removing the cortex, the medulla was homogenized andcentrifuged. The supernatant was subjected to 75% ammonium sulfateprecipitation and the pellet obtained by centrifugation was dissolved,dialyzed overnight at 4°C to remove salts and small peptides, andcentrifuged again. The supernatant was lyophilized. The proteins thusobtained were fractionated by RP-HPLC. Two MS approaches were usedfor profiling proteins. In the first approach, each HPLC fraction wassubjected to trypsin digestion. The MALDI-TOFMS analysis of this digestprovided a peptide mass map that was used for searching Swiss-Protdatabase. In the second approach, all RP-HPLC fractions were combined,subjected to trypsin digestion and analyzed by capillaryLC/nanospray/MS/MS. The expressed sequence tags thus obtained wereused to search nonredundant databases. For detection of phosphopeptides,the lyophilized sample was dissolved in buffer, passed through C4 ZipTip,digested with trypsin, passed through IMAC ZipTip, and analyzed withMALDI-TOFMS. A total of 64 proteins were identified, the prominent onesare Secretogranin I precursor, proenkephalin A precursor, 1-phosphatidylinisitol-4-5-biphosphateinisitol 1,4,5-triphosphate receptor type3, chromatogranin A (CMGA-BOVIN), hypothetical protein KIAA0373, andbeta-adrenergic receptor kinase 2.

Large-scale functional approach of proteins in human diseases

Christine Chavany and Moncef Jendoubi**Founder, President & CEOMilagen, Inc. 770 13th Street, Richmond, CA 94804

As proteins are the end product of gene expression and the ultimate effectors ofcellular processes, unraveling protein function is essential to understandingphenotype and finding new ways to diagnose and treat human diseases. We have developed a large-scale antibody-based approach designed to identifyessential proteins associated with disease. Our approach, globally referred to asAntibiomixTM is based on two major in house developments: i) a large collectionof high affinity polyclonal antibodies against known and unknown human geneproducts; ii) a protein array technology, named the matrix protein arraytechnologyTM, coupled to a readout and data analysis system. The combination ofthe above platform technologies enables the analysis in multiplex format of a largenumber of clinical specimens with a large number of antibodies generatingdifferential protein expression profiling We are presenting data in two major applications of the AntibiomixTM approach: i)protein expression profiling of matched normal and cancerous tissue and biologicalfluid specimens, in the context of diagnostic and therapeutic applications, and ii)protein expression profiling of human cell lines cultured in the presence or absenceof anti-cancer drugs, in the context of pharmacoproteomics. In both cases, theselection and identification of panels of antibodies that specifically recognizedifferentially expressed proteins further lead to the characterization and validationof the relevant proteins.Our approach allows for the identification of protein signatures in any givenbiological state, including disease, correlating gene products to phenotype. Theidentification of disease specific protein panels will help to develop more effectivetreatment strategies for individual patients and to develop improved diagnostics andtherapeutics.

New Insight of VEGF-induced Signaling Pathway inHUVEC by Proteomics and Bioinformatics

Young Mee Kim, Hee-Jung Kim, Eun Joo Song and Kong-Joo LeeFrom the Center for Cell Signaling Research, Division of MolecularLife Sciences and College of Pharmacy, Ewha Womans University,Seoul, 120-750, Korea

Vascular endothelial growth factor (VEGF) is an important angiogenicfactor. It mediates signaling by activating two receptors tyrosine kinase,Flt-1 (VEGFR-1) and KDR (VEGFR-2). It is known that endothelial cellstreated with VEGF increase the reactive oxygen species (ROS)production and activate MAPK pathway. To identify the target proteinsof VEGFR, we have investigated tyrosine-phosphorylated proteins inhuman umbilical vein endothelial cell (HUVEC). We treated HUVECwith VEGF or hydrogen peroxide. The phosphorylated proteins wereseparated by 2D-gel electrophoresis, detected with western analysisusing phospho-tyrosine antibody and identified with peptidefingerprinting using MALDI-TOF MS. We have detected 95 proteinsthat were differentially phosphorylated in HUVEC in response to VEGFand hydrogen peroxide. Also we can identify the proteins specificallyphosphorylated by VEGF, not by hydrogen peroxide. The obtainedresults were analyzed by bioinformatic tools. The combined resultsprovide the insight for new VEGF and H2O2-induced signalingpathways, and give us the direction of functional studies of targetproteins involved in angiogenesis. [Supported by MOST 21C Frontier Functional Human Genome Project(FG-4-14), by KOSEF through the Center for Cell Signaling Research(CCSR) at Ewha Womans University, and by IMT2000 project for IT-BT.Students (YM Kim, HJ Kim, EJ Song) were financially supported byBrain Korea 21 program]

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Preparation of Amniotic Fluid for Proteomic Analysis

Chatarina A Löfqvist1,3, Gunnel Hellgren1, Ann Hellström1,2 and Lois EH Smith3.1Department of Pediatrics, 2Department of Ophthalmology, The Sahlgrenska Academy,at Göteborg University, Göteborg, SE-416 85, Sweden, 3Department of Ophthalmology,Children’s Hospital, Harvard Medical School, Boston, US.

We have hypothesized that babies lose important sources of growth factors due topreterm birth.This loss is made up partly from growth factors and cytokines from amnioticfluid (AF), swallowed during fetal life, and partly from the placenta.In order to investigate the variance in growth factor levels in different gestational weeks(GA), AF will be taken at delivery (GA 23 – 40 weeks at weekly intervals) and stored. AsAF levels for cytokines has been found to vary depending on storage conditions weconducted a pilot study with the objective to evaluate stability of insulin-like growth factorsin AF.AF was collected in immediate connection to delivery. After centrifugation aliquots wasprepared with or without proteolytic enzyme inhibitor (PI). As PI either aprotinin or EDTAwas used and the aliquots was then stored at either –20o C or –80o degrees C. Analysisof insulin-like growth factor binding protein-1 (IGFBP-1) by Western blots using nativegels, showed fewer band in samples stored with addition of PI and at –80 degrees C.However, analyzing IGFBP-1 by Western blots using SDS gels showed no differencebetween the samples.So far a problem area has been elucidated 1) IGFBP-1 is the major growth factor in fetaland maternal circulation and we found very high levels of IGFBP-1 (>50.000 ng/ml) in oursamples. There could be a significant risk of influencing the protein detection in two-dimensional (2-D) gels with this level of binding protein present. Therefore IGFBP-1 mightneed to be removed. However removal of IGFBP-1 could also be associated withconcomitant removal of IGFs.The studies will consist of 2-D gel analysis of proteins in AF at each phase of fetaldevelopment. Selected protein spots will then be analyzed by mass spectrometry.Candidate molecules will be compared longitudinally with respect to gestationaldevelopment age. Furthermore data will be compared with respect to infant weight andprematurity.We anticipate that the proposed studies of amniotic fluid will reveal the changes thatoccur with development of the fetus. We know that IGF-I rises in the second/thirdtrimester and infants born prematurely miss this source of IGF-I. With the results from thisstudy we hope to establish the importance of growth factors in AF for the development ofblood vessel and neural tissue, and possible the protective impact of these factors.

Interactions of Cricket Paralysis Virus IGR-IRES with theHuman Ribosome

Anke Mulder (1,2,3), Christian Spahn (1), Robert A. Grassucci (1), Erik Jan(4), Peter Sarnow (4) and Joachim Frank (1,2) (1) Howard Hughes Medical Institute, HRI; (2) Wadsworth Center, EmpireState Plaza, Albany, NY 12201-0509; (3) Purdue University, West Lafayette,IN 47906; (4) Stanford University School of Medicine, Stanford, CA 94305.

Biochemical studies indicate that Cricket Paralysis Virus (CrPV) IGR-IRESutilizes a method of protein translation initiation that is distinct from thatpreviously observed in eukaryotes. We used cryo-electron microscopy (cryo-EM) and single-particle reconstruction techniques to investigate theinteractions between CrPV IGR-IRES and the human ribosome. HeLa 40Sand 80S ribosomal subunit samples with and without CrPV IGR-IRES wereapplied to glow-discharged copper EM grids, quick-frozen in liquid ethane,and examined on a FEI transmission electron microscope at 300 kV. Imageswere recorded at 37,700X magnification and digitized with a ZI scanner.Selected particles (18,511 for 80S-IGR, 21,626 for 80S control, 34,514 for40S-IGR) were subjected to reference-based reconstruction in SPIDER.Resolutions of the resulting reconstructions are 21Å, 23.1Å, and 20.2 Å,respectively. Analysis of these density maps suggests that CrPV IGR-IRESbinds the 80S ribosome in the inter-subunit space. IGR-IRES makes contactswith the 60S subunit that appear to mimic those made by P-site tRNA. CrPVIGR-IRES binding causes conformational changes in the ribosome that arerelated to the mechanism by which it initiates protein synthesis. Finally, thecontrol reconstruction represents the first structure of a human ribosome.Comparison with the cryo-EM map of the yeast ribosome reveals addeddensities on the 60S subunit of the human ribosome.

