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Lebenswissenschaftliche Innovationsplattform Dortmund

A Story of Success in applied Technology Transfer

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Editors TechnologieZentrumDortmund Management GmbH Zentrum fr Angewandte Chemische Genomik Zentrum fr Angewandte Proteomik Emil-Figge-Strae 7680 44227 Dortmund Germany Max-Planck-Institut fr molekulare Physiologie Zentrum fr Systembiologie Otto-Hahn-Strae 11 44227 Dortmund Germany Technische Universitt Dortmund Zentrum fr Angewandte Chemische Genomik Zentrum fr Angewandte Proteomik 44221 Dortmund Germany Ruhr-Universitt Bochum Medizinisches Proteom-Center ZKF E.143 Universittsstrae 150 44801 Bochum Germany

Layout and Design SEKTOR Werbeagentur Schwanenwall 31 44135 Dortmund Germany

Content

Lebenswissenschaftliche Innovationsplattform Dortmund . . . . . . . . . . . . . . . 4 Zentrum fr Angewandte Chemische Genomik (ZACG) . . . . . . . . . . . . . . . . . . 5 Nanobiotechnology Systems for Research in Active Agents . . . . . . . . . . . . . . 6 Chemical Biotechnology of Pharmaceutical Compounds . . . . . . . . . . . . . . . . 7 Substance Libraries in Drug Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Towards an Understanding of Diabetes Mellitus Type II Strategies for Inhibition of Protein Aggregation and Amyloid Formation . . . . . . . . . . . . . 10 Zentrum fr Angewandte Proteomik (ZAP) . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Quantitative Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Difference in Gel Electrophoresis (DIGE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Glycoproteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Protein Biochips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Biostatistics and Bioinformatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Zentrum fr Systembiologie (ZfS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Equipment platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Partners in Lebenswissenschaftliche Innovationsplattform Dortmund Max-Planck-Institut fr molekulare Physiologie . . . . . . . . . . . . . . . . . . . . . . 29 Technische Universitt Dortmund . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Medizinisches Proteom-Center . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 TechnologieZentrumDortmund GmbH/ TechnologieZentrumDortmund Management GmbH/ BioMedizinZentrum Dortmund . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Contact Persons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Supported by Ministry of Innovation, Science, Research and Technology of Northrhine-Westphalia

Co-financed by European Union Fonds for Regional Development (EFRE)

Lebenswissenschaftliche Innovationsplattform Dortmund


The Lebenswissenschaftliche Innovationsplattform Dortmund (LIP) was officially launched on 23rd June 2006 by the minister of innovation, science, research and technology of Northrhine-Westfalia, Prof. Dr. Andreas Pinkwart. LIP consists of three competence centres: Zentrum fr Systembiologie (ZfS), which is located at Max-Planck-Institut fr molekulare Physiologie (MPI), Zentrum fr Angewandte Chemische Genomik (ZACG) and Zentrum fr Angewandte Proteomik (ZAP), both located at BioMedizinZentrumDortmund (BMZ). Scientific partners are MPI, Medizinisches Proteom-Center (MPC) as part of Ruhr-Universitt Bochum (RUB) and Technische Universitt Dortmund (TUDo). LIP is to be understood as an interdisciplinary centre of excellence between science and industry and works in pre-competitive research and development, focussed on the optimisation of methods in target retrieval for new drugs. This leads to a reduction of the developmental period. The results of LIPs research will end in an optimisation both at the beginning of the value chain and at the end of the value chain, e.g. by biocatalysis. The funding is cofinanced by the European Union (European Fonds for regional development) and the state of Northrhine-Westfalia. Target of Lebenswissenschaftliche Innovationsplattform was to create pre-competitive research and development in the field of pharmaceutical diagnosis and therapy. The work was focused on strengthening the regional LifeScience Cluster promotion of innovation increasing the knowledge transfer between science and industry interdisciplinary exchange increasing scientific excellence Based on those targets TechnologieZentrumDortmund Management GmbH (TZM) was concerned with project management of Zentrum fr Angewandte Chemische Genomik and Zentrum fr Angewandte Proteomik. TZM GmbH was the virtual interface between academic project partners and potential application partners from industry, public and governmental sponsors as co-ordinator and administrator in the above mentioned fields. As one major task a communication- and transfer-platform was set up in order to enhance the contacts between academia and industry and among the academic partners. Parts of this infrastructure were regular jour fixe between all partners and later on between the sister centres, coachings of the academic partners in the fields of business administration, soft skills and intellectual property. Apart from this, internal communication, the external communication was rather important to transfer both, the idea and the results to industry and public. Visits of fairs, meetings and workshops had to be organised and carried out. Results were presented on a semestral event Science2Business (Spring and Fall) and a new biotechnological network was established. A couple of articles were published in well known journals. Public relation were replanished with precise marketing elements. All this leads to a unique platform with local co-operation partners and whitespread contacts all over the world. Dr. Petra Grnewald and Dr. Klaus F. Rammert, co-ordination of ZAP and ZACG resume: Our tasks were about to join a puzzle. It was a pleasure to see this excellence grow and to link all this together.

