Proteomics: Technologies and Applications, ANN KAREN

Tags: Speaker Abstracts, mass spectrometry, biological samples, peptide, Howard Hughes Medical Institute, Structural Proteomics, Proteomics Data, Ewha Womans University, identification, protein level, Poster Abstracts, PKA, gene expression, ectopic expression, trypsin digestion, Hee-Jung Kim, proteomics, Human Ribosome Anke Mulder, Seattle, WA, Structure of Proteins, Harvard Medical School, The Rockefeller University, Sahlgrenska Academy, University of Memphis, protein synthesis, Eun Joo Song, Chhabil Dass, expression profiling, Christian Spahn, growth factors, experimental methods, growth factor, hypothetical protein, cAMP-dependent protein kinase, Rockefeller University, mammalian cells, A. Sali, University of California, San Francisco, CA, regulatory subunits, Macromolecular Assemblies F. Alber, membrane proteins, protein function, computational methods, University of Washington, Kyle A. Serikawa1, binding properties, scoring function, Functional Proteomics Elizabeth Grayhack, Center for Cell Signaling Research, Division of Molecular Life Sciences and College of Pharmacy, protein kinase, The Scripps Research Institute, Howard Hughes Medical Institute., University of California, San Diego, Fred Hutchinson Cancer Research Center, Stanford University, transcription factor, protein complexes, prostate cells, proteomic analysis, transcription factors, binding protein, LNCaP, Arabidopsis thaliana, cell fractionation, Damon Runyon Cancer Research Foundation, ICAT, Prostate cancer, quantitative analysis, tandem mass spectrometry, Differential expression, peptide hormones, proteome analysis, Vagelos Research Laboratory University of Pennsylvania, high throughput screening, prostate cancer cells, protein expression profiling, Frontier Functional Human Genome Project, functional approach, human diseases, Robert A. Grassucci, IGR-IRES, Bioinformatics Young Mee Kim, Vascular endothelial growth factor, data analysis system, Center for Cell Signaling Research, protein expression, National Cancer Institute, chemical compounds, Frederick P. Roth2, U.K. Institute for Medicine and Engineering, fragmentation pattern, Purdue University, protein levels, DNA microarray, Department of Chemistry
Content: Program Proteomics: Technologies and Applications Ruedi Aebersold and Stanley Fields Supported by Keystone Symposia
Summary of Meeting
The completion of the genomic sequences of a number of prokaryotic and eukaryotic species has catalyzed new research approaches to study the structure, function, and control of biological processes. They are characterized by the systematic and, in many cases, quantitative analysis of all the molecules of a particular type expressed by a cell or tissue. The systematic analysis of proteins has been termed "proteomics." In an initial phase, most of the proteomics efforts were focused on large-scale protein identification. More recently, the objectives and technologies of proteomics have 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 protein abundance, 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 these properties on a proteome-wide scale, the interpretation of the large amounts of data obtained, the development of computational approaches to infer biological function, and the integration of different types of data into a coherent model describing a biological process. The goal of the meeting is to bring together leading experts from the different areas of proteomics research to discuss emerging technologies and their applications to biological and clinical research.
Tuesday, March 25
3 - 7pm 6:15 - 7:15pm 7:15 - 7:30pm 7:30 - 8:30pm
Registration Welcome Orientation KEYNOTE ADDRESSES *Ruedi H. Aebersold, Institute for Systems Biology Tony Pawson, Mount Sinai Hospital (002) Modular Protein Interactions and Signaling Networks
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* Session Chair · Number in ( ) equals speaker abstract number · Current as of February 20, 2003.
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Program
7 - 8am 8 - 11:30am
Wednesday, March 26 Breakfast PLENARY SESSION 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 Platform Marvin Vestal, Applied Biosystems (005) Trends in Mass Spectrometry Instrumentation for Proteomics Coffee Break John R. Yates, Scripps Research Institute Proteomics in Model Systems Short Oral Abstract Presentations of Selected Posters
Torreys Peak Shavano Peak Shavano Peak Foyer
11:30am - 1pm Poster Setup
1 - 10pm
Poster Viewing
4 - 4:30pm
SPECIAL LECTURE Gregory Milman, National Institutes of Health (008) Over $1 Billion in Biodefense Research Opportunities
4:30 - 5pm 5 - 7pm
Coffee & Snacks Available PLENARY SESSION 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 Data Pavel A. Pevzner, University of California, San Diego (011) New Algorithmic Approaches for the Analysis of Peptide Mass Spectral Data Bruno M. Domon, Celera Genomics Group (012) Generation and Analysis of Data for Target Discovery in an Industrial Setting
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7 - 8pm 8 - 10pm
Social Hour POSTER SESSION 1 Identification and Quantification of Proteins/Management of Proteomics Data
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* Session Chair · Number in ( ) equals speaker abstract number · Current as of February 20, 2003.
7 - 8am 8 - 11:30am
Thursday, March 27 Breakfast PLENARY SESSION 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 Microscopy Stanley Fields, University of Washington (015) Genetic and Genomic Approaches to Protein Interaction Coffee Break Marc Vidal, Dana Farber Cancer Institute (016) Toward a Proteome Atlas for C. elegans Bertrand Sйraphin, Centre National de la Recherche Scientifique (017) Large Scale Protein Linkage Maps via Purification of Protein Complexes and Mass Spectrometric Analysis Short Oral Abstract Presentations of Selected Posters
11:30am - 1pm Poster Setup
1 - 10pm
Poster Viewing
4 - 4:30pm
SPECIAL LECTURE Gregory Milman, National Institutes of Health Whom to Contact at NIH and When?
4:30 - 5pm 5 - 7pm
Coffee & Snacks Available PLENARY SESSION In vivo Analysis *Stanley Fields, University of Washington Stephen W. Michnick, Universitй de Montrйal (020) Mapping and Visualization of Biochemical Networks in living cells Mark Johnston, Washington University (021) Finding Regulatory Sequences and their Protein Partners in Yeast by Comparative and Functional Genomics Andrew Fraser, Wellcome Trust Sanger Institute (022) Functional Analysis of the C. elegans Genome by RNA-Mediated Interference
7 - 8pm 8 - 10pm
Social Hour POSTER SESSION 2 In vivo Approaches/Analysis of Protein Activities
Program Torreys Peak Shavano Peak Shavano Peak Foyer Torreys Peak Torreys Peak Shavano Peak Shavano Peak Foyer Shavano Peak Torreys Peak Torreys Peak
9
Program
7 - 8am 8 - 11:30am
Friday, March 28 Breakfast PLENARY SESSION 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 Proteome Dolores J. Cahill, Max Planck Institute of Molecular Genetics (025) Protein Arrays: Generation and Recent Applications Coffee Break Gavin MacBeath, Harvard University (026) Protein Microarrays: From Functional Genomics to Protein Profiling Larry Gold, SomaLogic Inc. (027) Aptamer Arrays and Proteomics Short Oral Abstract Presentations of Selected Posters
11:30am - 1pm Poster Setup
1 - 10pm
Poster Viewing
4:30 - 5pm
Coffee & Snacks Available
5 - 7pm
PLENARY SESSION
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 Assemblies Susan S. Taylor, University of California-San Diego (031) Dynamic Integration of Signaling by PKA
7 - 8pm 8 - 10pm
Social Hour POSTER SESSION 3 Structural Proteomics/Control of Protein Abundance and Activity/Computational Methods
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* Session Chair · Number in ( ) equals speaker abstract number · Current as of February 20, 2003.
7 - 8am 8 - 11:30am
Saturday, March 29 Breakfast PLENARY SESSION 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 Translation Brian T. Chait, Rockefeller University Probes of Protein Interaction and Phosphorylation Coffee Break Benjamin F. Cravatt III, The Scripps Research Institute (035) Activity-Based Protein Profiling: Chemical Approaches for Functional Proteomics Elizabeth J. Grayhack, University of Rochester School of Medicine (036) Functional Proteomics Short Oral Abstract Presentations of Selected Posters
4:30 - 5pm 5 - 7pm
Coffee & Snacks Available PLENARY SESSION 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
7 - 8pm 8 - 11pm
Social Hour Entertainment
Program Torreys Peak Shavano Peak Shavano Peak Foyer Shavano Peak Foyer Shavano Peak Torreys Peak Torreys Peak
* Session Chair · Number in ( ) equals speaker abstract number · Current as of February 20, 2003.
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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 modular interaction domains. Such interactions play a broad role, not only in conventional signaling, but also in processes such as cytoskeletal organization, vesicle/protein trafficking, cell polarity, and chromatin remodeling. Through their ability to recognize post-translational modifications, interaction domains control the dynamic response of cells to external and internal signals. The reiterated use of a limited set of interaction domains may have facilitated the evolution of complex cellular function, by the joining of domains in novel combinations. I will provide evidence for these ideas, and show that cellular behaviour can be re-wired by juxtaposing domains in new ways. The use of proteomic techniques such as mass spectrometry to analyze signaling networks in yeast and mammalian cells will be discussed, and a novel strategy to rapidly analyze protein function in the mouse embryo will be described. 24
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 Griffin Institute for Systems Biology, Seattle, WA 98103 The availability of the complete human genomic sequence has catalyzed the development of new technologies for the systematic and quantitative measurement of genomic and proteomic profiles to define comprehensive molecular signatures of tissues, cells and body fluids in health and disease. Such signatures are expected to impact a wide range of biological and clinical research questions, such as the systematic study 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 and accurately quantified. Since each study typically requires the analysis of multiple samples the generation of a high throughput, automated platform 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 coded affinity tags (ICATTM 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 ICATTM reagent method toward the generation of a robust, high throughput quantitative proteomics technology. The platform incorporates new methods for stable isotope labeling of proteins, new methods for high throughput, automated data collection by ESI and MALDI tandem mass spectrometry and a suite of software tools that provide probability-based scores for the determined protein identities and quantities and organize the data in relational databases. Specific applications of the platform illustrating its current performance will also be discussed. 25
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 Vestal Applied Biosystems, Framingham, MA, USA 01701 Since 1988 two powerful ionization techniques, electrospray and MALDI, have emerged for efficiently ionizing proteins, peptides, and other large nonvolatile molecules of biological importance. These ionization techniques have been combined with a variety of mass analyzers including several types of ion traps and time-of-flight systems for proteomic applications. Particularly important are systems employing tandem MS to first determine the accurate mass of an intact molecular ion, and then fragment that ion and employ a second MS to measure the masses of the fragments. These MS-MS spectra can be used for protein identification via database searching, or interpreted de novo to determine sequence and/or post-translational modifications. Recent developments in MS-MS systems for proteomics are reviewed, and results are presented that illustrate the strengths and weaknesses of some of the alternatives. Anticipated trends for the future are briefly discussed. 26
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 range of 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. Protein identification criteria and database `search space' can have considerable impact on the number of proteins incorrectly identified. To overcome some of these limitations, depletion and enrichment methods were applied to cellular and biological fluid samples prior to MS-based proteomic analysis. The selective removal of a set of high abundance proteins from plasma, serum, cerebrospinal fluid, and bronchioalveolar lavages enabled visualization and identification of minor components from two-dimensional gels. Cell-type enrichment was achieved from freshly-resected tissues and resulted in the identification of specific proteins and markers of histopathology in breast tumors. By combining subcellular fractionation and stable isotopic Cys- labeling, minor proteins in membranes and organelles were identified. As protoemics technology continues to improve, depletion and enrichment methods will remain key to identifying larger numbers of proteins from tissues, cells and biological fluids. 27
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 ongoing projects and establish new initiatives as part of a comprehensive and sustained biodefense research program. The NIAID biodefense research agenda focuses on studies of microbial biology and host responses to microbes; the development of new vaccines, therapies, and diagnostic tools; and the development of research resources such as appropriate laboratory facilities. We anticipate that the large investment in biodefense research will have many positive "spin-offs," similar to the manner in which HIV/AIDS research has advanced THE UNDERSTANDING and treatment of many other diseases. NIAID research on organisms with bioterrorism potential will almost certainly lead to an enhanced understanding of other more common and naturally occurring infectious diseases that afflict people here and abroad. In particular, the advancement of knowledge should have enormous positive impact on our ability to diagnose, treat and prevent major diseases such as malaria, tuberculosis, HIV/AIDS, and a spectrum of emerging 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 and Infectious 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 NIH Trans-Agency Complementary and Alternative Medicine Coordinating Committee (http://nccam.nih.gov/), the NIH Committee for Electronic Research Administration (http://era.nih.gov/), and the Board of Directors of the Biotechnology Industry Organization Council 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 House Office of Science and Technology Policy (http://www.ostp.gov/). In addition to his NIH "day job," Dr. Milman is a Visiting Professor at the University of Maryland. 28
Speaker Abstracts 010 · Wednesday, March 26 · Analysis, Interpretation and Management of Proteomics Data · Schwikowski Computational Tools and Challenges for Integrating Proteomic Data Benno Schwikowski Institute 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 the protein level. To unfold its full potential, this data needs to be interpreted together with other types of large-scale information, such as mRNA abundance measurements, protein-protein, and protein-DNA interaction data. As data volumes are enormous, the task of acquiring and integrating 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 and integrating proteomic data, and present a suite of open-source software tools under development at the ISB that support and automate this process. In human, the available data is much more sparse, and the biological systems are much more complex. This requires strategies that iterate between experiments and computational high-throughput analyses. This presentation will contain case studies in both yeast and human that illustrate these challenges. 29
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, the question 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/MS projects when the possibility of expert analysis is limited. Thus, algorithms that sort out reliable database hits from unreliable ones and identify mutated and modified peptides are sought. Most MS/MS database search algorithms rely on variations of the Shared Peaks Count approach that scores pairs of spectra by the peaks (masses) they have in common. Although this approach proved to be useful, it has a high error rate in identification 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 to identification of modified peptides and demonstrate their advantages over the Shared Peaks Count. We also use the spectral alignment approach as 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. 30
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 that include quantitative analysis of gene-specific mRNA levels and expressed proteins in normal and diseased cells. Differences in the expression levels of nucleic acid and protein gene products could suggest protein drug targets that are directly causative of disease, or reveal biochemical pathways that could be modulated by therapeutic molecules. Any effort based on mRNA or protein expression level comparisons could be confounded by a number of factors: (1) mRNA level in steady-state may not be correlated with actual encoded protein levels; (2) differentially expressed protein levels might be a result of disease process, and not causative of the process, and therapeutic intervention based on such a difference will be unproductive and (3) the differential expression of mRNA or protein may be the result of biological variation unrelated to the disease process under study. In order to address these possibly confounding factors, it is necessary to validate potential targets by establishing their firm association with disease, and their minimal distribution in non-diseased tissues of any type. This requirement suggests that emphasis on true and reproducible quantitation of protein expression levels in a variety of samples will be an effective and highly efficient method of generating drug targets with a high degree of utility. To achieve this aim, we have established an industrial-scale proteomics-based discovery platform consisting of cell biology, protein chemistry, and mass spectrometry technical groups. The analytical method used for quantitation employs isotope labeling for differential analysis (ICAT, Applied Biosystems, Inc.). With this technique, tryptic peptides are generated from labeled proteins that have been specifically captured from various subcellular locations or protein families. The resulting peptides are identified and quantified by mass spectrometry. To evaluate this approach on a large-scale, we have applied it to a study of continuous cell lines derived from human pancreatic adenocarcinomas. We have been able to establish processes for target discovery therapeutic antibody target identification for 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 an undertaking. 31
Speaker Abstracts 014 · Thursday, March 27 · Protein Interactions and Protein Linkage Maps · Frank Single-Particle Reconstruction of Molecular Machines Using Cryo-Electron Microscopy Joachim Frank Howard Hughes Medical Institute, Health Research, Inc. at the Wadsworth Center, and the Department of Biomedical Sciences, State University 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 reactions of 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 interacting molecules (Alberts, 1998). Three-dimensional visualization of molecular machines in different processing states poses a difficult challenge since many of the varying binding constellation do not allow formation of crystals suitable for X-ray crystallography. On the other hand, they are as a rule too large to be approached by NMR. Cryo-electron microscopy of single particles (see Frank, 1996), a relatively new technique, is able to overcome this problem, yielding three-dimensional density maps of the macromolecular assembly at close-to-native conditions. The success of the technique is exemplified by the ribosome. Molecular detail was revealed by cryo-EM well before the X-ray structure was solved. The current resolution, 7.8Е, 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 as three-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 routine achievement of ~10Е 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: 318322. J. Frank (1996). Three-dimensional Electron Microscopy of Macromolecular Assemblies. Academic Press, San Diego. 32
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 Hughes Medical 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-ligand interactions. 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 the yeast Saccharomyces cerevisiae. We generated an array of yeast transformants containing ~700 different membrane proteins fused to the Nterminal half of ubiquitin and screened it by mating to yeast bearing a membrane protein fused to the C-terminal half of ubiquitin. Interactions have been detected for oligosaccharyltransferase components, ER-associated degradation (ERAD) constituents, and a variety of other proteins as part of collaborative projects of the NCRR Yeast Resource Center. This assay has also been used with human Toll-like receptors (TLRs), which detect microbial antigens and activate an innate immune response. Initial experiments indicate that the assay can detect a ligand-dependent interaction of TLRs as well as an interaction of TLRs with a downstream signaling component. In another approach, we are developing a genetic screen to identify proteins that function in a common pathway or process. Interactions can be artificially forced via heterodimerizing leucine zippers, and the resulting yeast assayed for a gain-of-function phenotype. We have identified the minimal portions of Fos and Jun that will interact in yeast, and have shown that a Fos-GFP fusion can colocalize with Jun-protein fusions. In a third approach, we have initiated a systematic analysis of the chemical sensitivities of the haploid deletion strains generated by the Saccharomyces genome deletion project. The loss of a protective gene in a pathway affected by a drug can lead to a synthetic lethal effect in which the combination of a deletion and a normally sublethal dose of a chemical results in lethality. We have examined sensitivity to hydrogen peroxide and to several drugs. 33
Speaker Abstracts 016 · Thursday, March 27 · Protein Interactions and Protein Linkage Maps · Vidal Toward a Proteome Atlas for C. elegans Marc Vidal Dana Farber Cancer Institute, Boston, MA 02115 The availability of complete genome sequences suggests new approaches for biological research to complement conventional genetics and biochemistry. 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 phenotypic analysis (phenome), localization of expression projects (localizome), etc.... The resulting proteome atlas of integrated maps should be valuable for a systems biology approach to the study of development. 34
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 Sйraphin Centre de Gйnйtique Molйculaire, CNRS, 91198 Gif sur Yvette, France Many cellular processes are carried out by multiprotein complexes. Those include the ribosome, the proteasome, the spliceosome, RNA- and DNA-polymerases, etc.... To identify and analyse such assemblies, we have developed a generic procedure that we named TAP for Tandem Affinity Purification. The TAP tag allows for the rapid purification of proteins expressed at their natural level under native conditions permitting thereby protein complex identification from a relatively 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 given target protein providing insight into how the ensemble of expressed proteins (namely, proteome) is organised into functional units. The TAP method and mass spectrometry were used in a large-scale approach to characterise multiprotein 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 functions for many proteins. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Application of the TAP method to various organisms will be discussed. 35