Research was supported by the Howard Hughes Medical Institute and NSFgrant DBI-9987844.

Networks of transcription factors: proteomics approach.

Neuman, T. and Metsis, M.* Cemines, Inc. Los Angeles, CA 90048,USA, *Center for Genomics and Bioinformatics, Karolinska Institutet,SE-171 77 Stockholm, Sweden.

Individual transcription factors (TFs) regulate specific sets of targetgenes in different cell types in norm and pathology. This specificitydepends on the activity of signaling systems and composition oftranscriptional complexes. Helix-loop-helix (HLH) TFs have severalfunctions during development of the nervous system that requiresregulation of different sets of target genes. We characterized thenetwork of HLH TFs in developing neurons using: 1. Chromatinimmunoprecipitation followed by the cloning and sequencing ofgenomic binding regions (target genes) of HLH TFs, 2. Antibodymicroarrays of crosslinked chromatin, 3. Mass spectrometry analysesof immunoprecipitated crosslinked chromatin complexes, and 4. FRETanalyses in situ using labeled antibodies to HLH TFs and componentsof transcriptional complexes. Chemical cross-linking allows us toidentify chromatin complexes of HLH TFs under physiologicalconditions. Results of these analyses show that individual HLH TFsregulate different sets of target genes and form different chromatincomplexes during neurogenesis

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Switching Protein Interaction Networks by AlternativeSplicing of Protein Interaction Domains

Alissa Resch, Yi Xing, Barmak Modrek, Michael Gorlick, RobertRiley, Christopher Lee

Alternative splicing has emerged as a major mechanism forexpanding and regulating the repertoire of gene function. Alternativesplice data have the potential to contribute substantially to ourunderstanding of proteomic diversity and function. However, whilemost alternative splicing is studied at the nucleic acid level, manyfunctional questions can only be answered by analyzing the proteinproducts. To make this connection, we have constructed a databaseof alternatively-spliced protein forms (ASP database), consisting of11,907 protein isoform sequences of 4,204 human genes). Ourbioinformatics analysis indicates that the major impact of alternativesplicing is removal of protein-protein interaction domains thatmediate key linkages in protein interaction networks. 56% ofalternative splicing events removed protein-protein interactiondomains as opposed to DNA binding domains, RNA bindingdomains, or other domain types. We present a number of novelexamples (Kruppel transcription factors; Pbx2; Enc1) from the ASPdatabase, illustrating how this pattern of alternative splicing changesthe structure of a biological pathway, by redirecting proteininteraction networks at key switch points.

E. coli primase interaction with template DNA.

Anna Rodina, Nigel Godson, Department of Biochemistry, New YorkUniversity, New York, NY, USA, 10016.

All cellular organisms use specialized RNA polymerases called“primases” to synthesize RNA primers for the initiation of DNAreplication. Primase is a part of huge multi-protein complex called“replisome”. Primase activities are coupled to it by protein-proteininteractions with other replication factors.We have used DNA-protein crosslinking to define the path oftemplate DNA through E. coli primase during the reaction of pRNAsynthesis. We have found three sites on the primase surface, whichcome in close proximity to DNA. These are Zn-finger (92-120aa),active center region (120-187aa) and another region, which has yet tobe characterized. The data provide insight into the structure andfunctional properties of the enzyme.

Supported by NIH grant GM38292.

Novel Functions of FAF1 were Identified by Proteomics

Eun Joo Song, Seung-Hee Yim , Eunhee Kim, Kong-Joo Lee From the Center for Cell Signaling Research, Division of Molecular LifeSciences and College of Pharmacy, Ewha Womans University, Seoul120-750, Korea

Fas-associated factor 1 (FAF1) was first identified as another Fasassociating molecule, which lacks a characteristic death domain but canenhance Fas-mediated apoptosis. The biological role of FAF1 is notclearly defined. Recent our work indicates that FAF1 may be involvedin complicate procedures related to the apoptosis and ubiquitinpathway. Also FAF1 has several domains related to ubiquitin pathway,UBX, UBL, NLS etc. To examine the function of FAF1, we haveidentified FAF1 interacting proteins, Valosin containing protein (VCP)and hsp 70. VCP is necessary to degrade polyubiquitinated proteins inthe proteasome. In this study, we found that FAF1 regulates VCPactivity by various modifications. Protein modifications of FAF1including phosphorylation, methylation were detected and analyzedwith MALDI-TOF MS and tandem ESI-Q-TOF MS. These resultssuggest that FAF1 has multi-functions and each functions can beregulated by changing the interacting proteins and posttranslationalmodifications.[Supported by MOST 21C Frontier Functional Human Genome Project(FG-4-14), by KOSEF through the Center for Cell Signaling Research(CCSR) at Ewha Womans University, and by IMT2000 project for IT-BT.Student (EJ Song) were financially supported by Brain Korea 21program]

TOWARDS AN ANALYSIS OF MEMBRANE PROTEIN INTERACTIONS:THE MEMBRANE-BASED YEAST TWO-HYBRID TECHNOLOGY

Safia Thaminy1, Daniel Auerbach2, Anthony Arnoldo1, and Igor Stagljar 11 Institute of Vet. Biochemistry and Molecular Biology, University of Zurich, Switzerland2 Dualsystems Biotech Inc, Zurich, SwitzerlandEmail: [email protected]

Analysis of membrane protein interactions is difficult due to the hydrophobic natureof such proteins, which makes them ill-suited to use in the traditional two-hybrid assay. This is asubstantial problem since approximately one-third of all proteins in a typical eukaryotic cell arethought to be anchored in the lipid bilayer (1). Because of their accessibility, membrane proteins,in particular those spanning the plasma membrane, are also of considerable diagnostic andtherapeutic importance. Thus, understanding the physiology of membrane proteins and the waythese proteins communicate in the cell is of high biological importance (2).

We have recently developed a new genetic method for the in vivo detection ofmembrane protein interactions in Saccharomyces cerevisiae (3). The system uses the split-ubiquitin approach first described by Johnsson and Varshavsky (4) based on the observation thatcovalent addition of ubiquitin polypeptides can recruit particular proteases for the specificdegradation of target proteins. In our system, interaction between two membrane proteins resultsin ubiquitin reconstitution and leads to the proteolytic cleavage and subsequent release of atranscription factor which triggers the activation of a reporter system enabeling easy detection (3)

In the past two years, our central goal was to use a membrane-based yeast two-hybrid technology to identify proteins that interact with a given yeast or human membrane targetprotein. For that purpose, yeast and human cDNA libraries were constructed and fused either N-or C- terminally to the Ubiquitin (Nub) domain. We now show that various screenings of theselibraries identify the membrane and cytosolic proteins that specifically interact with theircorresponding bait protein (Thaminy, S., Auerbach, D., Arnoldo, A., and Stagljar, I., submitted toNat Biotechn). In addition, we are combining a membrane-based yeast-two hybrid approach to thereverse two-hybrid strategy to screen a library of peptides or small molecules in which preventingthe interaction between two membrane protein partners provides a selective advantage for yeast.Such an approach should enable identification of dissociating peptides or small molecules whichare able to dissociate the interaction between two membrane proteins X and Y.

A major advantage of our membrane-based yeast two-hybrid system is that it can beused to detect not only interactions between two membrane proteins, but also the interactionbetween membrane and cytoplasmic protein. The ability to monitor the activity and interactionsbetween membrane proteins in their native cellular compartment should prove useful in screeningfor proteins and drugs that alter their binding properties.REFERENCESAuerbach, A., Thaminy, S., Hottiger, M., and Stagljar, I. (2002) Proteomics 2, 611-623.Stagljar, I., and Fields, S. (2002), Trends Biochem Sci, in press.Stagljar, I., Korostensky, C., Johnsson, N., and te Heesen, S. (1998) Proc Natl Acad Sci USA 95,5187-5192.Johnsson, N. and Varshavsky, A. (1994) Proc Natl Acad Sci USA 91, 10340-10344.

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Immunoprecipitation and Western blot screening forphosphorylation statesof over 900 signal transduction proteins.