ZACG concentrates on target-orientated synthesis of small molecules driven by mostly natural materials. Those new targets are collected in substance libraries and will be performed into new active agents and into new innovative drugs. So, ZACG works on new antibiotics, which selectively destroy signal pathways of micro-organisms, so that resistances will become possibly avoidable. Furthermore, ZACG focuses on micro-structural methods to develop new biocatalytic hybrid systems from DNA-nucleotides and proteins especially in redox-systems. A smooth scale-up process will result from those researches in order to ease clinical studies. New nanostructural scaffolding methods will clarify the interaction between drug and organism directly in the cell. Later on, nano-chips, that are coated with bimolecular functional groups, could set free minimal doses of drug remoted by an external signal. Apart from this, ZACG is concerned about structure and dynamics of protein-protein and protein-membrane interactions in order to understand protein aggregations and misfoldings. These topics are related to several diseases, like diabetes mellitus II, Alzheimer disease, dementia and Parkinson disease.

Prof. Dr. Christof M. Niemeyer et al.

Nanobiotechnology Systems for Research in Active Agents


Biomolecular Hybrid Systems This project is concerned with the develpment of biomolecular hybrid systems, based on protein classes which are suitable for use as biocatalysts for drug synthesis. As promising candidates, heme redox-enzymes, in particular, monooxygenases, were chosen to be refined into addressable catalytes by conjugation with nucleic acids. A catalyst-library with more than 20 different enzyme-DNA conjugates was produced and kinetically and electrochemically characterized using, for instance, microelectrode arrays.[1] The potential for metabolization of these hybrids and their native enzyme analogues was tested using a small molecule library consisting of 30 drug candidates. In the course of this project, a new fluorescence-based screening method allowing highthroughput detection of new metabolic activities of P450 enzymes was developed. Addressable Micro- and Nanoarrays New lateral biostructuring methods for the develpment of high-quality protein- and small-molecule drug-arrays were developed. These arrays are being used in the screening of (i) chemical leads for drugs and (ii) biomolecular hybrid catalysts. In cooperation with the group of Prof. Waldmann several bioorthogonal coupling techniques for array-production were devised. In particular, the Orthogonal Chemical Lithography (OCL) for biofunctionalizing chemically activated Si-wafers was proven to be well-suited for directed photolithographic immobilization of various biomolecules.[2] The high quality the array platform was documented by mapping the biological activity of protein tyrosine phosphatases (PTP) using phosphopeptide microarrays.[3] Other studies in this project were concerned with the production of ultraflat substrates for protein arrays and the design of adressable DNA nanoarrays built through self-assembly of planar DNA tile motifs. Besides progress made directly on these two fields, a fundamentally new method based on Frster resonance energy transfer (FRET) for the complete thermodynamic characterization of the self-assembly process was developed.[4] Further, immobilization of proteins on glass and nanoparticle surfaces via DNA oligomers was proven to be reversible and applied to the selective adhesion of mammalian cells on planar glass surfaces, thereby opening up new avenues for phenotype-based screening assays.[5] Multifunctional Nanoprobes and carriers The modification of nanoscopic polymer-, metal-, and semiconductor-particles with biomolecular functional groups, in particular nucleic acids, peptides, and proteins, allows one for the utilization of such particles as reporters for the analysis of pharmacologically relevant processes inside the cell, such as the interaction of pharmacologically active reagents with intracellular target structures (proteins, organelles). In this context, lipid vesicle-stabilized CdSe-ZnS nanoparticles which remain stable in the intracellular medium were developed. Supramolecular FRET donor complexes consisting of fluorescent proteins and CdSe-ZnS particles have shown extraordinary fluorescence qualities allowing for far-reaching energy transfer processes.[6] In regard to refining nanoprobes such that reactions can be activated selectively inside the cell by an external stimulus, nanoparticle-induced generation of radicals was used to trigger reactivity of peroxidases and monooxygenases.[7] A cytochrome P450-based catalyst system was shown to be enzymatically active, stable and selectively addressable by irradiation with UV light.