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 organization in intact, living cells? We have developed a general experimental strategy that addresses this question by allowing the quantitative probing of molecular interactions in intact, living cells. The experimental strategy is based on Protein fragment Complementation Assays (PCA), a method whereby protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction. A biochemical machine or pathway is defined by grouping interacting proteins into those that are perturbed in the same way by common factors (hormones, metabolites, enzyme inhibitors, etc). I will describe how we go from descriptive to quantitative representations of biochemical networks at an individual to whole genome level and how our approach will lead ultimately to better descriptions of the biochemical machineries that underlie living processes. 36
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, MO 63130 Functional sequences in genomes are more conserved through evolution than surrounding non-functional sequences, and can be recognized by the `phylogenetic footprints' they make in alignments of sequences of related species. Functional protein-coding sequences make relatively obvious phylogenetic footprints; functional non-protein coding sequences, such as those regulating gene expression, are generally small and under less evolutionary constraint, causing their footprints to fade quickly in evolution. Therefore, detection of regulatory sequence elements requires comparison of genome sequences of relatively closely-related species. To identify functional non-protein coding sequences in the genome 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 motifs appear in alignments of orthologous promoters than in shuffled versions of these alignments. The distribution of conserved residues across a promoter 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 regulatory sequences 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 more genes. Since only about 40 of these were previously identified, we have identified hundreds of novel potential functional sequences in the yeast genome. We have developed and are employing high throughput genetic and biochemical methods for identifying the proteins that bind to these conserved sequence motifs. 37
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 Julie Ahringer2. 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 of predicted genes in any metazoan. One systematic approach to analyse gene function is to examine the loss-of-function phenotypes of every predicted gene in a genome -- this is the approach that we have taken in C. elegans. We have generated a reagent that uses RNA-mediated interference (RNAi) to individually inhibit ~90% of all ~19,000 predicted genes in the C. elegans genome. Using this reagent, we examined loss-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 essential genes lie in 13% of the genome. These multi-megabase regions are also enriched for genes with similar transcriptional profiles, suggesting that genes 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 currently carry out multiple screens to identify genes involved in modulating ras signalling in C. elegans and will present these data. 38
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, Scott Bidlingmaier2, Geeta Devgan1, Perry Miller2, Mark Gerstein2, Michael Snyder1,2, 1Department of Molecular, Cellular, and Developmental Biology, 2Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520 The genomes of a wide variety of organisms have now been sequenced; a major challenge ahead is to understand the function, regulation and modification of the many encoded gene products. We have been carrying out proteomics approaches to the identification and analysis of signalling pathways in yeast. 121 of 122 protein kinases were cloned and purifed from yeast as GST fusions and analyzed for their ability to phosphorylate 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 to phosphorylate Tyr. In addition, we have now cloned 6000 open reading frames and overexpressed their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with a variety of different proteins, nucleic acids and phospholipids. As examples, we have probed yeast proteome chips with calmodulin and six different phospholipids. These studies revealed many new calmodulin and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. They can also be used to screen protein-drug interactions and to detect posttranslational modifications. 39
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, IhnestraЯe 73, D-14195 Berlin, Germany and [email protected] AG, Emil-Figge-Strasse 76 A, D-44227 Dortmund, 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 a successful system for expression and purification of thousands of proteins in parallel. We have used these proteins to generate high density protein arrays on glass slides and demonstrates their potential applications in the profiling of the antibody repertoire of auto-immune patients, and the determination of the specificity or cross-reactivity of antibodies. The optimisation of antibody arrays will also be described. 40
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 Professor Department 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 are approaching this problem in two ways. First, we are developing and using protein microarray technology to elucidate molecular interactions within defined families of related proteins. In particular, we are focusing on families of protein domains ­ such as the PDZ domains ­ that mediate interactions by binding linear peptides in a sequence-specific fashion. Second, we are adapting protein microarray technology to screen 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 also developing methods to measure the abundance and post-translational modification state of multiple proteins in complex solutions (such as cellular lysates) using microarrays of antibodies. Our recent progress in these efforts will be reported. 41
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. The photocrosslinking of the photoaptamer's BrdU to an electron rich amino acid side chain of the protein adds a second dimension of specificity to 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 all proteins of interest without concern for crossreactivity of secondary binding agents. Since photoaptamers are covalently bound to their target analytes before signal detection, the arrays can be vigorously washed to remove background proteins, yielding superior signal-to-noise ratios and 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 key component for this change of paradigm to more informative multiplexed analyses. 42
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, San Francisco, 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 by experiment, 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 crystallography of the subunits, low-resolution electron microscopy of the assembly, and chemical cross-linking). Therefore, there is a need for a computational framework that can take into account all available information about the structure of an assembly and calculate at the appropriate resolution all models that are consistent with the given input. To this end, it is useful to express structure determination as an optimization problem. The three components 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 43
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). PKA is a prototype for the large protein kinase superfamily and the catalytic (C) subunit provides us with a template for the folding and dynamics of the protein kinase core. The very different properties of the two lobes, one highly dynamic and the other very stable, contribute to an extended active site, mostly preformed, for docking ATP and protein substrates. In addition, the extensive conserved networks that radiate outward from the site of phosphoryl transfer provide many opportunities for communication across long distances. The regulatory subunits harbor the cAMP binding modules that regulate the activity of the enzyme. The dynamic properties of the R subunits, specifically how the cAMP binding modules toggle between a cAMP bound state and the holoenzyme state has been probed by hydrogen/deuterium exchange coupled with mass spectrometry. 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-specific binding peptides that mimic the AKAP PKA binding site. These probes have been used in vitro and in cells to establish the importance of the binding of RI and/or RII subunits. In addition to probing the spatial organization of the type I and II holoenzymes in cells, we have used recombinant FRET-based activity reporters to probe the activity of PKA in living cells. These multiple strategies have allowed us to better appreciate the dyanmic features of the this enzyme and its targeting in living cells while high resolution structure analysis has provided an understanding 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.) 44
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 Roger Bumgarner3, Departments of 1Biochemistry and 3Microbiology, University of Washington, Seattle, WA 98193 and 2Division of Public Health Sciences, 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 mRNA species into their encoded proteins can be regulated, sometimes resulting in discordance between the level of an mRNA and the abundance of its protein product. We have reported a technology, Translation State Array Analysis or TSAA, that simultaneously assesses the level of an mRNA and its association with the translational machinery. Detailed descriptions through TSAA of the translational behavior of 4931 transcripts 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 mRNAs primarily located in polysomes, the average ribosome spacing on the message can vary by more than an order of magnitude. Thus, this spectrum of translational behavior provides an extraordinary dynamic range of regulation, exclusive of actual transcript levels. These diverse translational properties do not show a strong correlation with global mRNA properties such as transcript abundance, codon bias, initiator AUG context or estimated lengths of the untranslated regions. For example, abundant mRNAs tend on average to be better translated than the population as a whole, but some representatives of this class, such as the mRNA products of GCN4 and HAC1, are located only to a minor extent 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 its specific translation activity. The information garnered from this analysis allows one to estimate relative rates of protein production across the transcriptome, as well as providing new insights into this additional level of gene expression control. 45
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, CA 92037, 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 regulated by posttranslational mechanisms, limiting the utility of conventional proteomics techniques for the characterization of these proteins. Our research group has initiated a program aimed at generating chemical probes that interrogate the state of enzyme active sites in whole proteomes, thereby facilitating the simultaneous activity-based profiling of many enzymes in samples of high complexity. Progress towards the generation and utilization of active site-directed chemical probes for the proteomic characterization of several enzyme classes will be described. These enzyme classes fall into two general categories: 1) enzymes for which active site-directed affinity agents have been welldefined, and 2) enzymes for which active site-directed affinity agents have been lacking. The application of activity-based protein profiling to the functional characterization of enzyme activities that vary in human cancer specimens will be highlighted, as will be the use of this strategy as a screen to discover potent and selective reversible enzyme inhibitors. 46
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 fusing each ORF to GST. Purification and biochemical assay of pools of GST-ORF proteins allows analysis of the proteome for biochemical function. Assignment of biochemical activity is rapid, versatile, and sensitive using this method. Both binding and catalytic assays have been used to detect more than 35 proteins (and their cognate genes) with this method. The catalytic activities include a wide variety of different chemical reactions (ligation, group transfer, redox, isomerization, phosphorylation, and hydrolysis). Proteins that act in a complex have also been with this system. In collaboration with Mike Snyder, Erin O'Shea, and Jonathan Weissman, we are producing a new C-terminal fusion array that should be more useful for membrane proteins, and will have a number of improved features. 47
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 protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. We have shown that thiolysis of mutant intein-fusions leads to the generation of recombinant protein-thioester derivatives that can be chemically ligated to polypeptides (synthetic or recombinant) bearing an N-terminal Cys. This semisynthetic process, generally referred to as Expressed Protein Ligation (EPL), has been used to incorporate unnatural amino acids, posttranslational modifications and isotopic probes site-specifically into proteins. EPL has been applied to numerous systems over the last few years and has allowed a variety of biological questions to be addressed. Selected examples from our own work will be discussed. A second application of protein splicing that will be discussed is in the area of chemical genetics. We have recently developed a novel system that allows protein trans-splicing to occur only in the presence of the small molecule, rapamycin. This `conditional protein transsplicing' (CPS) technique provides a means to trigger the post-translational synthesis of a target protein from two fragments. In principle, CPS provides a level of temporal control over protein function that is difficult to achieve using standard genetic approaches. Recent progress in this area will be described. 48
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. Marcotte Institute 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 completely uncharacterized and of unknown function. There is a broad need for methods to discover the functions of these thousands of uncharacterized genes and how the corresponding proteins participate in networks, pathways, and systems in the cell. It has now become clear that the genomes themselves contain extensive information about the relationships between the proteins. To begin to reveal the functions of these proteins, and how the proteins associate into systems and pathways, we have developed computational approaches for inferring the functions of proteins that have never before been studied. These methods work by examining the contexts of genes, such as which organisms carry the genes, the occurrence of gene fusion events, and the relative positions of genes on the chromosomes. Proteins whose genes occur in similar contexts can be shown to work in similar cellular pathways. By using such methods, cellular networks consisting of thousands of proteins from a single organism can be reconstructed. The networks contain many known cellular systems, as well as potentially novel pathways and systems. Supported by grants from the Welch Foundation, the National Science Foundation, the Texas Advanced Research Program, a Dreyfus New Faculty Award, and a Packard Fellowship. 49
Speaker Abstracts 040 · Saturday, March 29 · Methods to Explore Function · Bork Comparative Analysis of Protein Interaction Networks Peer Bork EMBL, Meyerhofstr.1, 69012 Heidelberg and MDC, Berlin [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 interaction data [2] and finally try to identify biological meaningful units (e.g. pathway, cellular process) within interaction networks [3] that are derived from 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 50
poster abstracts Wednesday, March 26: Identification and Quantification of Proteins/Management of Proteomics Data
101 Analysis of redox sensitive proteins by tandem mass 102 Longitudinal Study of Rheumatoid Arthritis (RA) by
spectrometry
Mass Spectrometry: Serum Proteins and Metabolites
Nancy Andon1, Robert Cumming2, David R. Schubert2, John R.Yates III, and Paul A.Haynes1 1The Torrey Mesa Research Institute of Syngenta, San Diego, CA, USA, 92121; 2The Salk Institute for Biological Studies, San Diego, CA, USA, 92121 The toxicity of chemically reactive oxygen species is thought to make a significant contribution to the death of nerve cells in stroke, Parkinson's disease and Alzheimer's disease. We have developed an oxidative stress model in which murine hippocampal glutamate sensitive and resistant cell lines are exposed to glutamate, and then compared using a modified 2D system, which allows us to specifically identify proteins whose disulfide binding properties change with oxidative stress. We have also used this technique to test a severe oxidative stress model, in which the glutamate resistant and sensitive cell lines were exposed to either diamide or H2O2 . A number of changes in protein interactions were identified between the stressed and control cell lines. Of 117 unique proteins identified, the vast majority are heat shock proteins, 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 known to be involved in either oxidative stress or apoptosis were also identified, along with a number of other proteins implicated in these pathways, and, most interestingly, a number of proteins of as yet unknown function.
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 to collect serum, urine, and clinical outcome measures at multiple timepoints over the course of 3 years. An interim analysis of the baseline timepoint was performed on 20 subjects with chronic RA and 20 healthy subjects that were matched for age, gender, and comorbid conditions. RA is a chronic inflammatory disease. There is an unmet clinical need to identify biomarkers that are predictive of progression of disease as well as response to therapeutics. The interim analysis is a promising first step towards realizing that goal as it successfully revealed expected and novel molecules. With < 1 mL of serum, peptides and metabolites were quantitatively profiled on high-resolution time-of-flight instruments using on-line liquid chromatography and electrospray ionization (LC-ESI-MS) for nonvolatile molecules and GC-MS for volatile molecules. Using one-dimensional chromatography, 2500 molecular ions were quantified per sample yielding approximately 70 differences at p < 0.001 (~ 3 expected by chance). The analysis is currently being extended using two-dimensional chromatography. Molecular identification is made using tandem mass spectrometry with ion-trap and triple-quadrupole instruments along with computer analysis of the fragmentation spectra.
103 Identification of the surface interacting protein of decoy 104 receptor 3 Yung-Chi Chang, Shie-Liang Hsieh, Institute and Department of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan Decoy receptor 3 (DcR3) is a soluble receptor belonging to the tumor necrosis factor receptor (TNFR) superfamily and is readily detected in certain cancer patients. Recently, we further demonstrated that DcR3treated dendritic cells (DCs) skew T cells response to Th2 phenotype. Moreover, the presence of DcR3 also downregulated the differentiation and function of CD14+ monocytes into macrophages. We then want to address which surface molecule can exert such function. The monocytic cell line, THP-1 was lysed in lysis buffer and then enriching the glycoprotein fraction of cell lysate using ConA column. Finally the eluant of ConA column was further purified by DcR3.Fc affinity column and subjected to MALDI-TOF and LC MS/MS analysis. The potential molecule was about 67 kDa and high glycosylated, and the biological function of molecule should be further verified. 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
51
Poster Abstracts Wednesday, March 26: Identification and Quantification of Proteins/Management of Proteomics Data
105 Systematic, quantitative proteomic analysis of the
106 Novel Reagents for Quantitative Proteomics.
Saccharomyces cerevisiae cell cycle
Mark R. Flory, Hookeun Lee, Kyle Serikawa, Roger Bumgartner, David Morris, David Goodlett, Ruedi Aebersold Institute for Systems Biology, Seattle, WA 98103
M.H. Gelb, P. Bottari, Y. Li, D. Wang, F. Turecek Depts. of Chemistry and Biochemistry, Univ. of Washington, Seattle, WA 98195
Fundamental to a complete understanding of the structure, function and control of biological systems is the ability to quantitatively analyze proteins, the catalysts and effectors of nearly all biological activities. To this end, we are using quantitative mass spectrometry for the systematic measurement of cell cycle-regulated protein expression levels in Saccharomyces cerevisiae following release from mating factor synchronization. Protein samples harvested from both timepoint and reference (asynchronous) cultures were reduced and labeled with isotopically heavy or light ICAT (isotope-coded affinity tag) reagent prior to trypsin digestion. ICAT-labeled peptide mixtures separated by off-line liquid chromatography were analyzed by reverse-phase tandem mass spectrometry. Using a second-generation acid-cleavable ICAT reagent we achieve a consistent sampling depth over 1500 proteins. The proteomic profiles were integrated with translation and transcription expression profiles from the same yeast culture to create a composite, simultaneous description of three modes of expression. The data were analyzed using cluster analysis tools such as Cytoscape. The combination of three types of data representing gene expression at different levels extends traditional profiling approaches that measure only transcript levels. This dataset is expected to catalyze the development of informatics tools for the integration and analysis of large, diverse datasets and to increase the understanding of the eukaryotic cell cycle.
R. Aebersold, A. Tao, H. Zhang Institute for Systems Biology, Seattle, WA We have synthesized a variety of novel reagents for the quantitative analysis of enzymes in complex mixtures (cell lysates). The technique is based on biotinylated substrates combined with heavy isotope-containing product internal standars. Mass spectrometry is used for analysis after biotin conjugates are captured by solid phase streptavidin. It will be shown that the technique is applicable to the analysis of virtually any enzyme and is fully automable for application in clinical or research laboratories. Furthermore, several enzymes can be quantified in a signal analysis. A related technique is the development of Isotope Coded Affinity Tags (ICAT). New generation ICAT reagents have been designed and are being tested for quantitative analysis of specific protein functionalities.
107
A Protein Chemist's Guide To The Gel-Free Proteome
Galaxy
Kris Gevaert, An Staes, Marc Goethals, Jozef Van Damme, Lennart Martens, Hans Demol, Bart Ghesquiиre, Sara De Groot, Magda Puype, Grйgoire R. Thomas & Joлl Vandekerckhove, Department of Medical Protein Research, Ghent University, B-9000 Ghent, Belgium.
We have developed a highly versatile procedure for the isolation of subsets of peptides out of complex mixtures such as proteolytic digests of total cell or tissue extracts without the use of affinity tags. Because of its homology to diagonal electrophoresis and diagonal chromatography, we have called this novel procedure combined fractional diagonal chromatography or COFRADIC® (Gevaert et al., Mol. Cell. Proteomics, in press). Following a proteolytic digest, peptide mixtures are fractionated by reverse phase (RP) HPLC and in each fraction, representative peptides (i.e. peptides containing sufficiently rare amino acids) are altered such that their chromatographic behaviour changes. Upon a secondary RP-HPLC separation, these altered peptides elute differently and are specifically collected for further sequence analysis. COFRADIC® is a highly versatile technique since it allows to isolate any peptide containing a group that can be specifically altered. We here show that peptides containing methionine and/or cysteine as well as phosphorylated peptides and peptides spanning the amino terminus can be specifically isolated and used to identify their parent proteins. Furthermore, the complete procedure of peptide isolation, LC-MS/MS analysis and peptide/protein identification is hugely automated. The use of COFRADIC® for proteome studies will be addressed by the results of various proteome studies in which classes of proteins that frequently escape analysis in gelbased studies, are now clearly identified. These include highly hydrophobic proteins, low abundant proteins, extremely large proteins and highly charged ones.