Jennifer Stevenson, Allison Siemers, Pam Wethington and RobertoCampos. BD Biosciences-Lexington, KY

The importance of phosphorylation in signal transduction has been known formany years. Recently, tools have been made available that allow a morespecific, efficient and comprehensive study of protein phosphorylation. Thesetools include phospho-specific antibodies and monoclonal antibodiesrecognizing total protein whether phosphorylated or unphosphorylated. In thisstudy, we surveyed the phosphorylation states and general expression levels ofover 900 different signal transduction proteins during stimulation of EGF-responsive cells. Our methods include 1) multiplex Western blotting with>900 monoclonal antibodies 2) Western blotting using >30 phospho-specificmonoclonal antibodies and 3) anti-phospho-tyrosine immunoprecipitationfollowed by Western blot screening with >200 monoclonal antibodies. Wefound numerous phosphorylated proteins; many already described in theliterature, such as MAPK and Stats. Also, we detected novel tyrosinephosphorylation changes. One such novel change was identified on Bad, amember of the Bcl-2 family. Furthermore, we compared early phosphorylationevents in cells bearing a wild-type EGF receptor versus those containing anEGF-inactive receptor. Cells with the inactive receptor are unable toproliferate in response to EGF. Significant differences were detected in thephosphorylation of Stat1, ERK1/2, p38MAPK and Caveolin-1, implicatingthese proteins in a cell proliferation pathway. In summary, we were able toutilize an arsenal of cell biology reagents to examine changes in the generalexpression pattern and phosphorylation profile of EGF stimulated cells. Ourmethods provide a rapid and comprehensive view of the signaling pathwaysand proteins activated by EGF and will prove to be an invaluable tool forexamining cell states that have been perturbed by various compounds orcellular defects.

Reconstruction of protein interaction networks in bacteria.

de Juan D A; Devos D;, Blaschke C; Pazos F;Valencia A,National Center for Biotechnology, C.N.B. - C.S.I.C. , Madrid. SPAIN

The re-construction of protein interaction network in a model organism is one of the moreactive fields of molecular biology. Parallel to the application of experimental techniques forthe massive determination of protein interactions, the complementary set of computationalmethods, based on sequence and genomic information, have emerged.We have assessed the accuracy of the available experimental data on bacterial system (1),and of the three available computational tools for the prediction of interactions (2-4), plusour new two methods (5-7). We have used as standard of truth the presence of interactionsin the literature (8). The results shows that the predictions are of a quality similar to theexperimental data, and that the interactions detected by more than one method are of asubstantially better quality. Indeed the small sets of interactions predicted by three or moremethods seems to correspond in all cases to correct predictions of components of proteincomplexes.The probabilistic networks build for bacterial systems are compared with the onesproposed for yeast, in terms of coverage, accuracy and potential implications for themodeling of cellular systems.1. Rain JC, Selig L, De Reuse H, Battaglia V, Reverdy C, Simon S, Lenzen G, Petel F,

Wojcik J, Schächter V, et al.: The protein-protein interaction map ofHelicobacter pylori. Nature 2001, 409:211-215.

2. Pellegrini M, Marcotte EM, Thompson MJ, Eisenberg D, Yeates TO: Assigning proteinfunctions by comparative genome analysis: Protein pylogenetic profiles. ProcNatl Acad Sci USA 1999, 96:4285-4288.

3. Dandekar T, Snel B, Huynen M, Bork P: Conservation of gene order: a fingerprint ofproteins that physicaly interact. Trends Biochem Sci 1998, 23:324-328.

4. Enright AJ, Iliopoulos I, Kyrpides NC, Ouzounis CA: Protein interaction maps forcomplete genomes based on gene fusion events. Nature 1999, 402:86-90.

5. Pazos F, Valencia A: Similarity of phylogenetic trees as indicator of protein-proteininteraction. Protein Eng 2001, 14:609-614.

6. Pazos F, Valencia A: In silico two-hybrid system for the selection of physicallyinteracting protein pairs. Proteins 2002, .

7. Valencia A, Pazos F. Computational methods for the prediction of protein interactions.Curr Opin Struct Biol. 2002, 12:368-73.

8. Blaschke C, Andrade MA, Ouzounis C, Valencia A: Automatic extraction of biologicalinformation from scientific text: Protein-Protein Interactions. ISMB99 1999,60-67.

Quantitative Analysis of Glycoproteins: Applications toSerum and Membrane Proteins

Hui Zhang, Xiao-jun Li, Daniel B. Martin, and Ruedi AebersoldInstitute for Systems Biology1441 N 34th Street, Seattle, Washington 98103-8904

Abbreviated title: Quantitative analysis of glycoproteinsCorrespondence should be addressed to R.A. (Institute for SystemsBiology, 1441 N 34th Street, Seattle, Washington 98103-8904,[email protected])Quantitative proteome profiling using stable isotope protein tagging andautomated tandem mass spectrometry is an emerging technology with greatpotential for the functional analysis of biological systems and for thedetection of diagnostic or prognostic protein markers. Due to the enormouscomplexity of proteomes, their comprehensive analysis is an as yetunmatched technical challenge. However, biologically or clinicallyimportant information can be obtained if specific, information-rich proteinclasses or sub-proteomes are isolated and analyzed. Glycosylation is acommon post-translational modification. In particular, membraneproteins, secreted proteins, and proteins contained in body fluids are likelyto be N- glycosylated. Here we describe a method for the selectiveisolation, identification and quantification of peptides that contain N-linkedcarbohydrates. It was applied to the analysis of plasma membrane proteinsfrom cells and proteins from serum. It is expected that this method will findwide application for the identification of diagnostic biomarkers ortherapeutic targets in clinical specimens that are enriched in glycosylatedproteins.

A New Method for Identification of Protein Kinase SubstratesApplied to the Search for Novel JNK Targets in the Brain

Benny Björkblom1,2, Wenrui Li 1, Tatsiana Tararuk1, Jan-Jonas Filén1 ,Tuula A. Nyman1 , Michael J. Courtney3 and Eleanor T. Coffey11Turku Centre for Biotechnology, Åbo Akademi University & University ofTurku, BioCity, Turku, Finland. 2Department of Biochemistry & Pharmacy,Åbo Akademi University, BioCity, Turku, Finland. 3A.I.Virtanen Institute,University of Kuopio, Kuopio, Finland.

c-Jun amino-terminal kinases (JNK) typically respond strongly to stress, areimplicated in brain development and mediate neuronal apoptosis in response tonoxious stimuli. Given that JNK mediates apoptosis in response to stress, it isexpected that neurons display a low basal JNK activity that responds sensitivelyto apoptotic signals. Surprisingly however, JNK activity in primary culturedneurons and brain extracts is highly elevated in the absence of stress and isfurther upregulated during neuronal differentiation suggesting also non-apoptotic roles for JNKs in the brain. An increased knowledge of the JNK targetrepertoire would contribute greatly to our understanding of JNK function inneurons. We have taken a proteomics based approach to identify novel JNK substrates inthe brain. High specific activity kinases (JNK or other MAPKs) are incubatedwith brain extract, a rich source of potential target proteins. Proteins areseparated by 2-D gel electrophoresis and phosphoproteins located byautoradiography. JNK phosphorylated proteins are then identified by LC-MS/MS. Using this method we have detected several candidate JNK targets, anumber of which are expressed at low levels and are highly phosphorylated byJNK suggesting that they may be specific targets. One family of proteinsidentified by this method has been verified in vivo as JNK phosphorylatedproteins and others are currently being tested. This technique can be utilized formultiple kinases and has the advantage of combining a rich source of proteintogether with rapid identification by tandem MS.Acknowledgements - This work was supported by Academy of Finland grant72608 and the Informational and Structural Biology Graduate School

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Functional Proteomics of Heterotrimeric G-Protein RegulatedSignaling in Yeast

Metodi V. Metodiev, Sofia Zaichik, and David E. StoneLaboratory for Molecular Biology, University of Illinois at Chicago, Chicago,Illinois 60607

We are applying a targeted proteomic approach to discover novel proteininteractions in the pheromone response pathway of Saccharomycescerevisiae. In previously described experiments we detected interactionbetween the pheromone-responsive G-alpha protein (Gpa1) and the Fus3MAP kinase, and demonstrated the functional significance of this interactionwith respect to the overall efficiency of the mating process (1). Here wepresent new data concerning another important downstream effector of the G-alpha protein. Using high-resolution 2D electrophoresis and MALDI-Tofanalysis of proteins that co-purify with Gpa1, we identified the Kar3 kinesinas a protein that specifically associates with Gpa1 upon activation of thepheromone pathway. Yeast two-hybrid analysis revealed that Gpa1 bindsdirectly to the N-terminal globular domain of Kar3, and that the interaction isas strong as the two-hybrid interaction between Gpa1 and the yeast Gbeta subunit. Furthermore, this interaction is clearly important for the properregulation of microtubule dynamics during mating, and for pheromone-induced nuclear migration preparatory to cell fusion. Depletion of Gpa1protein caused severe defects in microtubule orientation and nuclearmigration, characteristics of Kar3 dysfunction. Thus, the application ofproteomics allowed us to uncover a completely unknown and potentially veryimportant regulatory mechanism in this extensively studied pathway. It linksa G-alpha protein, a kinesin motor protein, and possibly a MAP kinase, to theregulation of the microtubule cytoskeleton and nuclear migration duringsignal-induced cellular polarization and morphogenesis. Sequence andfunctional conservation data suggest that similar pathways might exist inhigher eukaryotic organisms. 1. Metodiev et al. 2002, Science, Vol. 296, pp. 1483-1486. NSF MCB-0218081 to DES and MVM.