Selected Publications [1] [2] [3] [4] [5] [6] [7] L. Fruk, J. Mller, G. Weber, A. Narvez, E. Domnguez, C. M. Niemeyer, Chem. Eur. J. 2007, 13, 5223. P. Jonkheijm, D. Weinrich, M. Khn, H. Engelkamp, P. C. M. Christianen, J. Kuhlmann, J. C. Maan, D. Nsse, H. Schroeder, R. Wacker, R. Breinbauer, C. M. Niemeyer, H. Waldmann, Angew. Chem. Int. Ed. 2008, 47, in press. M. Khn, M. Gutierrez-Rodriguez, P. Jonkheijm, S. Wetzel, R. Wacker, H. Schroeder, H. Prinz, C. M. Niemeyer, R. Breinbauer, S. E. Szedlacsek, H. Waldmann, Angew. Chem. Int. Ed. 2007, 46, 77007703. B. Sacca, R. Meyer, H. Schroeder, U. Feldkamp, C. M. Niemeyer, Angew. Chem. Int. Ed. 2008, 47, 2135. H. Schroeder, B. Ellinger, C. F. W. Becker, H. Waldmann, C. M. Niemeyer, Angew. Chem. Int. Ed. 2007, 46, 4180. H. Lu, O. Schoeps, U. Woggon, C. M. Niemeyer, J. Am. Chem. Soc. 2008, 130, 4815. L. Fruk, V. Rajendran, M. Spengler, C. M. Niemeyer, ChemBioChem 2007, 8, 2195.

Prof. Dr. Andreas Schmid et al.

Chemical Biotechnology of Pharmaceutical Compounds


Introduction Biocatalysis developed into an ecologically and economically interesting approach for the production of fine chemicals [Schmid et al, 2001, Nature]. Biotechnological processes are usually established very specifically for defined products and the technologies are typically not easily transferred to other reactions. Nevertheless, some progress has been made mainly concerning process development, but also biocatalyst optimization. In our work, we aimed at a technology platform by combining and optimizing different components such as host strain, expression system, enzyme, or reactor system, to broaden the applicability of biocatalysts to various reactions (kit-principle). Experimental Approaches To understand whole-cell biocatalysts, a systems oriented approach was followed including tools like genome-wide stoichiometric models and 13C-based metabolic flux analysis. Biocatalyst selection and engineering approaches focused on the expression system and the reaction enzyme as well as on carbon and energy metabolism. In parallel, reaction concepts were developed for the application of whole cells and isolated enzymes, focusing on the design of bioprocesses in a macro or micro scale. Main achievements In order to achieve specific carbon oxyfunctionalizations, biocatalysts and processes were constructed based on different oxidoreductases (P450s, Flavoenzymes, Molybdoenzymes) in recombinant microorganisms (E. coli, P.putida, S.cerevisiae). For analysis of the metabolic capacity of recombinant P.putida biocatalysts, a genome scale model was developed. Alternatively to suspended cells, continuous biotransformations based on biofilms were established. For analyzing the smallest catalytic unit, the single cell, a new bioreactor concept, the Envirostat, was developed. The necessary micro to macro interface for the microfluidic chip won the first price in the ideas competition G-DUR (Fig.1). Regarding isolated enzymes, an electroenzymatic cell was designed for the application of NAD(P)H or FADH dependent oxidoreductases. These concepts enable efficient cofactor recycling and allow the application of two-liquid phase systems and immobilization if desired. Outlook / Follow up projects This ZACG project paved the way for follow up projects in the field of biocatalyst and bioprocess design financed by the EU (Oxygreen in FP7) and CLIB/BMBF (Carboxyfun). Further project applications are in progress.

Fig. 1 New micro to macro interface for microfluidic chips. The design and beta-version won the 1st price of the ideas competition G-DUR (www.g-dur-online.de/aktuelles_ denk_dich_reich.htm).

Prof. Dr. Herbert Waldmann et al.