108
Parallel Peptide Tandem Mass Spectrometry on a Time-of-flight Mass
Analyzer
Eugene C. Yi, Ning Zhang, Marcello Marelli, John Aitchison, Benno Schwikowski and David R. Goodlett
Mass spectrometry (MS) is currently the most important discovery based tool in the field of Proteomics (1). It is used extensively for indirect descriptive analysis of proteins by analyzing peptides produced by proteolytic action on proteins. Two methods are commonly used to identify proteins through database search of MS data acquired on peptides: 1) mass fingerprinting and 2) tandem MS (MS/MS). In our laboratory microcapillary HPLC (µLC) MS/MS in an ion trap (IT) mass spectrometer is the preferred method. However, we have recently shown that standard data-dependent (DD) ion selection during µLC/MS/MS when applied to very complex samples reproducibly identifies the same proteins (~ same for peptides) in replicates 25% of the time (2). The reasons 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 collision induced dissociation (CID) of peptides in-source on an ESI-TOF-MS and in collision cell on an ESI-QTOF-MS. Parallel MS/MS of peptides rather than the current standard of serial MS/MS has been previously demonstrated using the high mass accuracy/resolution of Fourier transform ion cyclotron resonance MS (FTICRMS), referred to as multiplex MS (3), to de-convolute the mixed fragment ion mass spectra. We refer to the parallel MS/MS method carried out on lower mass accuracy/resolution TOF mass analyzers as shotgun MS/MS to distinguish it from the FTICRMS method. A combination of mass fingerprinting, MS/MS and chromatographic synchronization of parent to fragment ions is used to identify proteins from mixed fragment ion mass spectra of peptides. Additionally, the method may be used to quantify relative changes in protein expression by incorporating stable isotope labeling. When differential stable isotope labeling is incorporated, then sequence tags may be generated to aid cases of degeneracy at the protein level that arise from the lower mass accuracy of a TOF mass analyzer. In addition to proof of principle using standards, we will present some initial data using shotgun CID to characterize membrane proteins isolated from yeast peroxisomes and discuss bioinformatic approaches to identification of parent proteins from tandem MS data produced 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.
52
Poster Abstracts Wednesday, March 26: Identification and Quantification of Proteins/Management of Proteomics Data
109 Proteins induced by organophosphate exposure in OP-
110 Biomarkers of Inflammation from SELDI Analysis of
susceptible and -resistant strains of the southern cattle tick
Rat Serum after Acute Lipopolysaccharide (LPS) Treatment.
Felix D. Guerrero and Andrew Y. Li, USDA-ARS Livestock Insects Research Laboratory, Kerrville, TX, USA, 78028
JE Hartis, A Xu, ME Bruno, BA Wetmore, JA Taylor, BA Merrick. NIEHS, PO Box 12233, RTP, NC 27709; Ciphergen, Freemont, CA 94555
Organophosphate (OP) resistance has developed in Mexican populations of the southern cattle tick, Boophilus microplus. Reintroduction of this tick and its associated pathogens into the United States would cause severe economic losses to the cattle industry. We have begun a comparative study of the proteome of OP-susceptible and -resistant strains of B. microplus. As an initial step, 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 for each strain. Additionally, untreated larvae from each strain have also been collected. Soluble proteins were extracted from each sample and analyzed by 2-dimensional IEF-PAGE to identify OP-inducible proteins and strain-specific protein expression.
Bacterial LPS can induce septic shock by release of cytokines and vasoactive peptides. SELDI-MS (surface enhanced laser desorption ionization) analysis of serum was conducted for biomarker discovery of circulating bioactive peptides during acute inflammatory response to LPS in a rat model. Male rats at 12 rats per group were treated by i.p. with saline or 5 mg/kg, LPS for 2 and 6 hr. Serum TNF levels were high at 2 hr after LPS but returned to control after 6 hr. Organ histopathology was limited to vascular endothelial changes. Mass spectra (MS) were measured from surface retentates of whole serum or multidimensional fractionation (MDF) of serum by strong anion exchange and step-wise pH elution. SELDI analysis of whole serum showed 4 ion peak changes compared to 36 peak changes at p<0.01 in MDF serum at 2 hr. At 6 hr after LPS, 20 peak changes were found in MDF serum compared to 2 peak changes with whole serum. About two fold more ions were increased as were repressed in whole serum compared to MDF serum. Comparison of LPS treated rats at 2 and 6 hr revealed 17 ion changes, none of which were found in whole serum. Prominent ions in each fraction are being isolated for further MS analysis to obtain the identity of the underlying proteins. We conclude SELDI analysis of MDF serum fractions is a more sensitive means of detecting LPS responsive biopeptides than from whole serum alone.
111
Identification of Ubiquitinated ER Proteins
Amy L. Hitchcock, Kathryn Auld, Seth Frietze, Steven P. Gygi, and Pamela A. Silver Departments of Cancer Biology, Biological Chemistry and Molecular Pharmacology, and Cell Biology; The Dana Farber Cancer Institute and Harvard Medical School, Boston, MA USA 02115
The highly conserved Cdc48/p97-Npl4-Ufd1 protein complex is required for the proteasome-dependent degradation and processing of ubiquitinated endoplasmic reticulum (ER) proteins. As such, Saccharomyces cerevisiae cells containing the conditional npl4-1 mutation display a striking accumulation of ER-associated ubiquitinated proteins. However, it is unknown whether all ubiquitinated ER proteins require Npl4p function for proteasomal degradation, or if there might be an Npl4p-specific pathway for degradation of a subset of ubiquitinated ER proteins. In the current study, a combined biochemical/proteomic approach has been undertaken to identify (1) all ubiquitinated ER proteins in yeast, and (2) the ubiquitinated ER substrates of Npl4p-dependent proteasomal degradation. Using a yeast strain expressing 6xHis-tagged ubiquitin, we have purified ubiquitinconjugates from wild type and npl4-1 ER membrane fractions by immobilized Ni++ affinity chromatography. Proteins present in the bound fraction are identified by direct mixture analysis of tryptic peptides using nano-scale microcapillary LC-MS/MS. Currently we have identified >100 putative ubiquitinated ER proteins from wild type membranes. These proteins are qualitatively compared to those isolated from npl4-1 membranes, as well as to a negative control strain. To quantitatively identify ubiquitinated proteins that are specifically stabilized in npl4-1 ER membranes, we will directly compare wild type and npl4-1 ubiquitin conjugates by combining the above strategy with isotope-coded affinity tags (ICAT). Ultimately, this approach could be utilized to identify, for example, the substrates of specific E2 or E3 enzymes, or proteins differentially ubiquitinated in response to extracellular stimuli.
112 Quantitative Fungal Proteome Analysis Following Separation by Multidimensional Chromatography P. T Jedrzejewski, G. Ganshaw, A. Gaertner Genencor International, Inc., 925 Page Mill Road, Palo Alto, CA 94304, USA The filamentous fungus Trichoderma reesei is an important biological host for the production of industrially relevant enzymes, such as biomass degrading cellulases, and enzymes for food and beverage processing. In order to optimize enzyme production for this fungal host, and to design the appropriate enzyme mix for its respective application, a number of approaches can be utilized, for example, genes can be knocked out or added and fermentation conditions can be manipulated. Optimization of the fermentation conditions may be improved by gaining insight at the proteome level (protein identification, protein metabolism). We have applied multidimensional chromatography for the separation and purification of the exoproteome of a cellulase producing strain of the T. reesei. Following multidimensional chromatography (ion-exchange chromatography followed by various formats of reversed-phase chromatography) on the protein level, samples were digested and analyzed by nanoLC-MS/MS. Protein identification was performed using database search software. We successfully identified over 35 proteins from this fungal exoproteome. This compares favorably with other proteomic methods (e.g. 2D-PAGE) in terms of throughput as well as number of proteins identified. We show the results of quantitative characterization for a number of identified proteins across various fermentation conditions.
53
Poster Abstracts Wednesday, March 26: Identification and Quantification of Proteins/Management of Proteomics Data
113
PROTEOMIC STRATEGY FOR IDENTIFICATION OF GPI-
ANCHORED PROTEINS IN H. SAPIENS AND A. THALIANA PLASMA
114
Selective Chemical Profiling of PPAR Agonists Utilizing Proteomics
Technology
MEMBRANES.
Linda Kochanski, Anita Y. Lee, Patrick Griffin*, Margaret E. McCann, Ching H.
Felix Elortza1, Allan Stensballe1, Leonard Foster1, Thomas Nьhse2, Scott Peck2, and Ole N. Jensen1.
Chang, Ellen Rohde and William A. Hanlon. Departments of Molecular Profiling Proteomics Research, Basic Chemistry and Pharmacology. Merck Research Laboratories, Rahway, NJ 07065. ExSAR, Monmouth Junction, NJ 08852*
1Department of Biochemistry and Molecular Biology, University of Southern Denmark, 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 the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. There is substantial evidence suggesting that these GPI-anchored proteins are clustered in sphingolipid-sterol microdomains or lipid rafts. Rafts play a crucial role in many cellular processes including membrane traffic, cell signalling and human diseases. We have combined detergent-based phase separation methods and enzyme treatment to enrich and isolate GPI-proteins from lipid rafts from human cells and GPI-proteins from plasma membrane fractions isolated from Arabidopsis thaliana. The isolated GPI-protein fractions were precipitated with acetone, separated by SDS-PAGE and visualized by silver staining. Protein bands were excised for in-gel digestion and the resulting peptides were subsequently analyzed by nanoscale liquid chromatography coupled online to electrospray Q-TOF tandem mass spectrometry (nLC-MS/MS). Tandem mass spectra were interpreted automatically using the MASCOT sequence database search engine. With this combination of methods we have identified more than 50 GPI anchored proteins in H. sapiens and A. thaliana, validating this proteomic approach for analysis of GPI anchored membrane proteins and generating the largest experimental data set on GPI-proteins to date. The technique is expected to be generally applicable and highly useful for studying membrane proteins, for example for determination of molecular markers on the cell surface for diagnostic or therapeutic purposes. This research was supported by a post-doctoral fellowship to F.E. from the Basque government and by resources provided by the Danish Biotechnology Instrument Center.
Peroxisome Proliferator Activated Receptors (PPARs) are nuclear transcription factors that regulate genes involved in lipid and glucose homeostasis. Three PPAR isotypes exist. PPAR is ubiquitously expressed. PPAR, which is highly expressed in hepatocytes, results in peroxisomal proliferation and -oxidation of fatty acids in rodent liver when activated. PPAR, which is predominately expressed in adipose tissue, is responsible for adipocyte differentiation, fat storage and improved insulin signaling of mature adipocytes. PPAR and receptors may function as targets for the treatment of dyslipidemia and insulin resistance. It has been found that activation of these PPAR receptor isotypes causes different effects in the liver of rodents. Sustained induction of peroxisome proliferation and the peroxisomal -oxidation system in liver of rodents chronically treated with peroxisome proliferators leads to development of hepatocellular carcinomas. To better understand the toxicity associated with PPAR agonists we compared proteomic profiles of liver tissue from mice treated for up to 7 days with a selective agonist (Compound 1) and two selective agonists, rosiglitazone (Compound 2) 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 protein expression profile changes and identified proteins showing altered expression. Sixty eight proteins, induced by the PPAR agonist, had altered protein expression levels. Forty of these proteins were unique to the PPAR agonist. Treatment with the Merck TZD and rosiglitazone induced altered expression in 24 and 26 total proteins respectively. Thirteen of the proteins with altered expression were common between all three agonists. Proteins with altered expression were identified as enzymes involved in peroxisome proliferation and -oxidation of fatty acids. Various isoforms of these enzymes, which were differentially expressed in response to drug treatment, were identified as well. The identification of these proteins demonstrates that protein expression maps produced using 2D electrophoresis can be used to profile compounds of different chemical classes.
115
Use of discovery science-approach to elucidate biological effects induced by
mobile phone radiation: A pilot study
1Dariusz Leszczynski, 1Reetta Kuokka, 1Sakari Joenvддrд, 1Jukka Reivinen, 1Teemu Kallonen, 2Kari Jokela, 2Ari-Pekka Sihvonen, 2Tim Toivo, 3Niels Kuster and 3Juergen Schuderer; 1BioNIR 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. those
emitted by electrical power lines or mobile phones, has been done for decades but the reliable
answers are still missing. Extensive epidemiological studies are commonly expected to give the
answers. However, finding and scientific validation of any potential health hazard (whether it is
cancer or non-cancer effect) might not be possible using epidemiological approach alone because
of the "low sensitivity" of this method. Recently, it has been suggested that high-throughput
screening techniques (HTST) will be crucial in providing important information for thorough
determination 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 (global
gene expression) and proteomics (global protein expression) to enumerate the behavior of
biological system (Aebersold et al., Nature Biotechnology 18, 2000, 359). This approach seems
to be particularly suited for elucidation EMF health hazard issue because it might reveal a variety
of unpredictable biological effects. Also, it might provide valuable information about the potential
long 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 to
elucidate biological responses to EMF. We have used HTST to examine gene and protein changes
that occur in two human endothelial cell lines: fast proliferating EA.hy926 and its slow
proliferating variant EA.hy926v1. Cells were exposed for the 1-hour exposure to 900 or 1800
MHz GSM temperature
signal at an average specific absorption rate (SAR) of 37±0.1-0.3oC. We found that few tens of genes (out
of 2.0 - 2.4 W/kg and at of 3600 examined) alter their
expression 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 tens
of proteins (out of 1300 examined). Combination of the HTST data on gene and protein
expression and protein activity has revealed several signal transduction pathways that might be
affected by GSM signal exposure. E.g. activation of hsp27/p38MAPK stress response pathway
altered stability of stress fibers and caused endothelial cell shrinkage. This, if occurring in vivo in
brain 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 provide
direct evidence of health hazard or its absence, it will be essential in unraveling of all biological
effects induced by EMF exposure. Further elucidation of the physiological significance of these
biological effects for the health and well-being, in short- and long-term exposure conditions, will
allow determination whether any health hazard might be associated with the use of EMF-emitting
devices at the present safety levels.
Acknowledgements: Pia Kontturi and Hanna Tammio for help in performing biological
experiments. Funding support: the Finnish technology development Center ­ Tekes (LaVita project), the 5th Framework Programme of the European Union (REFLEX project), the Academy
of Finland (post-doc fellowship), the VerUm Foundation, Munich, Germany, and STUK, Finland.
54
116 Application proteomic approach toward mapping transregulatory elements of myosin heavy chain Qishan Lin, Proteomics Core Facility, CCFG, State University of New York at Albany, *Myomatrix Therapeutic, LLC. Rensselaer, NY12144 Rapid advances in genomic sequencing, bioinformatics, and analytical instrumentation have created the field of proteomics, which at present is based largely on two-dimensional electrophoresis separation of complex protein mixture and identification of individual protein using mass spectrometry. These analyses provide a wealth of data, which upon further evaluation lead to many questions regarding the structure and function of the proteins. The challenge of answering these questions creates a need for high-specificity approaches that may be used in the analysis of bio-molecular recognition events and interacting partners, and thereby places great demands on new methodology development. It has been shown that myosin heavy chain (MHC) plays an important role in the fetal gene program (One of the characteristic changes that occur in a failing heart is a change in gene expression wherein genes that were turned off shortly after birth are reactivated in the disease process. This reactivation of fetal genes is known as the induction of the fetal gene program). Thus, developing a highthroughput screening assay to understand the gene switch on/off machinery will aid us in our search for compounds that would reverse this change in gene expression and potentially reverse the progression of heart failure. In this study, nuclear fractionation, DNA promoter affinity chromatography, twodimensional electrophoresis, multiple dimension chromatography, and mass spectrometry were integrated toward mapping the trans-regulatory elements of myosin heavy chain in rat neonatal cardiomyocytes. To demonstrate proof of principle of the working platform, transcription factors including GATA 4, P300, and MEF2 were identified.
Poster Abstracts Wednesday, March 26: Identification and Quantification of Proteins/Management of Proteomics Data
117 Identification of androgen-regulated genes in Prostate Cancer cells using isotope-coded affinity tags and mass spectrometry
118 Functional characterization of transgenic Arabidopsis lines with ectopic expression of the
Katie L. Meehan and Marianne D. Sadar. Department of Cancer Endocrinology, BC Cancer Agency, Vancouver, BC, Canada, V5Z 4E6
transcription factor gene MYB13
Prostate cancer is the most common cancer diagnosed and the second leading cause of death from cancer for Canadian men. The molecular and biochemical mechanisms involved in prostate carcinogenesis are unknown. However, increasing reports suggest that androgens are involved in development and progression of prostate cancer. The objective of this study was to apply a new quantitative technology for the proteomic analysis of the mitogenic effect of androgens on prostate cancer cells. Nuclear, cytoplasmic and membrane protein fractions were isolated from androgen stimulated (10 nM of the synthetic androgen, R1881) and unstimulated LNCaP cells. Protein fractions were subjected to stringent denaturation, labelled with heavy and light ICAT reagents respectively then combined and proteolysed. Resulting peptides were first separated using a high-resolution cation exchange column and then further purified using an avidin affinity column. ICAT-labelled peptides were analysed by LC/MS/MS. Application of cell fractionation, ICAT labelling and LC/MS/MS enabled the concurrent quantification and identification of 2502 proteins in complex protein mixtures obtained from stimulated and un-stimulated LNCaP cells. Differential expression of proteins associated with stimulated LNCaP cells were revealed and consistent with previously identified proteins in prostate cancer cells. These proteins included members of the cyclin family, calreticulin, c-myc, Kruppellike factor, transforming growth factor-b, disintegrin-like metalloprotease, selenoprotein P and laminin. However, of considerable interest was the identification and quantitative differences in expression of a large number of proteins that have not been previously reported to be expressed in prostate cells. Some of these proteins included polyadenylate-binding protein 2, valosin, phosphoglycerate kinase 1, triosephosphate isomerase, tektin 3, sema domain, MUL-protein and PTPN13-like protein. In addition, many uncharacterised proteins were also detected and displayed aberrant expression in stimulated LNCaP cells compared to unstimulated. This comprehensive proteome analysis has generated a differential protein expression catalogue containing thousands of proteins differentially expressed in prostate cancer cells in response to androgens. Results from these studies will enhance our understanding of the molecular pathways involved in the proliferation of prostate cancer and give rise to potential therapeutic targets and novel biomarkers.