Integral Membrane Protein Interactions IdentifiedUsing a Modified Split-Ubiquitin Assay System

John Miller1, Russell Lo2, and Stanley Fields1,2 1Departments ofGenome Sciences and Medicine, University of Washington, 2HowardHughes Medical Institute, Seattle, WA, USA 98195

We have employed an assay system to analyze interactions betweenintegral membrane proteins of the yeast Saccharomyces cerevisiae inthe hopes that such information can provide clues as to the functionsof such proteins. The split-ubiquitin system of Johnsson andVarshavsky is based on the ability of interacting proteins to bring twofragments of ubiquitin into close proximity such that they are actedon by ubiquitin-specific proteases (Johnsson and Varshavsky PNAS91:10340-10344 (1994)). The original assay was modified byStagljar et al. (PNAS 95: 5187-5192 (1998)) such that proteolysisreleases a transcription factor that then enters the nucleus to activatereporter genes. We selected the proteins annotated in the YeastProteome Database as ‘integral membrane’ and created an array ofyeast colonies each expressing a different protein fused to the amino-terminal half of ubiquitin. We have screened 35 proteins to date andfrom these identified 261 putative interactions. Our analysis has thusfar centered around the oligosaccharyltransferase components andER-associated degradation (ERAD) constituents. This work isfunded by the NRCC/NIH Yeast Resource Center: PHS # P41RR11823.

IMAC/LC-MS/MS based strategy for phosphopeptideenrichment, detection and sequencing.

Allan Stensballe, , Thomas S. Nühse*, and Ole Nørregaard JensenDepartment of Biochemistry and Molecular Biology; University of SouthernDenmark, Odense, Denmark *The Sainsbury Laboratory, Norwich, UK

Detailed knowledge of the status of posttranslational modificationof proteins like phosphorylation is vital for understanding protein function.The application of nanoflow capillary liquid chromatography (LC) interfacedon-line to electrospray (ESI) mass spectrometers or off-line to MALDI massspectrometers are now valuable methods for phosphoproteincharacterization.

Detection of substoichiometric amounts of phosphopeptides incomplex peptide mixtures are often hindered by the presence of high levelsof non-modified peptides. Immobilized-Metal-Affinity-Chromatography(IMAC) is a powerfull technique for affinity enrichment of phosphopeptidesprior to analysis. We have investigated this technique as a front-end methodprior to analysis by nanoflow LC-MS/MS using data directed discovery ofmodified peptides. Strategies for selective analysis of phosphopeptides using LC-ESI-QTOFMS/MS includes the characteristic neutral loss of 97.98 Da per phosphatemoity (gas-phase beta-elimination of phosphoric acid). We have evaluatedData directed discovery triggered by these phospho-specific ions duringautomated nanoflow LC-MS/MS of complex peptide mixtures which allowsselection of the subset of modified peptides for fragmentation. This enablesselective detection and sequencing of modified peptides among co-elutingnormal peptides of higher abundance.

We have investigated these methods for analysis of simple orcomplex phosphoprotein mixtures. Analysis of a complex peptide mixturederived from an Arabidopsis thaliana membrane preparation revealed morethan 90 highly purified phosphopeptides in one IMAC/LC-MSMS experiment.

PROTEOMIC APPROACH TO LFA-1 MEDIATED SIGNALLINGIN T-CELL MIGRATION

Arun Chandran, Yuri Volkov, Aideen Long and Dermot KelleherTrinity College, Dublin and Dublin Molecular Medicine Centre, Ireland

Directed migration of T-lymphocytes towards tissue-specific destinationsrepresents a crucial step in immune system response to inflammation and pathogeninvasion. The interactions of LFA-1 integrin receptor with its ligand ICAM-1,contribute significantly to the molecular dynamics of active T-cell locomotion. Wehave previously reported the formation of a multi-molecular cytoskeleton/signallingcomplex commencing upon LFA-1 occupancy by its natural and imitatingrecombinant ligands. However, precise functional significance of the proteinsassociated with LFA-1 intracellular domain has not yet been established.

We utilized the original in situ immunoprecipitation technique in combinationwith proteomic analysis to further identify and characterize the individual proteinsthat are involved in the signalling cascade. In brief, T cells were activated viasubstrate-immobilized motility-inducing LFA-1 ligands either at physiological(37°C) or low temperature conditions (4°C), which block intracellular signallingprocesses. Live migrating cells were lysed in optimised membrane detergent bufferand only the stable protein complexes remaining attached to substrate vialigand/LFA-1 chains were collected for subsequent proteomic analysis.

2-D electrophoretic mapping of the LFA-1 associated complexes revealedsignificant differences in the cytoskeletal and signalling molecules recruitment underthese two experimental conditions. Western blotting and MALDI-TOF sequencing ofseparated proteins demonstrated that LFA-1 associated complex is enriched intubulin and vimentin cytoskeleton components and contains at least two signallingproteins of the protein kinase C family (PKC beta and delta). However, actin contentsof the complexes and PKC-theta involvement were not affected by blockingconditions, suggesting selective isoform-specific recruitment of PKC and certaincytoskeletal proteins in the process of active T-cell motility. These and otherpotentially novel LFA-1 associated molecules, which are currently beingcharacterized might be critically important for concerted intracellular signallingevents and changes of gene expression associated with cell migration.

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Poster Abstracts Friday, March 28: Structural Proteomics/Control of Protein Abundance and Activity/Computational Methods

Protein expression microarrays for proteomics

Kristi A. Miller, Daniel M. Yoshikawa, Todd Edwards, Matthew A.Coleman, and Joanna S. Albala.Biology & Biotechnology Research Program. Lawrence LivermoreNational Laboratory. Livermore, CA 94550.

Understanding the biology of an organism requires complexinvestigation. Unlike genes, proteins are a more diverse populationcomposed of many more elements having more widespreadinteractions within the cell. The key advantage to array-based methodsfor protein study is the potential for parallel analysis of thousands ofsamples in an automated, high-throughput fashion. Protein arrays areemerging as a practical format in which to study proteins in high-throughput using many of the same techniques as that of the DNAmicroarray. We have developed a method to utilize LLNL-I.M.A.G.E.cDNAs to generate recombinant protein libraries using a baculovirus-based paradigm. We have used this strategy to produce proteins foranalysis of protein/DNA and protein/protein interactions using proteinmicroarray techniques. Our efforts are focused on understanding thecomplex interactions of proteins involved in homologousrecombination and DNA repair. This work was performed under theauspices of the U.S. DOE by the UC-LLNL under Contract No. W-7405-Eng-48.

Reproducible Sample Preparation: A Key Step inProteome Analysis

J. Anders, R. HendriksMerck KGaA, Life Science Products R&D MDA, Frankfurter Str. 250,64293 Darmstadt, Germany

Proteome analysis implies the ability to separate proteins withhigh resolution and reproducibility prior to protease digestionand characterization by mass spectrometry or microsequencing.High-resolution protein separation for proteome analysis can beachieved by both liquid chromatography (LC) and two-dimensional gel electrophoresis (2DGE). The samplepreparation of any protein mixture for subsequent separation byeither LC or 2DGE is of major importance, as it will affect theoverall performance of the technique. Due to the very highdegree of complexity of the proteome especially in biologicalsamples from higher eukaryotes, standard operating proceduresfor sample preparation preferably including proteomeprefractionation must be employed in order to lower samplecomplexity prior to protease digestion of proteins. Differentapproaches to sample prefractionation including stepwiseextraction of partial proteomes from cells, sampleprefractionation by LC and depletion of highly abundant proteinsfrom body fluids will be discussed.

On de novo interpretation of peptide mass spectra

Vineet Bafna(*) and Nathan Edwards(!)(*) The Center for Advancement of Genomics, 1901 research Blvd., 6th Floor, Rockville, MD [email protected](!) Informatics research, Applied Biosystems45 W. Gude Drive, Rockville, MD [email protected]

The correct interpretation of tandem mass spectra is a difficult problem,even when it is limited to scoring peptides against a database. CorrectDe novo sequencing is considerably harder, but critical when sequencedatabases are incomplete or not available.

In this paper we build upon earlier work due to Dancik et al., and Chenet al. to provide a dynamic programming algorithm for interpreting denovo spectra. Our method can handle most of the commonly occuringions, including a,b,y, and their neutral losses. Additionally, we shift theemphasis away from sequencing to assigning ion types to peaks. Inparticular, we introduce the notion of core interpretations, which allowus to give confidence values to individual peak assignments, even in theabsence of a strong interpretation. Finally, we introduce a systematicapproach to evaluating de novo algorithms as a function of spectralquality. We show that our algorithm, in particular the core-interpretation,is robust in the presence of measurement error, and low fragmentationprobability.

A Protein-Domain Microarray Identifies Novel Protein-Protein Interactions

Alexsandra Espejo and Mark T. BedfordThe University of Texas M.D. Anderson Cancer Center, SciencePark-Research Division, Smithville, TX 78957.