Substance Libraries in Drug Discovery


Innovation through Anti-Infective Research Antibiotics have increased the average human life expectancy by at least 10 years. However, only very few fundamentally new antiinfective agents have been introduced during the last 40 years. Now serious threats through rapidly evolving bacterial strains need to be countered to prepare against future plagues. Additionally, research on bacteria often leads to new insights into more complex functions of the human cell not by simple comparison, but mostly by indirect reasoning and functional inference. Several projects have been successfully developed. Efforts concentrated on the chemical synthesis of novel promising candidates for antiinfective research. New access means to Biphenomycin B, Brunsvicamide A, Stevastelin B, Chondramide C, as well as their derivatization and modification have been devised, which allowed their biological testing. For the Biphenomycins a genuine set of design rules was established which will allow tailoring these compounds to their molecular target. Brunsvicamides and the Stevastelins were proven to be promising phosphatase inhibitors. A thorough antibacterial screening method was implemented and allowed uncovering new lead compounds from in-house compound collections, and a new high-throughput screening method for inhibitors of the bacterial ribosome was developed. During these investigations two additional discoveries were made. Firstly, by a skillful combination of biology and computational modeling techniques an atomic model for actin-stabilizing small molecules was developed. Secondly, lipid transferase inhibitors were discovered which show promising new selectivity within this widely important enzyme class, and X-Ray crystal structures of these could be obtained in house. Both discoveries now provide a solid basis for structure-based drug design. Natural Compound based Substance Libraries Natural products have influenced the development of chemical treatments of diseases or other pathological misconditions since ancient times and are still one of the major driving forces behind the invention of novel pharmaceuticals. Their importance for modern drug discovery stems from their inherent biological activity which nature has encoded in their molecular structures. Consequently, natural products have often been used as valuable starting points for the development of novel pharmaceuticals. With the advent of high-throughput and phenotypic screening, a need for high-quality compound collections (i.e. compound collections with a high chance of bioactivity) for initial screening applications arose. To obtain these compound libraries, we have developed Biology-Oriented Synthesis (BIOS) in which the underlying frameworks of natural products are employed as scaffolds for compound library synthesis. Using this concept, we have synthesized several natural product like compound libraries such as tetramic acids, ,-unsatured -lactones, tetrahydropyranes, [5.5] spiroketals, oxepanes, and -pyrones. Moreover, we have extended this concept to the synthesis of non-natural hybrid structures and natural product inspired oxygen and nitrogen heterocycles, resulting in the generation of a high-quality small molecule library as a basis for the development of novel pharmaceuticals. Assay Development for Drug Discovery We develop biochemical and cell-based assays and perform screens for automatic testing of small molecules as potential drug candidates. Using liquid handling robots we identify potential drug candidates from our in house library (approx. 32.000 compounds) in high throughput biochemical assays. A cell-based screen for substances that affect cell cycle or cytoskeleton is done in cells stained for chromatin and the

actin-microtubule-cytoskeleton. Images are acquired on an automated microscope in a 384-well format and analyzed for abnormalities. The Ras-MAPK pathway is an evolutionary conserved cascade that is involved in the control of many cellular processes e.g. cell proliferation, differentiation and apoptosis. Ras proteins and downstream effectors are mutated in many cancers. Therefore compounds that interfere in this pathway are of high interest. Two different screens are employed here: One determines the activity of the EGF induced MAPK pathway in the presence of different small molecules by indirect measuring of the expression levels of a luciferase reportergene. The second screen monitors the reversal of H-Ras transformed MDCK-fibroblasts from spindle-shaped cells to a normal phenotype characterized by adherent, rounded cells. Both screens identified small molecules that will be subjected to target identification. One approach towards target identification of small molecules is the analysis of phosphorylation states of key proteins in the Ras pathway. Other efforts for target identification and target validation will include the use of RNAi, three hybrid systems and chemical proteomics. So far, we could identify the potential targets of a biotinylated small molecule. Other active candidates identified in the Ras pathway screens are on their way to target identification. Modulation of Protein-Protein Interactions Protein-protein interactions lie at the heart of almost all cellular processes. Life-science research devotes much of its efforts to the identification and understanding of these protein-protein interactions. Disrupting or enabling proteinprotein interactions represents one of the most challenging subjects for life-science researchers today and this has resulted in an increased focus of both academic and industrial groups on this research area. Fascinating results and breakthroughs have been achieved the past years and the field is expanding. This increase in attention is necessary, as most of the aspects of protein-protein interactions are still not clarified. Increasingly it is becoming clear that many, if not most, diseases (cancer, Alzheimer, virus infections etc.) result from

a malfunctioning in protein-protein interactions. Molecules modulating these interactions offer entries for inhibiting undesired interactions or restoring proper functioning.