Schlesier, B., Bдumlein, 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 are involved in the control of a number of cellular functions such as developmental processes, hormone responses as well as phenylpropanoid metabolism. The completion of the Arabidopsis genome sequencing enables new approaches to define the functional significance of individual transcription factors in plants. The paper describes a proteome analysis of transgenic lines with ectopic expression of AtMYB13 in comparison to control plants. Plants were cultivated in a vessel under controlled environmental conditions to minimize biological variation. Protein extracts of roots and leaves were separated by 2-D gel electrophoresis. Image analysis revealed distinct changes in protein patterns for the transgenic lines. Proteins differentially expressed were identified using mass spectrometric techniques. Results from proteome analysis are compared with data obtained by transcript profiling. Funding by the Deutsche Forschungsgemeinschaft to H.-P. Mock is gratefully acknowledged.
119 Energy coupling between the PspF AAA+ protein and RNA-Polymerase
120 Quantitative analysis of protein complexes using solidphase isotope tags
J Schumacher; P Bordes; X Zhang and M Buck Department of Biological Sciences, Imperial College of Science, Technology and Medicine, London SW7 2AZ, UK Regulation of bacterial transcription by 54 dependent enhancer binding proteins critically involves their tri-nucleotide hydrolysis to restructure the 54 RNA polymerase and the promoter DNA to which it is bound, resulting in open promoter complex formation and transcription. We show by biochemical studies and cryo-electron microscopy that the activated enhancer binding protein PspF from Escherichia coli is organised in hexameric rings, demonstrating that PspF belongs to the AAA+ superfamily. AAA+ proteins are molecular motors that couple the energy derived from ATP hydrolysis to restructure their substrate. We have identified residues within PspF that are directly involved interacting and energy coupling between PspF and the closed promoter complex. These residues are mapped on a homology based PspF model to illustrate the energy coupling mechanism.
W. Andy Tao, Jeffrey Ranish, Ruedi Aebersold Institute for Systems Biology, Seattle, WA 98103 The identification and accurate quantification of proteins at high throughput are essential components of proteomic strategies for studying cellular functions and processes. Techniques largely based on stable isotope labeling and tandem mass spectrometry are increasingly being applied in quantitative proteomic studies. A solid-phase approach for the accurate quantification of individual proteins within complex mixtures is described. The method uses a class of new solid-phase chemical reagents to introduce a stable isotope tag on every peptide via its N-terminal for quantitative analysis by mass spectrometry. Using this approach, peptides derived from immunopurified STE12 protein complexes isolated from yeast cells in different states were analyzed for protein identification and changes in relative abundance. The method is redundant since multiple peptides from each protein are tagged, allowing better accuracy in quantification. In addition, the solid-phase based approach can be easily adapted for the automation and high throughput experiments, and for quantitative analysis of protein post-translational modifications. This work was supported by grants from NCI (R33, CA 93302), NHBLI Proteomics Initiative (N01-HV-28179), and from Oxford GlycoScience. W.A.T. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation (DRG 1740-02).
55
Poster Abstracts Wednesday, March 26: Identification and Quantification of Proteins/Management of Proteomics Data
121 Proteomic analysis of food-induced gastrointestinal hormones using SELDI proteinchip technology
122
Intact Mass Measurements Show That Current Bioinformatics
Programs Do Not Reliably Predict The Transit Peptide Cleavage Sites Of Integral
Membrane Proteins.
*Roel Vonk, *Han Roelofsen, *He Tao, *Renate Hagedoorn, Guido Rychen, Jean-Michel Antoine. *Dept.of Pediatrics, University of Groningen, Hanzeplein 1 9713 GZ Groningen,The Netherlands. Lab. Animal Science of INRA, Nancy, France. Danone Vitapole, Paris, France. The physiological functions of gastrointestinal hormones such as incretins (insulinotropic hormones e.g. GIP and GLP-1) are more and more recognized for their role in glucose intolerance and energy intake. In this respect interactions with other peptide hormones (resistin, leptin etc.), originating from different organs are not clear. A major drawback in this field is the complexity of the measurement of peptide hormones. To overcome this problem we applied SELDI proteinchip technology (Ciphergen) (see also www.medical proteomics.com). Methods: pigs provided with permanent canula's in the portal vein and artery were used as a model (Rerat et al. Br. J. Nutr. 1992; 68:111-138). The pigs received well characterized test meals. Blood samples of both vessels were taken before and after ingestion at time points up to 240 minutes. Sera were diluted 1:1 in 9 M urea/2% CHAPS containing protease inhibitor (Complete, Roche) and were diluted further in binding buffer. Weak cation exchange (WCX) and hydrophobic (H4) chips were used for analysis. Results: comparison of the obtained peptide profiles revealed at least 12 peptides in the range of 1 to 10 kDa that showed timedependent change in expression. A SWISSPROT database search with the found masses gave several hits for gastrointestinal hormones. Further identification of the peptides is necessary to validate this method. Conclusion: these data indicate that SELDI proteinchip technology is a promising tool for measurement and discovery of a whole range of food-induced peptide hormones.
Stephen M. Gуmeza, Karl Y. Bilb, John N. Nishioc, Kym F. Faulla and Julian P. Whiteleggea,1 a Pasarow Mass Spectrometry Laboratory - Departments of Psychiatry & Biobehavioral Sciences and Chemisty & Biochemisty, and the Neuropsychiatric Institute. University of California, 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. 310794-5156; fax 310-206-2161 Electrospray­ionization mass spectrometry coupled with reverse-phase liquid chromatography was used to identify 35 nuclear-encoded thylakoid-associated proteins from the chloroplasts of Arabidopsis thaliana. Identifications were made based upon intact mass measurements, cyanogen bromide mass tags and/or MS/MS sequencing. With the mature amino-terminus of each protein unequivocally assigned we constructed a data set to challenge several Web-based programs for predicting organelle targeting and/or the proteolytic processing site of the transit peptide. ChloroP and TargetP were found to be reliable at predicting chloroplast targeting, but were only able to reliably predict the transit peptide cleavage site for proteins targeted to the stroma. SignalP (eukaryote settings) was accurate at predicting the transit peptide cleavage site for proteins targeted to the lumen. SignalP (Gram-negative bacteria settings) was reliable at predicting peptide cleavage of thyakoid proteins inserted into the membrane via the "spontaneous" pathway, while the processing sites of thylakoid-integral proteins inserted by the SRP-dependent pathway were not well predicted by any of the programs tested. These results suggest that there is a second thylakoid processing protease that recognizes the transit peptide of proteins inserted via the "spontaneous" mechanism and that this mechanism may be related to the secretory mechanism of Gram-negative bacteria. None of the bioinformatics tools we tested were able to predict the transit peptide cleavage site from SRP-dependent thylakoid-integral proteins, however improvements in these tools could be made by more judicious selection of training data sets, including separate data sets for vascular plants and green algae and/or separate data sets for each sub-organellar compartment. Support from DOE (DE-FG03-01ER15251 to KFF/JPW) is gratefully acknowledged.
123
Quantitative Mass Spectrometry uncovers an androgen co-regulated
protein 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 has recently been accomplished by coupling microcapillary liquid chromatography (µLC) to tandem 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 has recently developed a new class of reagents termed isotope-coded affinity tags (ICAT) that selectively reacts and isotopically labels cysteine residues in proteins (Gygi, Rist et al. 1999). Combining the ICAT technology with µLC-MS/MS and database searching has allowed us to simultaneously sequence identify and accurately quantify individual proteins within complex mixtures (Gygi, Rist et al. 1999). In this report we demonstrate how the ICAT methodology can be used to quantify protein abundance changes in mammalian cells. We have successfully identified and quantified greater than 1,000 proteins from the microsomal fraction of LNCaP prostate cancer cells. Androgen increased the levels of a number of proteins involved in fatty acid biosynthesis, including fatty acid synthase (FAS), a known androgen-regulated target in prostate epithelia (Swinnen, Esquenet et al. 1997). Androgen also increased the levels of secreted and membrane-bound proteases, which included prostate-specific antigen (PSA), TMPRSS2, and kallikrein 2 (hK2). Several families of previously characterized membrane receptors and proteins involved in adhesion, secretion and vesicular trafficking were also androgen-regulated. We will integrate the results of this proteomic analysis with our cDNA microarray analyses and present a working model of androgen-regulated genes and 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 mass spectrometry." Nat Biotechnol 17(7): 676-82. Opiteck, G. J., K. C. Lewis, et al. (1997). "Comprehensive on-line LC/LC/MS of proteins." Anal Chem 69(8): 1518-24. Swinnen, J. V., M. Esquenet, et al. (1997). "Androgens stimulate fatty acid synthase in the 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-phase microextraction/multistep elution/capillary electrophoresis/tandem mass spectrometry." Anal Chem 71(13): 2270-8.
124
Proteomic analysis of skeletal muscle calcium homeostasis
Michael Zeece, Steve Jones, Ron Cerny, Joe Schultz, Nathan Palmer, & Boris Kornileav. University of Nebraska, Lincoln NE, USA, 68583-0919
Calcium homeostasis is a critical process in muscle because this cation represents the trigger for several physiological functions including metabolism, contraction and protein turnover. There is also evidence that calcium level is linked to apoptosis and programmed cell death. Abnormal regulation of calcium level is widely held as the principle cause of pathological conditions such as malignant hyperthermia (MH) and central core disease (CCD) in humans. Malignant hyperthermia is also manifested in some animals and often results in their sudden death.
Significant progress has been made in the past decade regarding our understanding of calcium regulation and in the identification of potential causes for its abnormalities. The discovery of a single mutation in ryanodine receptor (RyR1) gene stirred hopes that definitive testing would enable early detection of these potentially fatal diseases. However, a more complicated picture has emerged from recent research. For example, numerous (>20) mutations have been identified in RyR1 gene that correlate with MH in humans. It has also been shown that calcium homeostasis results from the coordinated interaction of several sarcoplasmic reticulum (SR)-associated proteins including: RyR1, dihydropyridine receptor, calsequestrin, tradin, junctin and calmodulin. These proteins form a functional complex that is responsible for calcium release. Several key proteins undergo reversible phosphorylation that modulates calcium flux. Thus there may be multiple mutations and/or modifications that collectively contribute to elevated calcium level and its adverse consequences.
Our group has taken a proteomic approach to investigate alterations in this complex protein system that are linked to abnormal calcium level. Twodimensional electrophoresis in combination with mass spectrometry methods are being used to separate, identify and characterize a targeted proteome of constituent SR proteins. This group of membrane proteins represents significant challenges to widely used proteomic separation methods. Thus we have developed appropriate 2-D techniques to separate these hydrophobic proteins. We are also developing non-gel based approaches to investigate potential polymorphisms in the 565 kDa RyR1 protein obtained from porcine skeletal muscle. The results of this work including separations of SR-associated proteins and their identification will be presented.
56
Poster Abstracts Wednesday, March 26: Identification and Quantification of Proteins/Management of Proteomics Data
125 Quantitative analysis of microsomal proteins from IL-12
126 Unbiased quantitative proteomics of lipid rafts
induced lymphoblasts
reveals high specificity for signaling factors
Jan-Jonas Filйn1,2, Tuula Nyman1, Juha Korhonen1 and Riitta Lahesmaa1 1 Turku Centre for Biotechnology, University of Turku and Еbo Akademi University, P.O.Box 123, 20521 Turku, Finland, 2 National Graduate School in Informational and Structural Biology T helper cells (Th) are divided into Th1 and Th2 subsets based upon their cytokine profiles and function. Naпve Th cells differentiate into Th1 and Th2 subsets depending on the antigens, costimulatory molecules, and cytokines they meet. Cytokines IL-12 and IL-4 play a major role in the regulation of the development and differentiation of the Th cell subsets. IL-12 enhances the generation of Th1 lymphocytes and inhibits the production of Th2 subset. Vice versa IL-4 enhances the generation of Th2 subset and inhibits the production of Th1 lymphocytes. The selective activation of Th1 and Th2 lymphocytes plays an important role in the pathogenesis of allergy, asthma and autoimmune diseases. In this study peripheral blood mononuclear cells obtained from healthy blood donors (Finnish Red Cross Blood Transfusion Service) were activated with phytohemagglutinin (PHA). After 48 hours IL-2 was added to cultures and Th1 cell differentiation was induced by addition of IL-12. After 48 hours cells were lysed and microsomal fraction was prepared. The microsomal fraction of IL-12 induced Th cells was studied using isotope-coded affinity tag (ICAT) reagent labelling and 2-dimensional chromatography followed by nanoLC-ESI-MS/MS analysis. The data was analysed with ProICAT software. As a preliminary result several proteins from microsomal fraction were identified and quantified.
Leonard J. Foster, Carmen L. de Hoog, Matthias Mann* Center for Experimental Bioinformatics (CEBI), Institute for Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark Membrane lipids were once thought to be homogenously distributed in the two-dimensional surface of a membrane but the lipid raft theory suggests that cholesterol and sphingolipids partition away from other membrane lipids. Lipid raft theory further implicates these cholesterolrich domains in many processes such as signaling and vesicle traffic. However, direct characterization of rafts has been difficult due to their small size and largely biochemical definition. In the first large-scale proteomic analysis of rafts we use quantitative high-resolution mass spectrometry to specifically detect proteins depleted from rafts by cholesterol-disrupting drugs, resulting in a set of 241 authentic lipid raft components from over 1000 identified proteins. We detect a large proportion of signaling molecules, highly enriched versus total membranes and detergent-resistant fractions, which thus far biochemically defined rafts. Our results provide the first large-scale and unbiased evidence for the connection of rafts with signaling and place limits on the fraction of plasma membrane composed by rafts.
127 Proteome analysis of lipid rafts subtracted from activated primary CD4+ T lymphocytes
128 Plasma Membrane (PM) Expression Profiles in Resting and Activated Murine Splenocytes
Juha T. Korhonen, Tuula A. Nyman, Jan-Jonas R. Filen, Riitta Lahesmaa Turku Centre for Biotechnology, University of Turku and Abo Akademi University, Turku, Finland, P.O. POX 123. The selective activation of functionally distinct human CD4+ T helper lymphocytes, Th1 and Th2 cells, plays an important role in the pathogenesis of asthma and many inflammatory diseases. T cell receptor mediated (TCR) signaling differs in these T helper cell subsets, but the molecular basis for this is currently unclear. However, it was found recently, that the composition of membrane microdomains, also referred to as lipid rafts, is distinct in Th1 and Th2 cells, leading to functional differences in TCR signaling in these T cell subsets. We have analyzed the proteome of lipid rafts subtracted from activated human primary CD4+ T lymphocytes in order to identify known and novel signaling molecules relevant for TCR signaling in these cells. Membranes resistant to 1% Brij 58 detergent were isolated using sucrose density gradient ultracentrifugation. Proteins co-purified with these lipid rafts were digested with trypsin, and tryptic peptide mixture was fractioned by cation exchange chromatography. Peptides were further separated and analyzed using nanoLCESI-MS/MS. Proteins were identified by comparison of tandem mass spectra to protein and DNA sequence databases using Mascot and ProID softwares. As a preliminary result many of the proteins known to be associated with lipid rafts in different cell lines were also identified from these primary CD4+ T lymphocytes. We were also able to identify some novel proteins co-purifying with lipid rafts and validation of these results is going on. In conclusion, the methodology described here is powerful in identifying novel proteins potentially important in cell signaling.
Matthew Peirce, Robin Wait, Shajna Begum, Andrew Cope and Jeremy Saklatvala, Kennedy Institute of Rheumatology Division, Imperial College London, 1, Aspenlea Road, Hammersmith, London W6 8LH, United Kingdom. PM proteins of mammalian cells are frequently refractory to analysis by two-dimensional electrophoresis. Using surface labelling with biotin followed by affinity capture, PM proteins were isolated (purity >50%) and separated by solution phase isoelectric focussing and SDSPAGE then identified by HPLC MS/MS. Using these methods we identified 75 PM proteins from a murine T cell hybridoma and murine splenocytes. These included one hypothetical protein and three proteins not previously reported in these cells. The expression of 12 PM proteins appeared sensitive to activation with phorbol ester and ionomycin. Activation-induced differences in the expression of MHCII, Glucocorticoid Induced TNF Receptor-related gene (GITR), CD69, IgM and IgD were independently verified by Western blot or FACS analyses. This approach may be applicable to the PMs of diverse mammalian cell types. This work was funded by the Arthritis Research Campaign, UK, the MRC and the Wellcome Trust.
57
Poster Abstracts Wednesday, March 26: Identification and Quantification of Proteins/Management of Proteomics Data
129 High-resolution and High-throughput Global Proteome Profiling by Three-dimensional LC-MS/MS
130 Low molecular weight proteomics in Arabidopsis thaliana
Analysis Jing Wei, Wen Yu, Jun Sun, Arianna Jones, Martin Keller, Jay Short Diversa Corp., San Diego, CA 92121
Jon Griffin, Prof. Johnathan Napier and Prof. Peter Shewry Crop Performance and Improvement, Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, UK, BS41 9AF
The complexity and dynamic range of the proteome is resolved by a novel approach involving on-line three-dimensional chromatography separation, mass spectrometry separation and detection, as well as novel bioinformatics software development. Comprehensive view of the yeast proteome, including semi-quantitative results, was obtained. Yeast cells at log-phase were homogenized and fractioned as soluble proteins, urea solubilized proteins, and SDS solubilized proteins. More than 2500 unique proteins were identified from these fractions with an average coverage of 11 peptides per protein by the 3D LC-MS/MS. Proteins of all classes were presented, including many lower abundance proteins. Majority of the primary metabolism pathways were fully identified. Posttranslational modifications including N-acetrylation and methylation were systematically surveyed. The protein
The number of predicted genes in the genome of Arabidopsis thaliana is 25,498 and analysis of these sequences revealed that 18% were low molecular weight (<20 kDa) proteins. Most current proteomic studies have been biased to proteins greater than 15 kDa. Using an adapted proteomic approach we aim to identify low molecular mass proteins and peptides that have either not been previously characterised or studied, or have not been identified by the use of bioinformatic algorithms when the Arabidopsis genome was analysed. Two different separation approaches have been undertaken. The first based on 2-Dimensional Electrophoresis using high percentage Tris-Tricine gels and the second using 2-Dimensional Chromatography with size exclusion and reverse phase chromatography. Identification of proteins was using MALDI-TOF MS and ESI-MS/MS following trypsin digestion. It is anticipated that some of the components identified will have biological activities (e.g. anti-microbial) or physical properties (e.g. surface activity), which will allow their exploitation. This poster details our findings so far.
abundance estimated by the "coverage index" correlated well with the published copy numbers of the corresponding genes. The higher sequence coverage and better separation will
PhD studentship supported by the BBSRC [email protected]
provide a powerful tool for detailed mapping of the proteome.