Protein domains mediate protein-protein interaction through binding toshort peptide motifs in their corresponding ligands. These peptiderecognition modules are critical for the assembly of multiproteincomplexes. We have arrayed GST fusion proteins, with a focus onprotein interaction domains, onto nitrocellulose coated glass slides togenerate a protein-domain chip. Arrayed protein-interacting modulesincluded WW, SH3, SH2, 14.3.3, FHA, PDZ, PH and FF domains.Here we demonstrate, using peptides, that the arrayed domains retaintheir binding integrity. Furthermore, we show that the protein-domainchip can “fish” proteins out of a total cell lysate, these domain-boundproteins can then be detected on the chip with a specific antibody, thusproducing an interaction map for a cellular protein of interest. Usingthis approach we have confirmed the domain binding profile of thesignaling molecule, Sam68, and have identified a new binding profilefor the core snRNP protein, SmB’. This protein-domain chip not onlyidentifies potential binding partners for proteins, but also promises torecognize qualitative differences in protein ligands (caused by post-translational modification), thus getting at the heart of signaltransduction pathways.

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Poster AbstractsFriday, March 28: Structural Proteomics/Control of Protein Abundance and Activity/Computational Methods

Printing chemical libraries on microarrays for fluidphase nanoliter reactions.

Dhaval N. Gosalia and Scott L. Diamond

Institute for Medicine and Engineering, Departments ofBioengineering and Chemical and Biomolecular Engineering, 1024Vagelos Research LaboratoryUniversity of Pennsylvania, Philadelphia, PA 19104

We printed chemical compounds within individual nanoliter dropletsof glycerol that remained adherent to the surface of a microarray at 400spots per cm2. Using ultrasonic-generated aerosol deposition, wemetered subsequent reagents (enzymes, substrates, buffer) into each200 µM diameter reaction center in order to rapidly assemblemulticomponent reactions without cross contamination or the need forchemical linkage to the surface. A volume of 2 uL of reagent wassufficient to activate 5000 reactions on a single slide. This proteomicstool allowed the profiling of protease mixtures and the detection offluorogenic substrate conversion in the presence of various inhibitors.A 352-compound library was microarrayed in quadruplicate on 100replicate slides. An inhibitor of caspases 2, 4, and 6 was identifiedusing five replicate 352-compound microarrays screened againstcaspase 2, 4, 6 as well as thrombin and chymotrypsin. From a singleprinting run, combinatorial libraries can be subjected to numeroushomogeneous separation-free reactions at volumes 103 to 104 smallerthan current high throughput methods.

Differential protein expression profiling on arrays

Easton P, Evans S, Wheeler C, Moody A, Cross T, Bamford A, CopseC, Smith C*Amersham Biosciences UK Limited, The Grove Centre, Amersham,Buckinghamshire, HP7 9LL, U.K.

The ability to profile the differences between biological samples is offundamental importance in biology. These differences may occur as aresult of a disease or the effects of a drug treatment and haveclassically been studied at both the gene and protein level using DNAmicroarray and 2-dimensional fluorescence difference gelelectrophoresis (2-D DIGE) technologies, respectively. However, 2-Delectrophoresis is not ideally suited to rapid, large-scale proteinexpression screening.

Recently, protein arrays have emerged as a technology with thepotential to fulfil the requirement for the high throughput screening ofdifferential protein expression profiles. There are a number of differentformats for protein arrays dependent on their specific application. Ingeneral, all array formats will require highly specific binders bound to asolid support and coupled to a method of detection.

We are developing a flexible technology for the production of highsensitivity protein arrays for differential protein expression profiling onglass slides. By using multi-colour fluorescent labelling of samples withCyDyeTM fluors and antibodies as capture agents, we are able toaccurately detect differences in protein levels within complex samples.Here, the utility of the technology is shown for the differential analysisof cytokines in biological samples.

Predicting MS/MS Peptide Fragmentation Patterns: Beyond SEQUEST

Joshua E. Elias1, Francis D. Gibbons2, Frederick P. Roth2, and Steven P. Gygi1. 1Departmentof Cell Biology, 2Department of Biochemistry and Molecular Physiology, Harvard MedicalSchool, Boston, MA, USA, 02115

Existing automated tandem mass spectrometry sequencing methods are limited: Their simplifiedtreatment of measured ions, often relying solely on mass, reduces their power. In reality,observed peptide fragmentation patterns depend on several factors. We hypothesize thatconsidering these factors will improve our ability to match spectra to sequence databases,ultimately allowing the acceptance or rejection of matches deemed marginal by other methods.Further, this practice may prove useful for evaluating de novo peptide sequencing.

More than 650,000 spectra from various sources were acquired by microcapillaryreverse-phase liquid chromatography coupled on-line to a tandem mass spectrometer (LCQDECA ion trap, ThermoFinnigan, San Jose, CA). Tandem mass spectra were searched againstappropriate sequence databases with the Sequest algorithm (Eng, 1994). From these spectra,more than 100,000 were selected as correct, since they meet filtering criteria more rigorous thanthose previously proposed (Washburn, 2001). For each peptide meeting these criteria, observedintensities were mapped to the predicted masses for each b and y fragment of the precursor ion.These data were used to populate a database linking several peptide features such as ion type (bor y), fractional position relative to peptide length, and intensity. This database was used toevaluate the probability that an observed fragmentation pattern came from the identified peptide.

Guided by more than 100,000 tandem mass spectra representing over 30,000 uniquepeptides as a training data set, we have developed an algorithm that evaluates the likelihood thata fragmentation pattern came from a given peptide. It is clear that fragmentation events andconsequent ion intensities are not random. For example, fragmentation events betweenparticular amino acid pairs such as P T are almost never detected for the b ion series (~ 4.6%).Conversely, fragmentation events are almost always observed between other amino acid pairs,such as T P for the y ion series (~74.1%), particularly when they occur near the midpoint of thepeptide (~95 of all occurrences), and are often the dominant features of their mass spectrum.By applying these and many similar criteria to additional peptides of known and unknownsequences, we suggest that in addition to augmenting established analytical tools, this methodwill have utility in de novo sequencing of peptides not yet found in a sequence database

Eng, JK. J Am Soc Mass Spectrom, 1994. 5: p. 976-989 Washburn, MDNat Biotechnol, 2001. 19: p. 242-247

This work was supported in part by NIH grant HG0041 (S.P.) and by NIH National ServiceResearch Award 5T32CA86878 from the National Cancer Institute (J.E.).

Development of Antibody Microarrays for Detectionand Quantitation of Specific Proteins

Lisa Gangi, Lionel Best, Lynn Rasmussen and David Munroe.Laboratory of Molecular Technology, SAIC-Frederick, NationalCancer Institute at Frederick, 915 Tollhouse Road, Suite 211,Frederick, MD, 21701, USA.

Protein microarrays hold the promise of a high-throughput approachto genome wide studies at the protein level. DNA microarrays haveproven to be an invaluable tool for profiling human cancers, but thedata is limited to observations at the transcriptional level. Sincealterations at the protein level also are important in key pathwaysleading to cancer, advances in proteomics have become a rapidlydeveloping field. Although complete proteome-wide array-basedresearch is not yet possible, practical applications of proteinmicroarrays are currently possible using standard microarrayinstrumentation. We have developed a platform that facilitates theproduction of high-density antibody microarrays. This platform isbeing utilized for both protein profiling and for specificity andaffinity screening of crude monoclonal hybridoma and single chainantibody arrays.

This research is supported by NCI contract # N01-C0-12400

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Rapid Peptide Identification by Recursive K-means Clustering ofAmino Acid Composition Data

Brian D. Halligan, Xin Feng, Simon N. Twigger, and Peter J. Tonellato,Bioinformatics Research Center, Medical College of Wisconsin, Milwaukee, WI53226

The functional proteomic analysis of complex biological samples presents asignificant bioinformatic challenge with respect to data analysis and reduction.High throughput proteomics experiments result in the production of large quantityof spectral data that need to be further analyzed to obtain biologically relevantinformation. The standard methods used to analyze this data, spectral matchingtechniques (SEQUEST) or denovo peptide sequence determination and sequencedatabase searching, require significant computing hardware resources to completethese task in real time.

To develop a real-time peptide identification system, we developed a methodthat uses the amino acid composition of a peptide to identify its parent protein.We have created a database of the predicted tryptic peptides derived from all ofthe mammalian entries in the SwissProt protein database and calculated a series of20 numerical attributes for each of these peptides, based on the frequency ofoccurrence of each amino acid in the peptide. A recursive K-means algorithm wasused to cluster the peptides into distinct groupings based on these amino acidfrequency attributes. By using this method recursively, we are able to generatelayers of clusters of peptides that ultimately correspond to the equivalent peptidein homologous and orthologous proteins. The purpose of clustering the aminoacid frequency data is to create an index to the peptide database that allows us toidentify a peptide based on its composition. To identify a peptide, its amino acidfrequency attributes are calculated and the distance of its attributes vector to thecenters of the clusters are measured. For each layer in the cluster database, theclosest cluster is identified and the attributes of individual members of the clustersare compared to those of the peptide and the closest match identified. In this way,the recursive clustering process forms a multi-layered index to the peptidedatabase that can be rapidly traversed. We are currently expanding this method toanalysis of primary proteomics data.