Innovative chemo- and bioinformatic Methods for Drug Discovery Turning the massive amount of data from high throughput initiatives into knowledge is a key challenge in life sciences and modern drug development. Within this project we analyzed large databases of bioactive compounds, i.e. natural products, to find promising, new molecular starting points for drug design. With a collaboration partner from the informatics department of the TU Dortmund, we developed an interactive tool to analyze chemical databases. This tool enables chemists to intuitively extract knowledge such as structure activity relationships from large amounts of complex chemical and biological data and thus to better direct further chemistry initiatives. In a second approach we grouped structurally similar proteins whose function may also be modulated by similar molecules. We hypothesized, that this approach will assist a) the identification of new protein targets b) the identification or novel starting points for inhibitor design and c) help to elucidate possible cross-inhibition activities causing unwanted off-target effects. The experimental application of these computational methods could be successfully validated by the study of lactones as inhibitors of an enzyme cluster based on lipases and phosphatases. Based on an additional cluster, formed by the mammalian protein kinase p56lck and the prokaryotic DD-Ligase, ATP competitive kinase inhibitors could be identified as inhibitors of DD-Ligase, a potential target protein for new antibiotics. Parts of our results have been published early in 2008. Additional publications will follow in due course. All our results thus be available for public use, also by small and medium sized companies which lack the resources for own efforts in this field.

Prof. Dr. Roland Winter

Towards an Understanding of Diabetes Mellitus Type II Strategies for Inhibition of Protein Aggregation and Amyloid Formation
Protein folding is one of the most crucial steps during the lifetime of a protein. If some failure occurs in achieving the native conformation, this will render the polypeptide totally inactive, or even worse, it can produce a misfolded molecule that can interfere or block components of the cellular machinery to the point of causing cell malfunction or even death. In recent years, it has become evident that a wide range of human diseases are associated with aberrations in the folding process. These diseases, which are also coined protein conformational diseases, include Alzheimers disease, Parkinsons disease, prion protein related encephalopathies, and type II diabetes mellitus (DMII). In our project, a series of biophysical techniques have been employed to unravel unknown structural and kinetic aspects of the aggregation and fibrillation process of amyloidogenic proteins, such as insulin, the prion protein, and IAPP (islet amyloid polypeptide). Using cell viability assays, the results were subsequently correlated with the cytotoxicity of the species that form in the various aggregation pathways. For example, in the case of insulin, beta-sheet-rich insulin assemblies of both fibrillar and amorphous nature have been shown to be toxic to pancreatic beta-cells. One particular focus of our project was on revealing the mechanism of the fibrillation reaction of IAPP, which is cosecreted with insulin and responsible for DMII. The disease is characterized by the deposition of amyloid plaques in pancreatic beta cells. In the assembly process, the monomeric peptide undergoes intramolecular and intermolecular conformational changes to form oligomers, protofibrils and finally mature fibrils. By NMR and MD computer simulation methods, the structure of the monomeric protein was solved. In a further step, we focussed on the mechanism of IAPP aggregation under various solution conditions. We could show that the presence of lipid membranes fosters the fibrillation reaction of the protein and drastically influences its aggregation mechanism. Time-lapse tapping mode atomic force microscopy revealed membrane disruption due to the interaction of IAPP oligomers with lipid bilayers, apparently through

Figure 1: The pathological aggregation of IAPP, which leads to type II-diabetes mellitus, is effectively inhibited by small-molecule rhodanine-based inhibitors at nanomolar concentrations.

an unspecific, detergent-like effect. Such IAPP-disruptive membrane interaction could contribute to the pathogenesis of type II diabetes mellitus. Inhibition of amyloid fibril formation is considered a key prospect therapeutic approach towards diabetes and other amyloid-related diseases. In collaboration with Prof. Waldmann, small molecule inhibitors of IAPP amyloid formation were found and evaluated. These inhibitors, which are based on a rhodanine scaffold, are of particular interest because of their biocompatibility and negligible cytotoxicity at the concentrations use (Fig. 1). To avoid the tendency of protein-based pharmaceuticals, such as insulin, to aggregate is also important for their production, storage and delivery. Since they are subjected to a variety of chemical and mechanical stresses, the investigation of protein stability is of primary importance for manufacturers and regulatory agencies. Hence, a further focus of our studies was to develop strategies for stabilizing aggregationprone protein solutions. Furthermore, in collaboration with TF Instruments GmbH, we explored and validated a new technique, based on ultrasonic resonator technology, for online monitoring of aggregation and fibril formation of proteins in industrial production plants.