58
Thursday, March 27: In vivo Approaches/Analysis of Protein Activities Poster Abstracts
201
Global analysis of changes in protein-phosphorylation
202 Protein Function Prediction Using Protein Interactions
upon TNF- signaling
Stephan Bek, Anja Pfenninger, Thomas Wendrich, Jochen Kruip. Aventis Pharma Germany, Industriepark Hцchst, 65926 Frankfurt am Main.
Minghua Deng, Ting Chen and Fengzhu Sun, Molecular and Computational Biology Program, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089-1113
Protein phosphorylation represents a major control step of protein function, which cannot be evaluated with conventional genomic tools. It plays central roles in signal transduction, metabolism, apoptosis and cell development. Among the most important signal transduction pathways, tightly regulated by phosphorylation, are the NFB pathways. They mediate inflammatory reactions in response to pathogenic mechanism that are involved in various diseases like osteoarthritis, rheumatoid arthritis, arteriosclerosis, ischemia, diabetes 2, and others. Kinases, phosphatases, and substrates involved in these pathways are therefore favoured targets for therapeutical strategies. Consequently tools to carefully analyse global changes in protein phosphorylation are essential for pharmaceutical drug research. Currently no general strategy is able to address these dynamic phoshorylation changes In order to obtain a quite sensitive and comprehensive overview about the phospho-proteome of a cell that has received inflammatory signals, we have established the 2D-separation of metabolically labelled celllysates. The acquired phosphorylation maps have been linked to overall protein pattern, and differentially phosphorylated proteins were identified by MALDI peptide mass fingerprint. In doing so, we were able to identify a significant number of proteins, that were differentially phosphorylated upon TNF- signalling. Whereas some of them are already known players in inflammatory pathways, others are not known up to know to be regulated by phosphorylation. In summary, this method allows the analysis of 32P-labelled proteins in definied biological/experimental backgrounds.
Assigning functions to novel proteins is one of the most important problems in the post-genomic era. We develop a novel approach that applies the theory of Markov random fields (MRF) to infer a protein's functions using protein-protein interaction data and the functional annotations of its interaction protein partners. For each function of interest and a protein, we predict the probability that the protein has that function using Bayesian approaches. Unlike in other available approaches for protein functions where a protein has or does not have a function of interest, we give a probability for having the function. We apply our method to predict cellular functions for yeast proteins defined in the Yeast Proteome Database (YPD), using the protein-protein interaction data from the Munich Information Center for Protein Sequences (MIPS, http://mips.gsf.de). We show that the MRF based approach outperforms other available methods for function prediction based on protein interaction data. This research is partially supported by the NSF ITR EIA-0112934 and NIH NIGMS 1-R01-RR16522-01.
203
Analysis of Mycobacterium tuberculosis cell wall glycoproteins
using a Novel Two-Dimesional Gel Electrophoresis System.
Karen M. Dobos1, John T. Belisle1, Regina Rooney2, Bradley Scott2, and Rene Nunez2 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 central to the development of diagnostic antigens, drug targets, new vaccines, and definition of pathogenic mechanisms; and thus crucial to the control and eradication of tuberculosis. Moreover, the proteome holds secrets that are not readily obtained or proven by direct analysis of the genome, including posttranslational modifications. Previously, we have described the glycosylation of secreted M. tuberculosis proteins through rigorous biochemical and molecular technologies. In contrast, the description of similar proteins that define the cellular envelope of M. tuberculosis is lacking. The study of these proteins is hindered by their poor solubility and recovery from mycobacteria lysates (restricting their availability for study) and the lack of technologies to study these macromolecules. We have employed traditional biochemical methods to purified cell wall preparations of M. tuberculosis to extract and isolate these proteins. This classical bio-separation methodology has been partnered with a novel vertical 2D-GE system (IPGZoom Runner) to afford high throughput analysis of this complex mixture of proteins. Thus far, we have been able to resolve over seventy unique protein spots from the mycobacterial cell wall, including four products of 19, 25, 26, and 38 kDa previously defined as mycobacterial lipoproteins; three of which may be glycosylated, and thus are likely to be prominent structural features of the tubercle bacillus. Concanavalin-A probing of these 2D-GE resolved products revealed the presence of four additional putative glycoproteins present in this fraction, 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 are underway. This work was funded in part by NIH,NIAID Contract NO1 A1-75320 and Grant RO1 A1-44042.
204 A Mass Spectrometric Approach to Identify Novel Active Protein Kinases Pasan Fernando*, Wen Ding+, John F. Kelly+ and Lynn A. Megeney*. *Ottawa Health Research Institute and the +National Research Council, Ottawa, Ontario, Canada. Protein phosphorylation is a primary mechanism for the regulation of cellular processes. As such, the characterization of protein kinases remains central to the understanding of phospho-signaling related events. However, the ability to screen active protein kinases on a large scale remains a daunting challenge. Here, we describe an adaptation of the previously established in-gel kinase method for screening the activities of protein kinases from murine tissue and cultured cells. Protein lysates from mice harbouring the activated MAP kinase kinase-6 (MKK6) gene were analyzed by IEF and 2D PAGE in gels crosslinked with myelin basic protein (MBP). Following electrophoresis, gels were denatured in a solution of guanidine-hydrochloride and then renatured in a solution of 2-mercaptoethanol. Incubation of the gels in a kinase buffer containing [-32P]ATP followed by autoradiography allowed the visualization of specific protein spots, ie. phosphorylated MBP by a putative protein kinase that co-migrated to the same location during IEF/PAGE. NanoflowLC/electrospray ionization mass spectrometry allowed for sensitive detection of numerous protein kinases. Importantly, the kinase profile generated with this approach allowed us to map a network of interactions within the cellular context of active MKK6. With additional refinements and alterations in sample preparation, we are able to demonstrate the utility of this method as a platform for the comparison of proteomes based on differences in kinase activities.
59
Poster Abstracts Thursday, March 27: In vivo Approaches/Analysis of Protein Activities
205 Characterization of the Phospho-Proteome of Human breast cancer Cells
206
Identification of in vivo phosphorylation changes in topoisomerase II
and in HL-60 cells that are differentially sensitive to topoisomerase II-poisons
Brie K. Fuqua and Dr. Edward M. Marcotte Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, Texas USA 78712
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 Oxford Cancer Centre, Oxford OX39DS, U.K.
Phytoestrogens are natural plant compounds that have a structure and function similar to that of the mammalian female sex hormone 17-estradiol. Studies have correlated a reduced risk of breast cancer development with diets rich in phytoestrogens, such as soy-based diets. However, the mechanism of phytoestrogen action on cells is not fully understood. Various cellular signaling cascades are triggered by exposure to estrogen and phytoestrogens. Classically, studies have concentrated on characterizing long-term transcriptional effects. However, rapid changes also occur via phosphorylation of serine, threonine, and tyrosine residues on key signaling molecules. We are using proteomic methods to identify proteins that have been modified by phosphorylation in response to phytoestrogen exposure. The total fraction of phosphorylated proteins, or phosphoproteome, is isolated from cancerous breast epithelial cell lysates with affinity chromatography following treatment with phytoestrogens. After further separation with HPLC, phosphorylated proteins are analyzed by ESI ion trap mass spectrometry. Global identification of proteins whose phosphorylation status is altered in response to estrogenic exposure will provide insight into the signaling processes shaping cancer cell growth and metastasis. Acknowledgements: Project supported by the University Co-Operative Society 2002-2003 Undergraduate Research Fellowship
Topoisomerase (topo) alters DNA topology for the processing of genetic material. Type II topo consists of two isoforms, topo II and topo II. Phosphorylation of topo II regulates its activity and sensitivity of cells to drugs that poison the enzyme. Resistance to topo IIpoisons is associated with hypophosphorylation of topo II and hyperphosphorylation of topo II. Decreasing intracellular Ca2+-transients can mimic this resistant phenotype. To locate specific phosphorylation site(s) on topo II and topo II that are altered in HL-60 cells resistant to topo-poisons we compared CNBr and tryptic phosphopeptide maps of these isozymes in cells: a) sensitive to topo II-poisons (HL-60/S), b) resistant to topo IIpoisons (HL-60/R), and c) treated with the intracellular calcium chelator, BAPTA-AM (HL60/S/B). CNBr or tryptic phosphopeptides generated from topo II or topo II immunoprecipitated from extracts of cells labeled with [32P]Pi were analyzed by 1D-gel electrophoresis or 2D-phosphopeptide mapping, respectively. CNBr phosphopeptide maps of topo II revealed hypophosphorylation of a 12 kDa peptide in HL-60/R and HL60/S/B cells, as compared to HL-60/S cells. This peptide was identified by N-terminal Edman sequencing to correspond to the 10.4 kDa CNBr peptide 34 (aa1041-1131), localized to the catalytic domain of topo II. Tryptic digestion of peptide 34 generated two phosphopeptides which migrated similarly to the two hypophosphorylated tryptic peptides observed in HL-60/R and HL-60/S/B cells. Comparison of CNBr phosphopeptide maps of topo II in HL-60/S and HL-60/R cells revealed hyperphosphorylation of a 65 kDa peptide in HL-60/R compared to HL-60/S cells. This peptide was one of two major phosphopeptides of MW 65 and 53 kDa detected in HL60 cells. Edman sequencing revealed that both peptides have identical N terminal sequences corresponding to the penultimate CNBr peptide starting at aa1248 . Since the last peptide consists of only two amino acids the difference in the size (12 kDa) is likely due to differential post translational modification(s). Tryptic phosphopeptide maps also identified 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 of topo II and in the C-terminus of topo II that are differentially phosphorylated in sensitive cells 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 by alterations in site specific phosphorylation of topo II and topo II, albeit by reciprocal mechanisms. (USPHS CA-749349 and DK56917)
207 Development of a system for the study of protein-protein interactions in planta, and application of the method to characterization of a TATA-box binding protein complex in Oryza sativa Paul A. Haynes, Jingping Zhong, Shiping Zhang, Xinping Yang, Nancy Andon, 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 vivo protein complex purification from plants using a tomato Biotin peptide as an affinity tag. The Biotin tag was fused in-frame to the carboxyl terminus of TATA-box binding protein and the fusion cassette was transformed into rice callus. The expressed chimeric protein was purified from rice suspension cell cultures, along with the associated protein complex components, using immobilized streptavidin. The proteins present in the purified complex were visualized by gel electrophoresis and 86 unique proteins were identified by tandem mass spectrometry. The bait protein and a number of proteins known to be associated with TBP proteins were identified, as well as many other proteins involved in pre-mRNA processing, chromatin remodeling, and translation. The identification of these novel protein-protein associations provides important new insights into the mechanisms of mRNA transcription and pre-mRNA processing, and demonstrates that this system can potentially be used for the identification of all proteins interacting with a given target protein under physiological conditions in rice. This system is also applicable to other tissues and organisms, and could be applied in a high-throughput fashion to enable systematic analysis of protein-protein interactions on a proteome-wide scale.
208 Identification of protein tyrosine kinases and tyrosine phosphorylated proteins overexpressed in transitional cell carcinoma 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 most prevalent disease leading to death in the developed countries. Since the up-regulated tyrosine kinase activity found in cancer cells is usually resulted from the altered structure of tyrosine kinase due to gene mutation and deletion, therefore techniques based on the detection of mRNA (such as DNA microarray, RT-PCR etc) can not detect the upregulated PTK activity under this situation, though PTK genes amplification and the increase of mRNA also occur in certain tumor cells. Therefore we use proteomic approach to study the altered kinase activity and the up-regulated tyrosine phosphorylated proteins in TCC. Several tyrosine phosphrylated proteins were identified via this approach, and their roles in TCC is under intensive investigation at this moment.
60
Thursday, March 27: In vivo Approaches/Analysis of Protein Activities Poster Abstracts
209 Proteomic analysis for oxidative stress-induced protein
210
Proteome and phosphoproteome Analysis in Bovine
modifications
Adrenal Medulla using MALDI-MS and LC/MS/MS
Narae Hwang, Ja Won Seo, Seung-Hee Yim, Eun Joo Song, Min Chung Kim, Ji Young Chung, and Kong-Joo Lee From the Center for Cell Signaling Research, Division of Molecular Life 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 various proteins. 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. To examine the physiological role of GADPH in oxidative stress, we have identified the protein modifications and interacting proteins using 2Dgel electrophoresis and MALDI-TOF MS and ESI-Q-TOF MS. Protein modifications including cystein oxidation, phosphorylation and lysineacetylation were detected and binding proteins having various cellular functions such as RNA binding, chaperone, and redox signal related proteins were identified. Redox dependent protein modifications regulate the protein functions. These results suggest that the various modifications and the change of binding partners in oxidative stress propagate differential signaling pathways. [Supported by MOST 21C Frontier Functional Human Genome Project (FG-4-14) by KISTEP, by KOSEF through the Center for Cell Signaling Research (CCSR) at Ewha Womans University and by IMT2000 project for IT-BT. Students (N Hwang, JW Seo, MC Kim, JY Chung and EJ Song) and Research Professor (SH Yim) were financially supported by Brain Korea 21 program]
Pegah R. Jalili and Chhabil Dass, Department of Chemistry, The University of Memphis, TN 38152, USA In this study, LC/MS-based proteomic approach is used to identify proteins and phosphoproteins in bovine adrenal medulla. This gland is a major source of hormones, opioids, neurotransmitters, and several important proteins. These biomolecules play a variety of roles in the functioning of hypothalamic-pituitary-adrenal axis (HPA), including pain suppression, influence on the immune system, maintenance of the vascular response, protection of the body against stress and in patho-physiological processes that lead to Alzheimer and Parkinson diseases. Phosphoproteins are involved in the control of cell growth, metabolism, differentiation, and in the function of many proteins, hormones, neurotransmitters and enzymes. After removing the cortex, the medulla was homogenized and centrifuged. The supernatant was subjected to 75% ammonium sulfate precipitation and the pellet obtained by centrifugation was dissolved, dialyzed overnight at 4°C to remove salts and small peptides, and centrifuged again. The supernatant was lyophilized. The proteins thus obtained were fractionated by RP-HPLC. Two MS approaches were used for profiling proteins. In the first approach, each HPLC fraction was subjected to trypsin digestion. The MALDI-TOFMS analysis of this digest provided a peptide mass map that was used for searching Swiss-Prot database. In the second approach, all RP-HPLC fractions were combined, subjected to trypsin digestion and analyzed by capillary LC/nanospray/MS/MS. The expressed sequence tags thus obtained were used 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 with MALDI-TOFMS. A total of 64 proteins were identified, the prominent ones are Secretogranin I precursor, proenkephalin A precursor, 1phosphatidylinisitol-4-5-biphosphateinisitol 1,4,5-triphosphate receptor type 3, chromatogranin A (CMGA-BOVIN), hypothetical protein KIAA0373, and beta-adrenergic receptor kinase 2.
211
Large-scale functional approach of proteins in human diseases
Christine Chavany and Moncef Jendoubi* *Founder, President & CEO Milagen, Inc. 770 13th Street, Richmond, CA 94804
As proteins are the end product of gene expression and the ultimate effectors of cellular processes, unraveling protein function is essential to understanding phenotype and finding new ways to diagnose and treat human diseases. We have developed a large-scale antibody-based approach designed to identify essential proteins associated with disease. Our approach, globally referred to as AntibiomixTM is based on two major in house developments: i) a large collection of high affinity polyclonal antibodies against known and unknown human gene products; ii) a protein array technology, named the matrix protein array technologyTM, coupled to a readout and data analysis system. The combination of the above platform technologies enables the analysis in multiplex format of a large number of clinical specimens with a large number of antibodies generating differential 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 biological fluid specimens, in the context of diagnostic and therapeutic applications, and ii) protein expression profiling of human cell lines cultured in the presence or absence of anti-cancer drugs, in the context of pharmacoproteomics. In both cases, the selection and identification of panels of antibodies that specifically recognize differentially expressed proteins further lead to the characterization and validation of the relevant proteins. Our approach allows for the identification of protein signatures in any given biological state, including disease, correlating gene products to phenotype. The identification of disease specific protein panels will help to develop more effective treatment strategies for individual patients and to develop improved diagnostics and therapeutics.
212 New Insight of VEGF-induced Signaling Pathway in HUVEC by Proteomics and Bioinformatics Young Mee Kim, Hee-Jung Kim, Eun Joo Song and Kong-Joo Lee From the Center for Cell Signaling Research, Division of Molecular Life Sciences and College of Pharmacy, Ewha Womans University, Seoul, 120-750, Korea Vascular endothelial growth factor (VEGF) is an important angiogenic factor. It mediates signaling by activating two receptors tyrosine kinase, Flt-1 (VEGFR-1) and KDR (VEGFR-2). It is known that endothelial cells treated with VEGF increase the reactive oxygen species (ROS) production and activate MAPK pathway. To identify the target proteins of VEGFR, we have investigated tyrosine-phosphorylated proteins in human umbilical vein endothelial cell (HUVEC). We treated HUVEC with VEGF or hydrogen peroxide. The phosphorylated proteins were separated by 2D-gel electrophoresis, detected with western analysis using phospho-tyrosine antibody and identified with peptide fingerprinting using MALDI-TOF MS. We have detected 95 proteins that were differentially phosphorylated in HUVEC in response to VEGF and hydrogen peroxide. Also we can identify the proteins specifically phosphorylated by VEGF, not by hydrogen peroxide. The obtained results were analyzed by bioinformatic tools. The combined results provide the insight for new VEGF and H2O2-induced signaling pathways, and give us the direction of functional studies of target proteins 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 by Brain Korea 21 program]
61
Poster Abstracts 213
Thursday, March 27: In vivo Approaches/Analysis of Protein Activities
214
Preparation of Amniotic Fluid for Proteomic Analysis
Chatarina A Lцfqvist1,3, Gunnel Hellgren1, Ann Hellstrцm1,2 and Lois EH Smith3. 1Department of Pediatrics, 2Department of Ophthalmology, The Sahlgrenska Academy, at Gцteborg University, Gцteborg, SE-416 85, Sweden, 3Department of Ophthalmology, Children's Hospital, Harvard Medical School, Boston, US.