Rapid Extract System: High throughputpurification of protein samples

Tariq A. Haq and Kevin M. HughesBD Biosciences, Two Oak Park, Bedford, MA, USA 01730

Abstract:As the field of Proteomics develops, there will be inevitablepressure to purify larger numbers of protein samples foranalysis. Current methodologies are well adapted forpurification of limited numbers of samples, but handling largernumbers—tens, hundreds or thousands—presents a logisticalproblem. The Rapid Extract System is a novel approach forquick affinity purification of protein samples in a highthroughput format. It allows rapid protein purification fromlarge number of samples without the requirement of changingplates or lengthy processing times. Multiple recombinantbacterial protein purifications were performed simultaneously inone of the applications described in this study. The systemhandled up to 96 samples at a time and was fully compatiblewith available automation equipment. Additional applicationsbeing developed for the system include: purification of taggedproteins, immunopurification, depletion of specific proteins etc.

Profiling Enzyme Activities in Models of Human Breast Cancer

Nadim Jessani, Benjamin F. Cravatt III. Departments of Cell Biology andChemistry, Scripps Research Institute, La Jolla, CA, USA, 92037

To compliment conventional genomic and proteomic methods that often focus onmeasuring abundance rather than activity, we have developed a chemicalproteomics method, referred to as “activity-based protein profiling” (ABPP). Thismethod employs chemical probes that covalently label the active sites of enzymesuperfamilies in a manner that provides a direct readout of changes in catalyticactivity. By providing a covalent link between the labeled proteins and a chemicaltag, ABPP permits the consolidated detection, isolation, and identification of activeenzymes directly from complex proteomes.

In a recent study, ABPP probes were applied to the proteomic analysis of a panelof human cancer cell lines. These studies resulted in the identification of multipleenzymes whose functional state correlated with tumor type as well as higher ordercellular phenotypes, such as invasiveness. Here, we extend the ABPP approach toa model that approximates the growth conditions of tumors in vivo, where hostfactors can be taken in to account. ABPP analysis of tumors and metastases derivedfrom human breast cancer line xenografts grown in immunodeficient mice havebeen carried out to assess whether the expression patterns of enzyme activitiespreviously identified in human cancer cell lines are maintained in vivo. Moreover,by taking advantage of the mixed species nature of the xenograft model, tumorderived (human) enzyme activities can be distinguished from host stromal (mouse)activities. Specifically, human and mouse enzymes, though similar, exhibitsufficiently different tryptic peptide maps providing a means to determine speciesof origin of enzymes identified by mass spectrometry methods. Notably, thesestudies demonstrate how proteomic approaches, like ABPP, can provide a distinctadvantage over mRNA profiling methods, which often rely on cDNA oroligonucleotide arrays that cannot distinguish orthologous transcripts with highsequence identity.

Supported by California Breast Cancer Research Program.

Comprehensive approach to acquire mouse KIAA cDNA clones and

to generate anti-mKIAA antibodies for establishment of the platform to analyze

function of KIAA proteins

Hisashi Koga1, 2, N Okazaki1, R Kikuno1, Y Hara2, K Shimada2, H Kohga2, K Imai2,

R Ohara1, S Inamoto2, 3, T Nagase1, Osamu Ohara1, Sigeki Yuasa4

1Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818,

Japan2Chiba Industry Advancement Center, 2-6 Nakase, Mihana-ku, Chiba261-7126, Japan3Institute of Research and Innovation, 1201 Takeda, Kashiwa, Chiba 277-0861, Japan4National Institute of Neuroscience, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187, Japan

Since 1994 we have been isolated and entirely sequenced long human cDNA clones

and have already registered to the public database more than 2000 genes (KIAA

genes) to date. From December 2001, we initiated a new project supported by JST

(Japan Science and Technology Corporation) to identify the functions of KIAA

proteins. However, there are obvious limitations in analysis of human genes primary

due to ethical issues. We therefore decided to use an animal model for accumulation

of the functional characterizations. For this purpose, we firstly started to isolate and

determine entire sequences of mouse cDNA clones which encode the polypeptides

corresponding human KIAA proteins (mKIAA), and subsequently to generate

antibodies against mKIAA proteins. As an extension of this project, we have begun to

prepare cDNA and antibody arrays. We herein present the strategy to isolate mKIAA

cDNA clones from size-fractionated mouse cDNA libraries and to generate mKIAA

antibodies using the in vitro recombination-assisted method.

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Proteomic Analysis of the Bacteriophage T7 Replisome

Jaya Kumar, Charles C. Richardson, Department of BiologicalChemistry and Molecular Pharmacology, Harvard Medical School,Boston, Massachusetts, USA, MA 02115.

We present a genome-wide analysis of proteins at the replication forkof bacteriophage T7. Bacteriophage T7 infection of Escherichia coliprovides an attractive model system to study how a chromosome isduplicated. Within twelve minutes after infection of E. coli, over ahundred copies of the 39,936 base pair linear T7 chromosome aresynthesized. The T7 DNA polymerase functions as part of a multi-protein complex that unwinds duplex DNA, primes lagging strandsynthesis, and coordinates leading and lagging strand synthesisduring DNA replication. Here we describe the tandem affinitypurification (TAP) of T7 DNA polymerase associated complexes.

T7 DNA polymerase has been tagged via its processivity factor, E.coli thioredoxin as thioredoxin binds T7 DNA polymerase with highaffinity (Kd = 5 nM). The gene encoding thioredoxin bearing a TAPtag at its C-terminus was inserted into the T7 chromosome.Functional protein complexes formed in vivo have been isolated fromphage-infected cell-lysates and characterized. The interactingpartners of T7 DNA polymerase and E. coli thioredoxin will bedescribed.

This work was supported by United States Public Health Grant NIH-GM54397.

MODULATION OF mRNA TRANSLATION IN YEAST PHEROMONERESPONSE.

MacKay, V.L.1, X. Li2, K. Plow1, G.L. Law1, K. Serikawa1, E. Turcott1, X.L.Xu2, L.P. Zhao2, and D.R. Morris1. 1Biochemistry Dept., Univ. of Washington,Seattle, WA 98195; 2Public Health Sciences Division, Fred Hutchinson CancerResearch Center, Seattle, WA 98109.

Cellular changes in response to external stimuli usually involve transcriptionalactivation of some key genes, but the rapidity of phenotypic change oftenobserved suggests that post-transcriptional alterations may also contribute. Wehave used microarrays to conduct a high-resolution analysis of the translationstate of all mRNAs in yeast mating-type a cells + acute treatment with themating pheromone α-factor. Lysates from treated and untreated cells were eachfractionated by sucrose gradient centrifugation. Purifed RNA from each fractionwas applied to microarrays, generating a profile of ribosome loading for eachmRNA and facilitating a comparison of mRNA translation state in control andtreated cells.

Although nearly all the mRNAs showed the same polysome profile for the twocell populations, for a few mRNAs the translation state was significantly altered.Several mRNAs well-loaded with ribosomes in the control gradients showeddramatic shifts to poor loading (1-2 ribosomes per mRNA) after α-factortreatment, while other mRNAs that were poorly loaded in the control shifted tohigher polysomes with pheromone treatment. Other mRNAs were affected lessdrastically by α-factor, shifting to either higher or lower ribosome density. Wehave initiated studies on a few genes to date and have determined that severalmechanisms seem to be represented. Some genes show promoter shifts withα-factor treatment, leading to mRNAs with longer or shorter 5’ leaders that aredifferentially translated. Short upstream open reading frames (uORFs) areincluded in some, but not all, of these 5’ leaders and may contribute totranslational regulation. In contrast, neither a promoter shift nor uORF areassociated with at least one mRNA that displays pheromone modulation oftranslation. Our results suggest that the yeast cell has evolved several distinctstrategies for regulating translation and, consequently, protein synthesis.Supported by NIH-CA-89807

In Vitro Analysis of Transcription and Expression of theTruncated Erythropoietin Receptor

Joseph Milano, Monicah A. Otieno and François Pognan. SafetyAssessment, AstraZeneca Pharmaceuticals, Wilmington, DE, USA,19850

Erythropoiesis is a multi-stage process requiring proliferation anddifferentiation of cells of the erythroid lineage to form matureerythrocytes. This process is controlled by erythropoietin (Epo) and iscommunicated by its cell surface receptor EpoR. There are two formsof EpoR – a full length (EpoR-F) expressed predominantly in matureerythrocytes and an alternatively spliced, truncated receptor (EpoR-T)expressed predominantly in immature erythrocytes. Expression ofEpoR-T is thought to have a dominant-negative effect on EpoR-F thusregulating erythropoiesis depending on circulating Epo levels.

We investigated the control of expression of EpoR-T in an in vitrosystem using murine erythroleukemia cells. Using semi-quantitativePCR, we have shown that these cell lines transcribe both the truncatedand full-length forms of the EpoR. The ratio between EpoR-F andEpoR-T ranged from 10:1 to 100:1 full-length vs. truncated. However,these ratios were not reflected in Western analysis of whole-celllysates. To determine the point of control of expression for the EpoRprotein we transfected Cos-1 cells with a cDNA plasmid construct thatconstitutively expresses the full-length receptor. Our results show thepresence of both the full-length and truncated forms of the protein.However, only the full-length cDNA was detected by RT-PCR. Theseobservations suggest mechanisms other than alternative splicing maycontrol expression of the erythropoietin receptor.

Application of statistical methods to database lookupfor protein identification via LCQ mass spectrometry

Aleksey Nakorchevskiy, Edward Marcotte. Department ofChemistry and Biochemistry, University of Texas, Austin.

Mass spectrometry is one of the most powerful toolsavailable for protein identification from whole-cell digests. Thequality of the peptide/protein identification currently relies only onthe quality of the match between experimental and theoreticalfragmentation spectra. We introduce a computational method tostrengthen protein identification quality and coverage by includingother types of data into the database lookup. Such data includeadditional measurements collected from the peptides during a typicalmass spectrometry experiment, including chromatographic elutiontimes and previously collected mass spectra. We construct aprobabilistic framework for protein identification that incorporatethese additional data, and thereby gain many additional constraints onthe peptides’ identities, allowing extension of mass spectrometricpeptide lookup beyond spectral alignment.

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ROSPath: a database of Reactive Oxygen Species mediated cell-signaling PATHways

Jisook Park1, Joonkyu Park2, Sang-Hyuk Lee3, Seung-Rock Lee3, Kong-Joo Lee3, andEunok Paek4, 1Seoul Women’s University, Seoul, Republic of Korea, 139-774, 2InterfaceInfotech, Co., 3Ewha Womans University, 4University of Seoul, Seoul, Republic of Korea.

Recent massive data generation by genomics and proteomics requires bioinformatic toolsto extract the biological meaning from the massive results. Here we introduce ROSPath, adatabase system to deal with information on reactive oxygen species (ROS)-mediated cellsignaling pathways. It provides a structured repository for handling pathway related dataand tools for querying, displaying, and analyzing pathways.

ROSPath data model provides the extensibility for representing incomplete knowledge andthe accessibility for linking the existing biochemical databases via the Internet. Forflexibility and efficient retrieval, hierarchically structured data model is defined by usingentity-relation model and following an object-oriented paradigm. There are two major datatypes in ROSPath data model: ‘Data Entity’ and ‘Interaction’. ‘Data Entity’ represents asingle biochemical entity: a protein or protein state involved in ROS cell-signalingpathways. ‘Interaction’, characterized by a list of inputs and outputs, describes the type ofrelationships between data entities. Typical interactions are state transitions, chemicalreactions, and reactions. Then, a complex network can be constructed from the two maintypes and this allows describing and viewing various biochemical processes.

ROSPath provides flexible and intuitive visualization for biochemical entities and/orcurated pathways given a simple keyword query. More complex queries such as sub-graphextraction can also be visually answered by pathway queries. Another type of complexquery(searching a knock-out component on pathways, for example) is conducted by asimple simulation. These tools together with extensive annotation on ROS signalingmolecules make ROSPath database an innovative addition to information on signalingmolecules, thus opening up new possibilities for biological pathway analysis and pathwaydiscovery.

Supported by IMT2000 project for IT-BT and by KOSEF through the Center for CellSignaling Research (CCSR) at Ewha Womans University.

Accuracy and Variance in the Identification andQuantification of Proteins in Mass Spectrometry BasedProteomics

John Prince, Institute for Cellular and Molecular Biology, TheUniversity of Texas, Austin, Texas, USA, 78712

Mass Spectrometry (MS) offers researchers a powerful technique formeasuring the proteome. Databases with fully sequenced genomesand techniques for resolving complex samples (e.g. MuDPIT) haveallowed the identification and quantification of large numbers ofproteins. Still, the analysis of MS data remains one of the greatestchallenges to the field. Better analyses promise larger numbers ofidentified proteins at explicit levels of confidence.

We are using two-dimensional liquid chromatography with ESI Ion-Trap MS on the E. coli proteome to explore methods of analysis andquantification. We measure the variation introduced at different stepsin a proteomics experiment including sample preparation,chromatography, and spectrometry. Examination of sub-cellularfractions has allowed us to quantify the accuracy of the identificationprocess and allowed comparison of protein look-up strategies.Finally, we present results from an isotope labeling experimentapplied to an E. coli proteome, and compare these results withalternative methods for measuring protein concentration on aproteome-wide scale.

Dissection of guard cell ABA signal transduction mechanisms andComplexes in Arabidopsis

Julian Schroeder, June M. Kwak, Izumi Mori, Nathalie Leonhardt, Yoshiyuki Murata,Alison DelongCell and Developmental Biology Section, Division of Biological Sciences, University ofCalifornia, San Diego, La Jolla, CA 92093-0116

Guard cells have become a well-suited model system for dissecting early signaltransduction mechanisms in Arabidopsis (Schroeder et al., 2001, Nature 410:327-330).Previous pharmacological research suggested that type 2A protein phosphatases (PP2As)act as both negative and positive regulators of ABA signaling. To dissect the molecularmechanisms responsible for these proposed counteracting PP2As, gene chip experimentswere performed with Arabidopsis guard cell RNA together with degenerate oligo-basedPCR screening of Arabidopsis guard cell cDNA libraries. A knock-out mutant in a guardcell-expressed PP2A gene, rcn1, was obtained and showed ABA insensitivity in stomatalmovements and anion channel activation. Calcium imaging analyses show a reducedsensitivity of ABA-induced cytosolic Ca2+ ([Ca2+]cyt) elevations in rcn1, whereasmechanisms that are stimulated after [Ca2+]cyt increases show wild-type responses,suggesting that RCN1 functions upstream of [Ca2+]cyt increases. rcn1 shows ABAinsensitivity in ABA inhibition of seed germination and ABA-induced gene expression.ßThe PP1/2A inhibitor, okadaic acid, phenocopies the rcn1 phenotype in wild-type plants.These data show that RCN1 is a positive transducer of early ABA signaling. Researchtowards identifying the signaling complex of RCN will be presented. Hyperpolarization-activated Ca2+-permeable channels (ICa) were identified as a component of ABAsignaling (Pei et al., 2000, Nature 406:731-734). ROS were shown to activate ICa channelsin Arabidopsis guard cells. Data will be presented analyzing NADPH oxidase disruptionmutations that suggest a central role for these genes in ABA activation of ICa channels andstomatal closure. Additional mechanisms in this signaling complex will be reported.

Development of an Arabidopsis Protein Chip: Design,Production and Applications

Montrell Seay, Biaqing Lin, Michael Snyder and S.P. Dinesh-KumarMCDB Department, Yale University, New Haven, CT, 06520

Plants have evolved elaborate defense mechanisms in response to awide range of pathogens. Phosphoproteome studies have implicatedchanges in protein phosphorylation as playing key regulatory roles inresistance pathways, however the lack of existing technologies haslimited further studies. Proteomics in yeast has allowed researchers toevaluate protein-protein interactions, kinase activity and substratespecificity using protein chip technologies. On the other hand,proteomics in plants has been limited to the isolation and identificationof phosphoproteins without the identification of the responsiblekinases. Despite the tremendous power of protein arrays to uncoversubstrate-kinase combinations for a given phosphoproteome, to date aprotein chip from higher eukaryotes has not emerged.

Here we present the development of a protein chip using recombinantArabidopsis proteins arrayed on nickel coated-slides and immobilizedin nanowells. Arabidopsis ORFs were cloned into various modifiedGateway expression vectors for protein expression in yeast, insect cellsand Nicotiana plants. Several strategies were used to optimize proteinexpression, purification and catalytic activity. Our preliminaryfindings outline the optimal expression system and method for highthroughput protein production for the plant protein chip.

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GeneAtlas – A Software Tool for Proteome-WideFunctional Annotation

Mikhail Velikanov, Lisa Yan, and Sandór SzalmaAccelrys, Inc., 9685 Scranton Road, San Diego, CA 92121

GeneAtlas is an automated high-throughput software pipelinedesigned for functional annotation and 3D structure prediction of largesets of protein sequences. It uses a combination of sequence similaritysearches (PSI-BLAST), fold recognition (SeqFold) and homologymodeling (MODELER) methods to identify homologues in PDBdatabase and create 3D models of the input protein sequences. Theprotein function is predicted based on the quality of these models,estimated by Profile3D and statistical potentials-of-mean-force (PMF)methods. This information is augmented by the domain identificationusing HMMER and PFAM database, transmembrane segmentprediction by TMHMM, secondary structure prediction by DSC, aswell as a variety of residue-based annotations (e.g. active site, bindingpockets, etc.) using both public and Accelrys’ proprietary databases.The annotations and 3D models are stored in an Oracle database,AtlasStore.