Withdrawn 215 Interactions of Cricket Paralysis Virus IGR-IRES with the Human 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, Empire State 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-IRES utilizes a method of protein translation initiation that is distinct from that previously observed in eukaryotes. We used cryo-electron microscopy (cryoEM) and single-particle reconstruction techniques to investigate the interactions between CrPV IGR-IRES and the human ribosome. HeLa 40S and 80S ribosomal subunit samples with and without CrPV IGR-IRES were applied to glow-discharged copper EM grids, quick-frozen in liquid ethane, and examined on a FEI transmission electron microscope at 300 kV. Images were 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 for 40S-IGR) were subjected to reference-based reconstruction in SPIDER. Resolutions of the resulting reconstructions are 21Е, 23.1Е, and 20.2 Е, respectively. Analysis of these density maps suggests that CrPV IGR-IRES binds the 80S ribosome in the inter-subunit space. IGR-IRES makes contacts with the 60S subunit that appear to mimic those made by P-site tRNA. CrPV IGR-IRES binding causes conformational changes in the ribosome that are related to the mechanism by which it initiates protein synthesis. Finally, the control reconstruction represents the first structure of a human ribosome. Comparison with the cryo-EM map of the yeast ribosome reveals added densities on the 60S subunit of the human ribosome. Research was supported by the Howard Hughes Medical Institute and NSF grant DBI-9987844.
We have hypothesized that babies lose important sources of growth factors due to preterm birth. This loss is made up partly from growth factors and cytokines from amniotic fluid (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. As AF levels for cytokines has been found to vary depending on storage conditions we conducted a pilot study with the objective to evaluate stability of insulin-like growth factors in AF. AF was collected in immediate connection to delivery. After centrifugation aliquots was prepared with or without proteolytic enzyme inhibitor (PI). As PI either aprotinin or EDTA was used and the aliquots was then stored at either ­20o C or ­80o degrees C. Analysis of insulin-like growth factor binding protein-1 (IGFBP-1) by Western blots using native gels, 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 difference between the samples. So far a problem area has been elucidated 1) IGFBP-1 is the major growth factor in fetal and maternal circulation and we found very high levels of IGFBP-1 (>50.000 ng/ml) in our samples. There could be a significant risk of influencing the protein detection in twodimensional (2-D) gels with this level of binding protein present. Therefore IGFBP-1 might need to be removed. However removal of IGFBP-1 could also be associated with concomitant removal of IGFs. The studies will consist of 2-D gel analysis of proteins in AF at each phase of fetal development. Selected protein spots will then be analyzed by mass spectrometry. Candidate molecules will be compared longitudinally with respect to gestational development age. Furthermore data will be compared with respect to infant weight and prematurity. We anticipate that the proposed studies of amniotic fluid will reveal the changes that occur with development of the fetus. We know that IGF-I rises in the second/third trimester and infants born prematurely miss this source of IGF-I. With the results from this study we hope to establish the importance of growth factors in AF for the development of blood vessel and neural tissue, and possible the protective impact of these factors. 216 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 target genes in different cell types in norm and pathology. This specificity depends on the activity of signaling systems and composition of transcriptional complexes. Helix-loop-helix (HLH) TFs have several functions during development of the nervous system that requires regulation of different sets of target genes. We characterized the network of HLH TFs in developing neurons using: 1. Chromatin immunoprecipitation followed by the cloning and sequencing of genomic binding regions (target genes) of HLH TFs, 2. Antibody microarrays of crosslinked chromatin, 3. Mass spectrometry analyses of immunoprecipitated crosslinked chromatin complexes, and 4. FRET analyses in situ using labeled antibodies to HLH TFs and components of transcriptional complexes. Chemical cross-linking allows us to identify chromatin complexes of HLH TFs under physiological conditions. Results of these analyses show that individual HLH TFs regulate different sets of target genes and form different chromatin complexes during neurogenesis
62
Thursday, March 27: In vivo Approaches/Analysis of Protein Activities Poster Abstracts
217 Switching Protein Interaction Networks by Alternative
218 E. coli primase interaction with template DNA.
Splicing of Protein Interaction Domains
Anna Rodina, Nigel Godson, Department of Biochemistry, New York
Alissa Resch, Yi Xing, Barmak Modrek, Michael Gorlick, Robert Riley, Christopher Lee
University, New York, NY, USA, 10016.
Alternative splicing has emerged as a major mechanism for expanding and regulating the repertoire of gene function. Alternative splice data have the potential to contribute substantially to our understanding of proteomic diversity and function. However, while most alternative splicing is studied at the nucleic acid level, many functional questions can only be answered by analyzing the protein products. To make this connection, we have constructed a database of alternatively-spliced protein forms (ASP database), consisting of 11,907 protein isoform sequences of 4,204 human genes). Our bioinformatics analysis indicates that the major impact of alternative splicing is removal of protein-protein interaction domains that mediate key linkages in protein interaction networks. 56% of alternative splicing events removed protein-protein interaction domains as opposed to DNA binding domains, RNA binding domains, or other domain types. We present a number of novel
All cellular organisms use specialized RNA polymerases called "primases" to synthesize RNA primers for the initiation of DNA Replication. Primase is a part of huge multi-protein complex called "replisome". Primase activities are coupled to it by protein-protein interactions with other replication factors. We have used DNA-protein crosslinking to define the path of template DNA through E. coli primase during the reaction of pRNA synthesis. We have found three sites on the primase surface, which come in close proximity to DNA. These are Zn-finger (92-120aa), active center region (120-187aa) and another region, which has yet to be characterized. The data provide insight into the structure and functional properties of the enzyme. Supported by NIH grant GM38292.
examples (Kruppel transcription factors; Pbx2; Enc1) from the ASP database, illustrating how this pattern of alternative splicing changes the structure of a biological pathway, by redirecting protein
interaction networks at key switch points.
219 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 Life Sciences and College of Pharmacy, Ewha Womans University, Seoul 120-750, Korea Fas-associated factor 1 (FAF1) was first identified as another Fas associating molecule, which lacks a characteristic death domain but can enhance Fas-mediated apoptosis. The biological role of FAF1 is not clearly defined. Recent our work indicates that FAF1 may be involved in complicate procedures related to the apoptosis and ubiquitin pathway. Also FAF1 has several domains related to ubiquitin pathway, UBX, UBL, NLS etc. To examine the function of FAF1, we have identified FAF1 interacting proteins, Valosin containing protein (VCP) and hsp 70. VCP is necessary to degrade polyubiquitinated proteins in the proteasome. In this study, we found that FAF1 regulates VCP activity by various modifications. Protein modifications of FAF1 including phosphorylation, methylation were detected and analyzed with MALDI-TOF MS and tandem ESI-Q-TOF MS. These results suggest that FAF1 has multi-functions and each functions can be regulated by changing the interacting proteins and posttranslational modifications. [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 21 program]
220
TOWARDS AN ANALYSIS OF MEMBRANE PROTEIN INTERACTIONS:
THE MEMBRANE-BASED YEAST TWO-HYBRID TECHNOLOGY
Safia Thaminy1, Daniel Auerbach2, Anthony Arnoldo1, and Igor Stagljar 1 1 Institute of Vet. Biochemistry and Molecular Biology, University of Zurich, Switzerland 2 Dualsystems Biotech Inc, Zurich, Switzerland Email: [email protected]
Analysis of membrane protein interactions is difficult due to the hydrophobic nature of such proteins, which makes them ill-suited to use in the traditional two-hybrid assay. This is a substantial problem since approximately one-third of all proteins in a typical eukaryotic cell are thought 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 and therapeutic importance. Thus, understanding the physiology of membrane proteins and the way these proteins communicate in the cell is of high biological importance (2). We have recently developed a new genetic method for the in vivo detection of membrane protein interactions in Saccharomyces cerevisiae (3). The system uses the splitubiquitin approach first described by Johnsson and Varshavsky (4) based on the observation that covalent addition of ubiquitin polypeptides can recruit particular proteases for the specific degradation of target proteins. In our system, interaction between two membrane proteins results in ubiquitin reconstitution and leads to the proteolytic cleavage and subsequent release of a transcription 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 twohybrid technology to identify proteins that interact with a given yeast or human membrane target protein. For that purpose, yeast and human cDNA libraries were constructed and fused either Nor C- terminally to the Ubiquitin (Nub) domain. We now show that various screenings of these libraries identify the membrane and cytosolic proteins that specifically interact with their corresponding bait protein (Thaminy, S., Auerbach, D., Arnoldo, A., and Stagljar, I., submitted to Nat Biotechn). In addition, we are combining a membrane-based yeast-two hybrid approach to the reverse two-hybrid strategy to screen a library of peptides or small molecules in which preventing the interaction between two membrane protein partners provides a selective advantage for yeast. Such an approach should enable identification of dissociating peptides or small molecules which are 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 be used to detect not only interactions between two membrane proteins, but also the interaction between membrane and cytoplasmic protein. The ability to monitor the activity and interactions between membrane proteins in their native cellular compartment should prove useful in screening for proteins and drugs that alter their binding properties. REFERENCES Auerbach, 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.
63
Poster Abstracts Thursday, March 27: In vivo Approaches/Analysis of Protein Activities
221 Immunoprecipitation and Western blot screening for phosphorylation states
222
Reconstruction of protein interaction networks in bacteria.
of over 900 signal transduction proteins. Jennifer Stevenson, Allison Siemers, Pam Wethington and Roberto Campos. BD Biosciences-Lexington, KY The importance of phosphorylation in signal transduction has been known for many years. Recently, tools have been made available that allow a more specific, efficient and comprehensive study of protein phosphorylation. These tools include phospho-specific antibodies and monoclonal antibodies recognizing total protein whether phosphorylated or unphosphorylated. In this study, we surveyed the phosphorylation states and general expression levels of over 900 different signal transduction proteins during stimulation of EGFresponsive cells. Our methods include 1) multiplex Western blotting with >900 monoclonal antibodies 2) Western blotting using >30 phospho-specific monoclonal antibodies and 3) anti-phospho-tyrosine immunoprecipitation followed by Western blot screening with >200 monoclonal antibodies. We found numerous phosphorylated proteins; many already described in the literature, such as MAPK and Stats. Also, we detected novel tyrosine phosphorylation changes. One such novel change was identified on Bad, a member of the Bcl-2 family. Furthermore, we compared early phosphorylation events in cells bearing a wild-type EGF receptor versus those containing an EGF-inactive receptor. Cells with the inactive receptor are unable to proliferate in response to EGF. Significant differences were detected in the phosphorylation of Stat1, ERK1/2, p38MAPK and Caveolin-1, implicating these proteins in a cell proliferation pathway. In summary, we were able to utilize an arsenal of cell biology reagents to examine changes in the general expression pattern and phosphorylation profile of EGF stimulated cells. Our methods provide a rapid and comprehensive view of the signaling pathways and proteins activated by EGF and will prove to be an invaluable tool for examining cell states that have been perturbed by various compounds or cellular defects.
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 more active fields of molecular biology. Parallel to the application of experimental techniques for the massive determination of protein interactions, the complementary set of computational methods, 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), plus our new two methods (5-7). We have used as standard of truth the presence of interactions in the literature (8). The results shows that the predictions are of a quality similar to the experimental data, and that the interactions detected by more than one method are of a substantially better quality. Indeed the small sets of interactions predicted by three or more methods seems to correspond in all cases to correct predictions of components of protein complexes. The probabilistic networks build for bacterial systems are compared with the ones proposed for yeast, in terms of coverage, accuracy and potential implications for the modeling of cellular systems. 1. Rain JC, Selig L, De Reuse H, Battaglia V, Reverdy C, Simon S, Lenzen G, Petel F, Wojcik J, Schдchter V, et al.: The protein-protein interaction map of Helicobacter pylori. Nature 2001, 409:211-215. 2. Pellegrini M, Marcotte EM, Thompson MJ, Eisenberg D, Yeates TO: Assigning protein functions by comparative genome analysis: Protein pylogenetic profiles. Proc Natl Acad Sci USA 1999, 96:4285-4288. 3. Dandekar T, Snel B, Huynen M, Bork P: Conservation of gene order: a fingerprint of proteins that physicaly interact. Trends Biochem Sci 1998, 23:324-328. 4. Enright AJ, Iliopoulos I, Kyrpides NC, Ouzounis CA: Protein interaction maps for complete genomes based on gene fusion events. Nature 1999, 402:86-90. 5. Pazos F, Valencia A: Similarity of phylogenetic trees as indicator of protein-protein interaction. Protein Eng 2001, 14:609-614. 6. Pazos F, Valencia A: In silico two-hybrid system for the selection of physically interacting 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 biological
information from scientific text: Protein-Protein Interactions. ISMB99 1999,
60-67.
223 Quantitative Analysis of Glycoproteins: Applications to Serum and Membrane Proteins Hui Zhang, Xiao-jun Li, Daniel B. Martin, and Ruedi Aebersold Institute for Systems Biology 1441 N 34th Street, Seattle, Washington 98103-8904 Abbreviated title: Quantitative analysis of glycoproteins Correspondence should be addressed to R.A. (Institute for Systems Biology, 1441 N 34th Street, Seattle, Washington 98103-8904, [email protected]) Quantitative proteome profiling using stable isotope protein tagging and automated tandem mass spectrometry is an emerging technology with great potential for the functional analysis of biological systems and for the detection of diagnostic or prognostic protein markers. Due to the enormous complexity of proteomes, their comprehensive analysis is an as yet unmatched technical challenge. However, biologically or clinically important information can be obtained if specific, information-rich protein classes or sub-proteomes are isolated and analyzed. Glycosylation is a common post-translational modification. In particular, membrane proteins, secreted proteins, and proteins contained in body fluids are likely to be N- glycosylated. Here we describe a method for the selective isolation, identification and quantification of peptides that contain N-linked carbohydrates. It was applied to the analysis of plasma membrane proteins from cells and proteins from serum. It is expected that this method will find wide application for the identification of diagnostic biomarkers or therapeutic targets in clinical specimens that are enriched in glycosylated proteins.
224 A New Method for Identification of Protein Kinase Substrates Applied to the Search for Novel JNK Targets in the Brain Benny Bjцrkblom1,2, Wenrui Li 1, Tatsiana Tararuk1, Jan-Jonas Filйn1 , Tuula A. Nyman1 , Michael J. Courtney3 and Eleanor T. Coffey1 1Turku Centre for Biotechnology, Еbo Akademi University & University of Turku, BioCity, Turku, Finland. 2Department of Biochemistry & Pharmacy, Еbo 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, are implicated in brain development and mediate neuronal apoptosis in response to noxious stimuli. Given that JNK mediates apoptosis in response to stress, it is expected that neurons display a low basal JNK activity that responds sensitively to apoptotic signals. Surprisingly however, JNK activity in primary cultured neurons and brain extracts is highly elevated in the absence of stress and is further upregulated during neuronal differentiation suggesting also nonapoptotic roles for JNKs in the brain. An increased knowledge of the JNK target repertoire would contribute greatly to our understanding of JNK function in neurons. We have taken a proteomics based approach to identify novel JNK substrates in the brain. High specific activity kinases (JNK or other MAPKs) are incubated with brain extract, a rich source of potential target proteins. Proteins are separated by 2-D gel electrophoresis and phosphoproteins located by autoradiography. JNK phosphorylated proteins are then identified by LCMS/MS. Using this method we have detected several candidate JNK targets, a number of which are expressed at low levels and are highly phosphorylated by JNK suggesting that they may be specific targets. One family of proteins identified by this method has been verified in vivo as JNK phosphorylated proteins and others are currently being tested. This technique can be utilized for multiple kinases and has the advantage of combining a rich source of protein together with rapid identification by tandem MS. Acknowledgements - This work was supported by Academy of Finland grant 72608 and the Informational and Structural Biology Graduate School
64
Thursday, March 27: In vivo Approaches/Analysis of Protein Activities Poster Abstracts
225 Functional Proteomics of Heterotrimeric G-Protein Regulated
226 Integral Membrane Protein Interactions Identified
Signaling in Yeast
Using a Modified Split-Ubiquitin Assay System
Metodi V. Metodiev, Sofia Zaichik, and David E. Stone Laboratory for Molecular Biology, University of Illinois at Chicago, Chicago, Illinois 60607 We are applying a targeted proteomic approach to discover novel protein interactions in the pheromone response pathway of Saccharomyces cerevisiae. In previously described experiments we detected interaction between the pheromone-responsive G-alpha protein (Gpa1) and the Fus3 MAP kinase, and demonstrated the functional significance of this interaction with respect to the overall efficiency of the mating process (1). Here we present new data concerning another important downstream effector of the Galpha protein. Using high-resolution 2D electrophoresis and MALDI-Tof analysis of proteins that co-purify with Gpa1, we identified the Kar3 kinesin as a protein that specifically associates with Gpa1 upon activation of the pheromone pathway. Yeast two-hybrid analysis revealed that Gpa1 binds directly to the N-terminal globular domain of Kar3, and that the interaction is as strong as the two-hybrid interaction between Gpa1 and the yeast Gbeta subunit. Furthermore, this interaction is clearly important for the proper regulation of microtubule dynamics during mating, and for pheromoneinduced nuclear migration preparatory to cell fusion. Depletion of Gpa1 protein caused severe defects in microtubule orientation and nuclear migration, characteristics of Kar3 dysfunction. Thus, the application of proteomics allowed us to uncover a completely unknown and potentially very important regulatory mechanism in this extensively studied pathway. It links a G-alpha protein, a kinesin motor protein, and possibly a MAP kinase, to the regulation of the microtubule cytoskeleton and nuclear migration during signal-induced cellular polarization and morphogenesis. Sequence and functional conservation data suggest that similar pathways might exist in higher eukaryotic organisms. 1. Metodiev et al. 2002, Science, Vol. 296, pp. 1483-1486. NSF MCB-0218081 to DES and MVM.
John Miller1, Russell Lo2, and Stanley Fields1,2 1Departments of Genome Sciences and Medicine, University of Washington, 2Howard Hughes Medical Institute, Seattle, WA, USA 98195 We have employed an assay system to analyze interactions between integral membrane proteins of the yeast Saccharomyces cerevisiae in the hopes that such information can provide clues as to the functions of such proteins. The split-ubiquitin system of Johnsson and Varshavsky is based on the ability of interacting proteins to bring two fragments of ubiquitin into close proximity such that they are acted on by ubiquitin-specific proteases (Johnsson and Varshavsky PNAS 91:10340-10344 (1994)). The original assay was modified by Stagljar et al. (PNAS 95: 5187-5192 (1998)) such that proteolysis releases a transcription factor that then enters the nucleus to activate reporter genes. We selected the proteins annotated in the Yeast Proteome Database as `integral membrane' and created an array of yeast colonies each expressing a different protein fused to the aminoterminal half of ubiquitin. We have screened 35 proteins to date and from these identified 261 putative interactions. Our analysis has thus far centered around the oligosaccharyltransferase components and ER-associated degradation (ERAD) constituents. This work is funded by the NRCC/NIH Yeast Resource Center: PHS # P41 RR11823.
227
IMAC/LC-MS/MS based strategy for phosphopeptide
enrichment, detection and sequencing.
Allan Stensballe, , Thomas S. Nьhse*, and Ole Nшrregaard Jensen Department of Biochemistry and Molecular Biology; University of Southern Denmark, Odense, Denmark *The Sainsbury Laboratory, Norwich, UK
Detailed knowledge of the status of posttranslational modification of proteins like phosphorylation is vital for understanding protein function. The application of nanoflow capillary liquid chromatography (LC) interfaced on-line to electrospray (ESI) mass spectrometers or off-line to MALDI mass spectrometers are now valuable methods for phosphoprotein characterization. Detection of substoichiometric amounts of phosphopeptides in complex peptide mixtures are often hindered by the presence of high levels of non-modified peptides. Immobilized-Metal-Affinity-Chromatography (IMAC) is a powerfull technique for affinity enrichment of phosphopeptides prior to analysis. We have investigated this technique as a front-end method prior to analysis by nanoflow LC-MS/MS using data directed discovery of modified peptides. Strategies for selective analysis of phosphopeptides using LC-ESI-QTOF MS/MS includes the characteristic neutral loss of 97.98 Da per phosphate moity (gas-phase beta-elimination of phosphoric acid). We have evaluated Data directed discovery triggered by these phospho-specific ions during automated nanoflow LC-MS/MS of complex peptide mixtures which allows selection of the subset of modified peptides for fragmentation. This enables selective detection and sequencing of modified peptides among co-eluting normal peptides of higher abundance. We have investigated these methods for analysis of simple or complex phosphoprotein mixtures. Analysis of a complex peptide mixture derived from an Arabidopsis thaliana membrane preparation revealed more than 90 highly purified phosphopeptides in one IMAC/LC-MSMS experiment.
228
PROTEOMIC APPROACH TO LFA-1 MEDIATED SIGNALLING
IN T-CELL MIGRATION
Arun Chandran, Yuri Volkov, Aideen Long and Dermot Kelleher Trinity College, Dublin and Dublin Molecular Medicine Centre, Ireland
Directed migration of T-lymphocytes towards tissue-specific destinations represents a crucial step in immune system response to inflammation and pathogen invasion. The interactions of LFA-1 integrin receptor with its ligand ICAM-1, contribute significantly to the molecular dynamics of active T-cell locomotion. We have previously reported the formation of a multi-molecular cytoskeleton/signalling complex commencing upon LFA-1 occupancy by its natural and imitating recombinant ligands. However, precise functional significance of the proteins associated with LFA-1 intracellular domain has not yet been established. We utilized the original in situ immunoprecipitation technique in combination with proteomic analysis to further identify and characterize the individual proteins that are involved in the signalling cascade. In brief, T cells were activated via substrate-immobilized motility-inducing LFA-1 ligands either at physiological (37°C) or low temperature conditions (4°C), which block intracellular signalling processes. Live migrating cells were lysed in optimised membrane detergent buffer and only the stable protein complexes remaining attached to substrate via ligand/LFA-1 chains were collected for subsequent proteomic analysis. 2-D electrophoretic mapping of the LFA-1 associated complexes revealed significant differences in the cytoskeletal and signalling molecules recruitment under these two experimental conditions. Western blotting and MALDI-TOF sequencing of separated proteins demonstrated that LFA-1 associated complex is enriched in tubulin and vimentin cytoskeleton components and contains at least two signalling proteins of the protein kinase C family (PKC beta and delta). However, actin contents of the complexes and PKC-theta involvement were not affected by blocking conditions, suggesting selective isoform-specific recruitment of PKC and certain cytoskeletal proteins in the process of active T-cell motility. These and other potentially novel LFA-1 associated molecules, which are currently being characterized might be critically important for concerted intracellular signalling events and changes of gene expression associated with cell migration.
65
Poster Abstracts Friday, March 28: Structural Proteomics/ Control of Protein Abundance and Activity/Computational Methods
301 Protein expression microarrays for proteomics
302 Reproducible Sample Preparation: A Key Step in
Proteome Analysis
Kristi A. Miller, Daniel M. Yoshikawa, Todd Edwards, Matthew A. Coleman, and Joanna S. Albala. Biology & Biotechnology Research Program. Lawrence Livermore National Laboratory. Livermore, CA 94550.
J. Anders, R. Hendriks Merck KGaA, Life Science Products R&D MDA, Frankfurter Str. 250, 64293 Darmstadt, Germany
Understanding the biology of an organism requires complex investigation. Unlike genes, proteins are a more diverse population composed of many more elements having more widespread interactions within the cell. The key advantage to array-based methods for protein study is the potential for parallel analysis of thousands of samples in an automated, high-throughput fashion. Protein arrays are emerging as a practical format in which to study proteins in highthroughput using many of the same techniques as that of the DNA microarray. We have developed a method to utilize LLNL-I.M.A.G.E. cDNAs to generate recombinant protein libraries using a baculovirusbased paradigm. We have used this strategy to produce proteins for analysis of protein/DNA and protein/protein interactions using protein microarray techniques. Our efforts are focused on understanding the complex interactions of proteins involved in homologous recombination and DNA repair. This work was performed under the auspices of the U.S. DOE by the UC-LLNL under Contract No. W7405-Eng-48.
Proteome analysis implies the ability to separate proteins with high resolution and reproducibility prior to protease digestion and characterization by mass spectrometry or microsequencing. High-resolution protein separation for proteome analysis can be achieved by both liquid chromatography (LC) and twodimensional gel electrophoresis (2DGE). The sample preparation of any protein mixture for subsequent separation by either LC or 2DGE is of major importance, as it will affect the overall performance of the technique. Due to the very high degree of complexity of the proteome especially in biological samples from higher eukaryotes, standard operating procedures for sample preparation preferably including proteome prefractionation must be employed in order to lower sample complexity prior to protease digestion of proteins. Different approaches to sample prefractionation including stepwise extraction of partial proteomes from cells, sample prefractionation by LC and depletion of highly abundant proteins from body fluids will be discussed.
303 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 20850 [email protected] (!) Informatics research, Applied Biosystems 45 W. Gude Drive, Rockville, MD 20855 [email protected] The correct interpretation of tandem mass spectra is a difficult problem, even when it is limited to scoring peptides against a database. Correct De novo sequencing is considerably harder, but critical when sequence databases are incomplete or not available. In this paper we build upon earlier work due to Dancik et al., and Chen et al. to provide a dynamic programming algorithm for interpreting de novo spectra. Our method can handle most of the commonly occuring ions, including a,b,y, and their neutral losses. Additionally, we shift the emphasis away from sequencing to assigning ion types to peaks. In particular, we introduce the notion of core interpretations, which allow us to give confidence values to individual peak assignments, even in the absence of a strong interpretation. Finally, we introduce a systematic approach to evaluating de novo algorithms as a function of spectral quality. We show that our algorithm, in particular the core-interpretation, is robust in the presence of measurement error, and low fragmentation probability.
304 A Protein-Domain Microarray Identifies Novel ProteinProtein Interactions Alexsandra Espejo and Mark T. Bedford The University of Texas M.D. Anderson Cancer Center, science park-Research Division, Smithville, TX 78957. Protein domains mediate protein-protein interaction through binding to short peptide motifs in their corresponding ligands. These peptide recognition modules are critical for the assembly of multiprotein complexes. We have arrayed GST fusion proteins, with a focus on protein interaction domains, onto nitrocellulose coated glass slides to generate a protein-domain chip. Arrayed protein-interacting modules included WW, SH3, SH2, 14.3.3, FHA, PDZ, PH and FF domains. Here we demonstrate, using peptides, that the arrayed domains retain their binding integrity. Furthermore, we show that the protein-domain chip can "fish" proteins out of a total cell lysate, these domain-bound proteins can then be detected on the chip with a specific antibody, thus producing an interaction map for a cellular protein of interest. Using this approach we have confirmed the domain binding profile of the signaling molecule, Sam68, and have identified a new binding profile for the core snRNP protein, SmB'. This protein-domain chip not only identifies potential binding partners for proteins, but also promises to recognize qualitative differences in protein ligands (caused by posttranslational modification), thus getting at the heart of signal transduction pathways.
66
Poster Abstracts Friday, March 28: Structural Proteomics/ Control of Protein Abundance and Activity/Computational Methods
305 Printing chemical libraries on microarrays for fluid 306 Differential protein expression profiling on arrays
phase nanoliter reactions.
Easton P, Evans S, Wheeler C, Moody A, Cross T, Bamford A, Copse
Dhaval N. Gosalia and Scott L. Diamond
C, Smith C* Amersham Biosciences UK Limited, The Grove Centre, Amersham,
Buckinghamshire, HP7 9LL, U.K. Institute for Medicine and Engineering, Departments of
Bioengineering and Chemical and Biomolecular Engineering, 1024 Vagelos Research Laboratory University of Pennsylvania, Philadelphia, PA 19104
The ability to profile the differences between biological samples is of fundamental importance in biology. These differences may occur as a result of a disease or the effects of a drug treatment and have classically been studied at both the gene and protein level using DNA
microarray and 2-dimensional fluorescence difference gel We printed chemical compounds within individual nanoliter droplets electrophoresis (2-D DIGE) technologies, respectively. However, 2-D
of glycerol that remained adherent to the surface of a microarray at 400 electrophoresis is not ideally suited to rapid, large-scale protein spots per cm2. Using ultrasonic-generated aerosol deposition, we expression screening.
metered subsequent reagents (enzymes, substrates, buffer) into each 200 µM diameter reaction center in order to rapidly assemble multicomponent reactions without cross contamination or the need for
Recently, protein arrays have emerged as a technology with the potential to fulfil the requirement for the high throughput screening of differential protein expression profiles. There are a number of different
chemical linkage to the surface. A volume of 2 uL of reagent was sufficient to activate 5000 reactions on a single slide. This proteomics tool allowed the profiling of protease mixtures and the detection of
formats for protein arrays dependent on their specific application. In general, all array formats will require highly specific binders bound to a solid support and coupled to a method of detection.
fluorogenic substrate conversion in the presence of various inhibitors. We are developing a flexible technology for the production of high A 352-compound library was microarrayed in quadruplicate on 100 sensitivity protein arrays for differential protein expression profiling on
replicate slides. An inhibitor of caspases 2, 4, and 6 was identified using five replicate 352-compound microarrays screened against caspase 2, 4, 6 as well as thrombin and chymotrypsin. From a single printing run, combinatorial libraries can be subjected to numerous homogeneous separation-free reactions at volumes 103 to 104 smaller
glass slides. By using multi-colour fluorescent labelling of samples with CyDyeTM fluors and antibodies as capture agents, we are able to accurately detect differences in protein levels within complex samples. Here, the utility of the technology is shown for the differential analysis of cytokines in biological samples.
than current high throughput methods.
307
Predicting MS/MS Peptide Fragmentation Patterns: Beyond SEQUEST
Joshua E. Elias1, Francis D. Gibbons2, Frederick P. Roth2, and Steven P. Gygi1. 1Department of Cell Biology, 2Department of Biochemistry and Molecular Physiology, Harvard Medical School, Boston, MA, USA, 02115
Existing automated tandem mass spectrometry sequencing methods are limited: Their simplified treatment of measured ions, often relying solely on mass, reduces their power. In reality, observed peptide fragmentation patterns depend on several factors. We hypothesize that considering 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 microcapillary reverse-phase liquid chromatography coupled on-line to a tandem mass spectrometer (LCQ DECA ion trap, ThermoFinnigan, San Jose, CA). Tandem mass spectra were searched against appropriate 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 than those previously proposed (Washburn, 2001). For each peptide meeting these criteria, observed intensities 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 (b or y), fractional position relative to peptide length, and intensity. This database was used to evaluate 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 unique peptides as a training data set, we have developed an algorithm that evaluates the likelihood that a fragmentation pattern came from a given peptide. It is clear that fragmentation events and consequent ion intensities are not random. For example, fragmentation events between particular 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 the peptide (~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 unknown sequences, we suggest that in addition to augmenting established analytical tools, this method will 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 Service Research Award 5T32CA86878 from the National Cancer Institute (J.E.).
308 Development of Antibody Microarrays for Detection and Quantitation of Specific Proteins Lisa Gangi, Lionel Best, Lynn Rasmussen and David Munroe. Laboratory of Molecular Technology, SAIC-Frederick, National Cancer Institute at Frederick, 915 Tollhouse Road, Suite 211, Frederick, MD, 21701, USA. Protein microarrays hold the promise of a high-throughput approach to genome wide studies at the protein level. DNA microarrays have proven to be an invaluable tool for profiling human cancers, but the data is limited to observations at the transcriptional level. Since alterations at the protein level also are important in key pathways leading to cancer, advances in proteomics have become a rapidly developing field. Although complete proteome-wide array-based research is not yet possible, practical applications of protein microarrays are currently possible using standard microarray instrumentation. We have developed a platform that facilitates the production of high-density antibody microarrays. This platform is being utilized for both protein profiling and for specificity and affinity screening of crude monoclonal hybridoma and single chain antibody arrays. This research is supported by NCI contract # N01-C0-12400
67
Poster Abstracts Friday, March 28: Structural Proteomics/ Control of Protein Abundance and Activity/Computational Methods
309
Rapid Peptide Identification by Recursive K-means Clustering of
310 Rapid Extract System: High throughput
Amino Acid Composition Data
purification of protein samples
Brian D. Halligan, Xin Feng, Simon N. Twigger, and Peter J. Tonellato, Bioinformatics Research Center, Medical College of Wisconsin, Milwaukee, WI 53226
Tariq A. Haq and Kevin M. Hughes BD Biosciences, Two Oak Park, Bedford, MA, USA 01730
The functional proteomic analysis of complex biological samples presents a significant bioinformatic challenge with respect to data analysis and reduction. High throughput proteomics experiments result in the production of large quantity of spectral data that need to be further analyzed to obtain biologically relevant information. The standard methods used to analyze this data, spectral matching techniques (SEQUEST) or denovo peptide sequence determination and sequence database searching, require significant computing hardware resources to complete these task in real time. To develop a real-time peptide identification system, we developed a method that 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 of the mammalian entries in the SwissProt protein database and calculated a series of 20 numerical attributes for each of these peptides, based on the frequency of occurrence of each amino acid in the peptide. A recursive K-means algorithm was used to cluster the peptides into distinct groupings based on these amino acid frequency attributes. By using this method recursively, we are able to generate layers of clusters of peptides that ultimately correspond to the equivalent peptide in homologous and orthologous proteins. The purpose of clustering the amino acid frequency data is to create an index to the peptide database that allows us to identify a peptide based on its composition. To identify a peptide, its amino acid frequency attributes are calculated and the distance of its attributes vector to the centers of the clusters are measured. For each layer in the cluster database, the closest cluster is identified and the attributes of individual members of the clusters are 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 peptide database that can be rapidly traversed. We are currently expanding this method to analysis of primary proteomics data.
Abstract: As the field of Proteomics develops, there will be inevitable pressure to purify larger numbers of protein samples for analysis. Current methodologies are well adapted for purification of limited numbers of samples, but handling larger numbers--tens, hundreds or thousands--presents a logistical problem. The Rapid Extract System is a novel approach for quick affinity purification of protein samples in a high throughput format. It allows rapid protein purification from large number of samples without the requirement of changing plates or lengthy processing times. Multiple recombinant bacterial protein purifications were performed simultaneously in one of the applications described in this study. The system handled up to 96 samples at a time and was fully compatible with available automation equipment. Additional applications being developed for the system include: purification of tagged proteins, immunopurification, depletion of specific proteins etc.
311
Profiling Enzyme Activities in Models of Human Breast Cancer
Nadim Jessani, Benjamin F. Cravatt III. Departments of Cell Biology and Chemistry, Scripps Research Institute, La Jolla, CA, USA, 92037
To compliment conventional genomic and proteomic methods that often focus on measuring abundance rather than activity, we have developed a chemical proteomics method, referred to as "activity-based protein profiling" (ABPP). This method employs chemical probes that covalently label the active sites of enzyme superfamilies in a manner that provides a direct readout of changes in catalytic activity. By providing a covalent link between the labeled proteins and a chemical tag, ABPP permits the consolidated detection, isolation, and identification of active enzymes directly from complex proteomes.
In a recent study, ABPP probes were applied to the proteomic analysis of a panel of human cancer cell lines. These studies resulted in the identification of multiple enzymes whose functional state correlated with tumor type as well as higher order cellular phenotypes, such as invasiveness. Here, we extend the ABPP approach to a model that approximates the growth conditions of tumors in vivo, where host factors can be taken in to account. ABPP analysis of tumors and metastases derived from human breast cancer line xenografts grown in immunodeficient mice have been carried out to assess whether the expression patterns of enzyme activities previously identified in human cancer cell lines are maintained in vivo. Moreover, by taking advantage of the mixed species nature of the xenograft model, tumor derived (human) enzyme activities can be distinguished from host stromal (mouse) activities. Specifically, human and mouse enzymes, though similar, exhibit sufficiently different tryptic peptide maps providing a means to determine species of origin of enzymes identified by mass spectrometry methods. Notably, these studies demonstrate how proteomic approaches, like ABPP, can provide a distinct advantage over mRNA profiling methods, which often rely on cDNA or oligonucleotide arrays that cannot distinguish orthologous transcripts with high sequence identity.
Supported by California Breast Cancer Research Program.
312 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, Japan 2Chiba Industry Advancement Center, 2-6 Nakase, Mihana-ku, Chiba261-7126, Japan 3Institute of Research and Innovation, 1201 Takeda, Kashiwa, Chiba 277-0861, Japan 4National 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.