To date, Accelrys has annotated 33 publicly available proteomes usingGeneAtlas, including human, mouse, C. elegans, A. thaliana, etc.The details of GeneAtlas methodology as well as some of the resultsfrom the annotated proteomes will be discussed.

A Sperm-Associated WD Repeat Protein Orthologous toChlamydomonas PF20 Associates with Spag6, the MammalianOrthologue of Chlamydomonas PF16

Zhibing Zhang, Jerome F. Strauss IIICenter for Research on Reproduction and Women’s Health,University ofPennsylvania Medical Center, Philadelphia, Pennsylvania 19104

cDNAs were cloned for the murine and human orthologues ofChlamydomonas PF20, a component of the alga axoneme central apparatusthat is required for flagellar motility. The mammalian genes encodetranscripts of 1.4 and 2.5 kb that are highly expressed in testis. The twotranscripts appear to arise from alternative transcription start sites. Themurine Pf20 gene was mapped to chromosome 1, syntenic with thelocation of the human gene on chromosome 2. An antibody generatedagainst an N-terminal sequence of mouse Pf20 recognized a 71-kDaprotein in sperm and testis extracts. Immunocytochemistry localized Pf20to the tails of permeabilized sperm; electron microscopeimmunocytochemistry showed that Pf20 was located in the axonemecentral apparatus. A murine Pf20-green fluorescent protein fusion proteinexpressed in Chinese hamster ovary cells accumulated in the cytoplasm.When coexpressed with Spag6, the mammalian orthologue ofChlamydomonas PF16, Pf20 was colocalized with Spag6 on polymerizedmicrotubules. Yeast two-hybrid assays demonstrated interaction of thePf20 WD repeats with Spag6. Pf20 was markedly reduced in spermcollected from mice lacking Spag6, which are infertile due to a motilitydefect. Our observations provide the first evidence for an associationbetween mammalian orthologues of two Chlamydomonas proteins knownto be critical for axoneme structure and function.

These studies were supported by NIH grant R01-HD37416-02.

Structure of the Protein Kinase A Regulatory-Catalytic SubunitComplex by Amide H/2H Exchange and Protein Docking

Ganesh S. Anand‡,1, Dennis Law2,3‡, Jeff Mandell, Aaron N. Snead2, IgorTsigelny3, Susan S. Taylor1,2, Elizabeth A. Komives 2 and Lynn Ten Eyck2,3*

1 Howard Hughes Medical Institute 2Department of Chemistry and Biochemistry,University of California, San Diego 9500 Gilman Dr. La Jolla, CA 92093-0654 3

San Diego Supercomputer Center, P.O. Box 85608, San Diego, CA, 92186-5608,USA

A model of the quaternary structure of PKA using coordinates of the individualsubunits in solution has been achieved using backbone hydrogen/deuterium (H/2H)exchange with mass spectrometry and the docking program DOT. The changes inbackbone hydrogen/deuterium (H/2H) exchange in the regulatory subunit (RIα(94-244)) and the catalytic subunit of cyclic AMP-dependent protein kinase A (PKA)upon complexation to form holoenzyme (R-C) were probed by MALDI-TOF massspectrometry. Protection from amide exchange upon C-subunit binding wasobserved for the helical subdomain, including the A and B-helices, one of whichhad previously been shown to be important by mutagenesis for C-subunit binding.Using the same approach we also identified a contiguous surface on the C-terminallobe of the C-subunit that contributes part of the intersubunit interface with RIαsubunit. The results from amide exchange experiments were then used to designdistance filters to constrain the solutions of the PKA holoenzyme obtained by theprotein docking program DOT. This enabled identification of a limited set ofsolutions that satisfy amide exchange data. Site-directed mutagenesis was appliedto test each of the solutions to identify a single solution that best represents thestructure of the PKA holoenzyme. Our approach can be used to determine testablemodels of higher order protein-protein interaction complexes from the structures ofinteracting proteins by a combination of amide exchange and docking.

The use of “LADDERS” to detect protein changes in living cells in livetime.

Benson, R.S.P.*; Embleton, M.J.?; Elliott A.C.*?Cancer Research UK, Christie Hospital, Wilmslow Road, Manchester, UK*School of Biological Science, University of Manchester, G38 Stopford, Building, OxfordRoad, Manchester, M13 9PT, UK

Antibodies provide a valuable tool for examining the proteome. In the last decade, newtechnologies have been developed which allow the expression of antibody fragments(intrabodies) inside living cells. However, the use of intrabodies to detect protein changesin living cells has not been realised because of the problem of distinguishing whichintrabodies are bound to antigen. Live Antigen Detection Dual Epitope Reporter System(LADDERS) is a potential solution to this problem. The technology consists of fusing cyan(CFP) and yellow (YFP) fluorescent protein respectively to an intrabody and the peptideantigen it binds. When these entities interact Fluorescent Resonance Energy Transfer(FRET) takes place between CFP and YFP, so that excitation of CFP (440 nm) results inYFP (530 nm) fluorescence. When endogenous cellular antigen is expressed, it competeswith the YFP – tagged peptide antigen for intrabody binding. CFP-tagged intrabody boundto endogenous protein is no longer in proximity to YFP, so excitation of CFP now leads toCFP (480 nm) emission. Thus the net effect of an increase in protein expression is anincrease in the fluorescent ratio of cyan to yellow fluorescence. We constructed a“LADDERS” probe using an intrabody raised against a core sequence of human mucin I(MUC I). We demonstrated, using ELISA, that the binding of this intrabody was unalteredwhen CFP was attached to the C-terminus. Likewise, we successfully created the peptideepitope that the anti-MUC intrabody binds, attaching YFP to its C-terminus. This proteinwas detected by Western analysis using anti-MUC intrabodies and also interacted with theanti-MUC1 scFv. However, despite the fact that CFP-labelled anti-MUC intrabodycomplexed with YFP-labelled MUC epitope, we were unable to demonstrate significantFRET between the two entities, indicating that the molecular distance between the proteinswas too great. Consequently, we have constructed a second generation of LADDERSprobes reducing the linking sequences between the fluorescent protein and theintrabody/epitope in order to decrease the molecular distance between the two fluorescentproteins when the two chimaeric proteins are complexed.

Supported by Wellcome Trust UK Showcase Award GR064075MA

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Construction Use of a Protein Trap to Probe the Impact of ClpXPPprotease on the E.coli Pproteome

Julia M. Flynn, Saskia B. Neher, Robert T. Sauer, Tania A. Baker

Protein synthesis and degradation are partners in restructuring the cellularproteome in response to shifts in nutrients and other environmental conditions.In E. coli, five intracellular proteases, ClpXP, ClpAP, HslUV, Lon and FtsH, playroles in regulating cellular processes by terminating the activitycontrolling theavailability of a variety of proteins.. Studying the full impact of degradation onthe bacterial proteome requires methods for identifying substrates for each ofthese proteases under a varriety of environmental conditions. Here, we describethe use of an inactive variant of the ClpP protease as an intracellular trap toidentify new ClpXP substrates.

ClpXP is a ATP-dependent protease comprised of two components: ClpX, ahexameric ATPase responsible for substrate recognition and unfolding, and ClpP,a peptidase whose serine active sites face an internal chamber. Substrates ofthis protease are recognized by ClpX and subsequently translocated into theClpP chamber where they are degraded. To identify new ClpXP substrates, weconstructed a variant of ClpP (ClpPtrap) in which substrates are captured but notdegraded. By expressing ClpPtrap in vivo, substrates of this protease werecaptured; these trapped proteins were purified, and identified by massspectrometry.

We identified about 50 ClpXP substrates under normal logarithmic growthconditions and 40 additional substrates following the stress of DNA damage.Analysis of substrate sequences revealed five reoccurring peptide motifs thatdirected protein allow proteins to be recognitionzed by ClpXP. This definition ofClpX recognition signals and the role of ClpXP degradation during stressresponses provides a foundation for understanding strategies for regulatingprotein turnover. For example, we find that recognition of two substrates, RseAand LexA, dependenteach depend on an initial protein-processing event toreveal an internal recognition signal. Thus, regulating the availability of crypticprotease recognition signals provides one way to control degradation in responseto environmental change. Identification of additional ClpXP substrates byintracellular trapping under a wide range of cellular conditions should permitfurther definition of the full impact of ClpXP on the bacterial proteome.

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proteomics: technologies and applications · ruedi h. aebersold, institute for systems biology...

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