68
Poster Abstracts Friday, March 28: Structural Proteomics/ Control of Protein Abundance and Activity/Computational Methods
313 Proteomic Analysis of the Bacteriophage T7 Replisome
314
MODULATION OF mRNA TRANSLATION IN YEAST PHEROMONE
RESPONSE.
Jaya Kumar, Charles C. Richardson, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA, MA 02115.
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 Cancer Research Center, Seattle, WA 98109.
We present a genome-wide analysis of proteins at the replication fork of bacteriophage T7. Bacteriophage T7 infection of Escherichia coli provides an attractive model system to study how a chromosome is duplicated. Within twelve minutes after infection of E. coli, over a hundred copies of the 39,936 base pair linear T7 chromosome are synthesized. The T7 DNA polymerase functions as part of a multiprotein complex that unwinds duplex DNA, primes lagging strand synthesis, and coordinates leading and lagging strand synthesis during DNA replication. Here we describe the tandem affinity purification (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 high affinity (Kd = 5 nM). The gene encoding thioredoxin bearing a TAP tag at its C-terminus was inserted into the T7 chromosome. Functional protein complexes formed in vivo have been isolated from phage-infected cell-lysates and characterized. The interacting partners of T7 DNA polymerase and E. coli thioredoxin will be described. This work was supported by United States Public Health Grant NIHGM54397.
Cellular changes in response to external stimuli usually involve transcriptional activation of some key genes, but the rapidity of phenotypic change often observed suggests that post-transcriptional alterations may also contribute. We have used microarrays to conduct a high-resolution analysis of the translation state of all mRNAs in yeast mating-type a cells + acute treatment with the mating pheromone -factor. Lysates from treated and untreated cells were each fractionated by sucrose gradient centrifugation. Purifed RNA from each fraction was applied to microarrays, generating a profile of ribosome loading for each mRNA and facilitating a comparison of mRNA translation state in control and treated cells. Although nearly all the mRNAs showed the same polysome profile for the two cell populations, for a few mRNAs the translation state was significantly altered. Several mRNAs well-loaded with ribosomes in the control gradients showed dramatic shifts to poor loading (1-2 ribosomes per mRNA) after -factor treatment, while other mRNAs that were poorly loaded in the control shifted to higher polysomes with pheromone treatment. Other mRNAs were affected less drastically by -factor, shifting to either higher or lower ribosome density. We have initiated studies on a few genes to date and have determined that several mechanisms seem to be represented. Some genes show promoter shifts with -factor treatment, leading to mRNAs with longer or shorter 5' leaders that are differentially translated. Short upstream open reading frames (uORFs) are included in some, but not all, of these 5' leaders and may contribute to translational regulation. In contrast, neither a promoter shift nor uORF are associated with at least one mRNA that displays pheromone modulation of translation. Our results suggest that the yeast cell has evolved several distinct strategies for regulating translation and, consequently, protein synthesis. Supported by NIH-CA-89807
315 In Vitro Analysis of Transcription and Expression of the Truncated Erythropoietin Receptor Joseph Milano, Monicah A. Otieno and Franзois Pognan. Safety Assessment, AstraZeneca Pharmaceuticals, Wilmington, DE, USA, 19850
316 Application of statistical methods to database lookup for protein identification via LCQ mass spectrometry Aleksey Nakorchevskiy, Edward Marcotte. Department of Chemistry and Biochemistry, University of Texas, Austin.
Erythropoiesis is a multi-stage process requiring proliferation and differentiation of cells of the erythroid lineage to form mature erythrocytes. This process is controlled by erythropoietin (Epo) and is communicated by its cell surface receptor EpoR. There are two forms of EpoR ­ a full length (EpoR-F) expressed predominantly in mature erythrocytes and an alternatively spliced, truncated receptor (EpoR-T) expressed predominantly in immature erythrocytes. Expression of EpoR-T is thought to have a dominant-negative effect on EpoR-F thus regulating erythropoiesis depending on circulating Epo levels. We investigated the control of expression of EpoR-T in an in vitro system using murine erythroleukemia cells. Using semi-quantitative PCR, we have shown that these cell lines transcribe both the truncated and full-length forms of the EpoR. The ratio between EpoR-F and EpoR-T ranged from 10:1 to 100:1 full-length vs. truncated. However, these ratios were not reflected in Western analysis of whole-cell lysates. To determine the point of control of expression for the EpoR protein we transfected Cos-1 cells with a cDNA plasmid construct that constitutively expresses the full-length receptor. Our results show the presence of both the full-length and truncated forms of the protein. However, only the full-length cDNA was detected by RT-PCR. These observations suggest mechanisms other than alternative splicing may control expression of the erythropoietin receptor.
Mass spectrometry is one of the most powerful tools available for protein identification from whole-cell digests. The quality of the peptide/protein identification currently relies only on the quality of the match between experimental and theoretical fragmentation spectra. We introduce a computational method to strengthen protein identification quality and coverage by including other types of data into the database lookup. Such data include additional measurements collected from the peptides during a typical mass spectrometry experiment, including chromatographic elution times and previously collected mass spectra. We construct a probabilistic framework for protein identification that incorporate these additional data, and thereby gain many additional constraints on the peptides' identities, allowing extension of mass spectrometric peptide lookup beyond spectral alignment.
69
Poster Abstracts Friday, March 28: Structural Proteomics/ Control of Protein Abundance and Activity/Computational Methods
317 ROSPath: a database of Reactive Oxygen Species mediated cell- 318 Accuracy and Variance in the Identification and
signaling PATHways
Quantification of Proteins in Mass Spectrometry Based
Jisook Park1, Joonkyu Park2, Sang-Hyuk Lee3, Seung-Rock Lee3, Kong-Joo Lee3, and Eunok Paek4, 1Seoul Women's University, Seoul, Republic of Korea, 139-774, 2Interface Infotech, Co., 3Ewha Womans University, 4University of Seoul, Seoul, Republic of Korea.
Proteomics John Prince, Institute for Cellular and Molecular Biology, The
Recent massive data generation by genomics and proteomics requires bioinformatic tools University of Texas, Austin, Texas, USA, 78712
to extract the biological meaning from the massive results. Here we introduce ROSPath, a database system to deal with information on reactive oxygen species (ROS)-mediated cell signaling pathways. It provides a structured repository for handling pathway related data and tools for querying, displaying, and analyzing pathways.
Mass Spectrometry (MS) offers researchers a powerful technique for measuring the proteome. Databases with fully sequenced genomes and techniques for resolving complex samples (e.g. MuDPIT) have
ROSPath data model provides the extensibility for representing incomplete knowledge and the accessibility for linking the existing biochemical databases via the Internet. For flexibility and efficient retrieval, hierarchically structured data model is defined by using entity-relation model and following an object-oriented paradigm. There are two major data types in ROSPath data model: `Data Entity' and `Interaction'. `Data Entity' represents a single biochemical entity: a protein or protein state involved in ROS cell-signaling pathways. `Interaction', characterized by a list of inputs and outputs, describes the type of relationships between data entities. Typical interactions are state transitions, chemical
allowed the identification and quantification of large numbers of proteins. Still, the analysis of MS data remains one of the greatest challenges to the field. Better analyses promise larger numbers of identified proteins at explicit levels of confidence. We are using two-dimensional liquid chromatography with ESI Ion-
reactions, and reactions. Then, a complex network can be constructed from the two main types and this allows describing and viewing various biochemical processes.
Trap MS on the E. coli proteome to explore methods of analysis and quantification. We measure the variation introduced at different steps
ROSPath provides flexible and intuitive visualization for biochemical entities and/or in a proteomics experiment including sample preparation,
curated pathways given a simple keyword query. More complex queries such as sub-graph extraction can also be visually answered by pathway queries. Another type of complex query(searching a knock-out component on pathways, for example) is conducted by a simple simulation. These tools together with extensive annotation on ROS signaling molecules make ROSPath database an innovative addition to information on signaling molecules, thus opening up new possibilities for biological pathway analysis and pathway discovery.
chromatography, and spectrometry. Examination of sub-cellular fractions has allowed us to quantify the accuracy of the identification process and allowed comparison of protein look-up strategies. Finally, we present results from an isotope labeling experiment applied to an E. coli proteome, and compare these results with alternative methods for measuring protein concentration on a
Supported by IMT2000 project for IT-BT and by KOSEF through the Center for Cell proteome-wide scale. Signaling Research (CCSR) at Ewha Womans University.
319
Dissection of guard cell ABA signal transduction mechanisms and
Complexes in Arabidopsis
320 Development of an Arabidopsis Protein Chip: Design, Production and Applications
Julian Schroeder, June M. Kwak, Izumi Mori, Nathalie Leonhardt, Yoshiyuki Murata, Alison Delong Cell and Developmental Biology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093-0116 Guard cells have become a well-suited model system for dissecting early signal transduction 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 molecular mechanisms responsible for these proposed counteracting PP2As, gene chip experiments were performed with Arabidopsis guard cell RNA together with degenerate oligo-based PCR screening of Arabidopsis guard cell cDNA libraries. A knock-out mutant in a guard cell-expressed PP2A gene, rcn1, was obtained and showed ABA insensitivity in stomatal movements and anion channel activation. Calcium imaging analyses show a reduced sensitivity of ABA-induced cytosolic Ca2+ ([Ca2+]cyt) elevations in rcn1, whereas mechanisms that are stimulated after [Ca2+]cyt increases show wild-type responses, suggesting that RCN1 functions upstream of [Ca2+]cyt increases. rcn1 shows ABA insensitivity in ABA inhibition of seed germination and ABA-induced gene expression. ЯThe 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. Research towards identifying the signaling complex of RCN will be presented. Hyperpolarizationactivated Ca2+-permeable channels (ICa) were identified as a component of ABA signaling (Pei et al., 2000, Nature 406:731-734). ROS were shown to activate ICa channels in Arabidopsis guard cells. Data will be presented analyzing NADPH oxidase disruption mutations that suggest a central role for these genes in ABA activation of ICa channels and stomatal closure. Additional mechanisms in this signaling complex will be reported.
Montrell Seay, Biaqing Lin, Michael Snyder and S.P. Dinesh-Kumar MCDB Department, Yale University, New Haven, CT, 06520 Plants have evolved elaborate defense mechanisms in response to a wide range of pathogens. Phosphoproteome studies have implicated changes in protein phosphorylation as playing key regulatory roles in resistance pathways, however the lack of existing technologies has limited further studies. Proteomics in yeast has allowed researchers to evaluate protein-protein interactions, kinase activity and substrate specificity using protein chip technologies. On the other hand, proteomics in plants has been limited to the isolation and identification of phosphoproteins without the identification of the responsible kinases. Despite the tremendous power of protein arrays to uncover substrate-kinase combinations for a given phosphoproteome, to date a protein chip from higher eukaryotes has not emerged. Here we present the development of a protein chip using recombinant Arabidopsis proteins arrayed on nickel coated-slides and immobilized in nanowells. Arabidopsis ORFs were cloned into various modified Gateway expression vectors for protein expression in yeast, insect cells and Nicotiana plants. Several strategies were used to optimize protein expression, purification and catalytic activity. Our preliminary findings outline the optimal expression system and method for high throughput protein production for the plant protein chip.
70
Poster Abstracts Friday, March 28: Structural Proteomics/ Control of Protein Abundance and Activity/Computational Methods
321 GeneAtlas ­ A Software Tool for Proteome-Wide Functional Annotation
322 A Sperm-Associated WD Repeat Protein Orthologous to Chlamydomonas PF20 Associates with Spag6, the Mammalian Orthologue of Chlamydomonas PF16
Mikhail Velikanov, Lisa Yan, and Sandуr Szalma Accelrys, Inc., 9685 Scranton Road, San Diego, CA 92121 GeneAtlas is an automated high-throughput software pipeline designed for functional annotation and 3D structure prediction of large sets of protein sequences. It uses a combination of sequence similarity searches (PSI-BLAST), fold recognition (SeqFold) and homology modeling (MODELER) methods to identify homologues in PDB database and create 3D models of the input protein sequences. The protein 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 identification using HMMER and PFAM database, transmembrane segment prediction by TMHMM, secondary structure prediction by DSC, as well as a variety of residue-based annotations (e.g. active site, binding pockets, 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 using GeneAtlas, including human, mouse, C. elegans, A. thaliana, etc. The details of GeneAtlas methodology as well as some of the results from the annotated proteomes will be discussed.
Zhibing Zhang, Jerome F. Strauss III Center for Research on Reproduction and Women's Health,University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104 cDNAs were cloned for the murine and human orthologues of Chlamydomonas PF20, a component of the alga axoneme central apparatus that is required for flagellar motility. The mammalian genes encode transcripts of 1.4 and 2.5 kb that are highly expressed in testis. The two transcripts appear to arise from alternative transcription start sites. The murine Pf20 gene was mapped to chromosome 1, syntenic with the location of the human gene on chromosome 2. An antibody generated against an N-terminal sequence of mouse Pf20 recognized a 71-kDa protein in sperm and testis extracts. Immunocytochemistry localized Pf20 to the tails of permeabilized sperm; electron microscope immunocytochemistry showed that Pf20 was located in the axoneme central apparatus. A murine Pf20-green fluorescent protein fusion protein expressed in Chinese hamster ovary cells accumulated in the cytoplasm. When coexpressed with Spag6, the mammalian orthologue of Chlamydomonas PF16, Pf20 was colocalized with Spag6 on polymerized microtubules. Yeast two-hybrid assays demonstrated interaction of the Pf20 WD repeats with Spag6. Pf20 was markedly reduced in sperm collected from mice lacking Spag6, which are infertile due to a motility defect. Our observations provide the first evidence for an association between mammalian orthologues of two Chlamydomonas proteins known to be critical for axoneme structure and function. These studies were supported by NIH grant R01-HD37416-02.
323
Structure of the Protein Kinase A Regulatory-Catalytic Subunit
Complex by Amide H/2H Exchange and Protein Docking
Ganesh S. Anand,1, Dennis Law2,3, Jeff Mandell, Aaron N. Snead2, Igor Tsigelny3, 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 individual subunits in solution has been achieved using backbone hydrogen/deuterium (H/2H) exchange with mass spectrometry and the docking program DOT. The changes in backbone hydrogen/deuterium (H/2H) exchange in the regulatory subunit (RI(94244)) and the catalytic subunit of cyclic AMP-dependent protein kinase A (PKA) upon complexation to form holoenzyme (R-C) were probed by MALDI-TOF mass spectrometry. Protection from amide exchange upon C-subunit binding was observed for the helical subdomain, including the A and B-helices, one of which had 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-terminal lobe of the C-subunit that contributes part of the intersubunit interface with RI subunit. The results from amide exchange experiments were then used to design distance filters to constrain the solutions of the PKA holoenzyme obtained by the protein docking program DOT. This enabled identification of a limited set of solutions that satisfy amide exchange data. Site-directed mutagenesis was applied to test each of the solutions to identify a single solution that best represents the structure of the PKA holoenzyme. Our approach can be used to determine testable models of higher order protein-protein interaction complexes from the structures of interacting proteins by a combination of amide exchange and docking.
324 time.
The use of "LADDERS" to detect protein changes in living cells in live
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, Oxford Road, Manchester, M13 9PT, UK
Antibodies provide a valuable tool for examining the proteome. In the last decade, new technologies have been developed which allow the expression of antibody fragments (intrabodies) inside living cells. However, the use of intrabodies to detect protein changes in living cells has not been realised because of the problem of distinguishing which intrabodies 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 peptide antigen 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 in YFP (530 nm) fluorescence. When endogenous cellular antigen is expressed, it competes with the YFP ­ tagged peptide antigen for intrabody binding. CFP-tagged intrabody bound to endogenous protein is no longer in proximity to YFP, so excitation of CFP now leads to CFP (480 nm) emission. Thus the net effect of an increase in protein expression is an increase 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 unaltered when CFP was attached to the C-terminus. Likewise, we successfully created the peptide epitope that the anti-MUC intrabody binds, attaching YFP to its C-terminus. This protein was detected by Western analysis using anti-MUC intrabodies and also interacted with the anti-MUC1 scFv. However, despite the fact that CFP-labelled anti-MUC intrabody complexed with YFP-labelled MUC epitope, we were unable to demonstrate significant FRET between the two entities, indicating that the molecular distance between the proteins was too great. Consequently, we have constructed a second generation of LADDERS probes reducing the linking sequences between the fluorescent protein and the intrabody/epitope in order to decrease the molecular distance between the two fluorescent proteins when the two chimaeric proteins are complexed.
Supported by Wellcome Trust UK Showcase Award GR064075MA
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Poster Abstracts Friday, March 28: Structural Proteomics/ Control of Protein Abundance and Activity/Computational Methods
325
Construction Use of a Protein Trap to Probe the Impact of ClpXP
Pprotease 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 cellular proteome in response to shifts in nutrients and other environmental conditions. In E. coli, five intracellular proteases, ClpXP, ClpAP, HslUV, Lon and FtsH, play roles in regulating cellular processes by terminating the activitycontrolling the availability of a variety of proteins.. Studying the full impact of degradation on the bacterial proteome requires methods for identifying substrates for each of these proteases under a varriety of environmental conditions. Here, we describe the use of an inactive variant of the ClpP protease as an intracellular trap to identify new ClpXP substrates.
ClpXP is a ATP-dependent protease comprised of two components: ClpX, a hexameric ATPase responsible for substrate recognition and unfolding, and ClpP, a peptidase whose serine active sites face an internal chamber. Substrates of this protease are recognized by ClpX and subsequently translocated into the ClpP chamber where they are degraded. To identify new ClpXP substrates, we constructed a variant of ClpP (ClpPtrap) in which substrates are captured but not degraded. By expressing ClpPtrap in vivo, substrates of this protease were captured; these trapped proteins were purified, and identified by mass spectrometry.
We identified about 50 ClpXP substrates under normal logarithmic growth conditions and 40 additional substrates following the stress of DNA damage. Analysis of substrate sequences revealed five reoccurring peptide motifs that directed protein allow proteins to be recognitionzed by ClpXP. This definition of ClpX recognition signals and the role of ClpXP degradation during stress responses provides a foundation for understanding strategies for regulating protein turnover. For example, we find that recognition of two substrates, RseA and LexA, dependenteach depend on an initial protein-processing event to reveal an internal recognition signal. Thus, regulating the availability of cryptic protease recognition signals provides one way to control degradation in response to environmental change. Identification of additional ClpXP substrates by intracellular trapping under a wide range of cellular conditions should permit further definition of the full impact of ClpXP on the bacterial proteome.
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ANN KAREN

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