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ZAP is focussed on the development of new techniques in the fields of proteomics, protein-biochips, glycoanalytics, bioinformatics and -statistics. New procedures of label-free quantitative proteome analysis are intended to lead to time- and cost savings, enabling companies to carry out far more targeted proteome analyses than has so far been possible. The interdisciplinary exchange provides a self-contained research and development approach that enables companies to engage in rapid development to marketability of the findings gained. The aim was to understand the correlation between protein function and a certain disease. In order to generate detailed protein profiles, ZAP develops new platform technologies consisting of links between two dimensional gel electrophoresis and mass spectroscopy. This leads to a standardized method of quantitative label free and reproducible proteome analysis and so to time and cost savings. Biostatistic and bioinformatic methods will help to analyse high data volumes. Later, new biochips will result from that research and development. Within ZAP, scientists develop new strategies in 5 subprojects as quantitative proteomics, difference in gel electrophoresis (DIGE), glycoproteomics, protein biochips und bioinformatics. The Medizinische ProteomCenter (MPC), Ruhr-Universitt Bochum, as well as the Institutes of Biostatistics (Prof. Katja Ickstadt, Prof. Jrg Rahnenfhrer) und Bioinformatics (Prof. Petra Mutzel, Prof. Bernhard Steffen), all Technische Universitt Dortmund, care of the scientific part. ZAP successfully transferred academic ideas into industrial applications. It provides a suitable infrastructure as well as potential academic and industrial partners, respectively, thus growing to a cluster of applied biotechnology.

Prof. Dr. Helmut E. Meyer et al.

Aims and Perspectives of ZAP Subprojects


One corner stone of the Lebenswissenschaftliche Innovationsplattform is the Zentrum fr Angewandte Proteomik (ZAP). Within ZAP, scientists develop new strategies in the 5 subprojects 1. 2. 3. 4. 5. Quantitative Proteomics Difference in Gel Electrophoresis (DIGE) Glycoproteomics Protein Biochips Biostatistics and Bioinformatics The Medizinische Proteom-Center (Prof. Helmut E. Meyer), Ruhr-Universitt Bochum, as well as the Institutes of Biostatistics (Prof. Katja Ickstadt, Prof. Jrg Rahnenfhrer) and Bioinformatics (Prof. Petra Mutzel, Prof. Bernhard Steffen), all Technische Universitt Dortmund, care of the scientific part. ZAP aims at transferring academic ideas into industrial applications. It provides a suitable infrastructure as well as potential academic and industrial partners, respectively, thus growing to a cluster of applied biotechnology. In the following, the aims and perspectives of the different subprojects will be highlighted.

Quantitative Proteomics
Quantitative Proteomics with Stable Isotope Labeling Mass spectrometry (MS)-based proteomics is a powerful tool for the identification of (sub)cellular proteomes, for the characterization of posttranslational modifications in proteins as well as for the mapping of functional protein complexes and the establishment of protein networks. In recent years, increasing efforts have been made to develop efficient MSbased methodologies for comparative, relative quantitative proteomics. Applying such quantitative methods, proteomics researchers aim at determining those proteins that undergo changes in abundance in, for example, healthy tissue versus disease tissue, which additionally offers them the possibility to place distinct proteins into a functional context. MS-based quantification of proteins is facilitated by chemical labeling of functional groups in proteins/peptides with stable isotope reagents. This labeling approach can be universally applied to all kinds of samples, including tissue samples from higher organisms. In order to minimize errors due to different sample processing, differentially isotope labeled samples are directly combined in equal ratio and jointly processed for MS analysis. The aim of this project was to develop a new chemical labeling reagent which is universally applicable to MS-based relative quantitative proteomics. The ICPT (Isotope-Coded Protein Tag) reagent developed in this project contains an N-hydroxy succinimide ester that covalently modifies the free amino groups in proteins/peptides; a light version and as heavy version containing 5x 13C atoms of ICPT are available (s. Fig. 1).

Figure 1: The 12C / 13C5-coded ICPT reagent for relative quantitative MS analysis.

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Prof. Dr. Helmut E. Meyer et al.

Aims and Perspectives of ZAP Subprojects

Figure 2: Standard workflow for relative quantitative proteomics analysis using ICPT.

A standard workflow was developed as depicted in Figure2. For relative quantitative analysis of two distinct cellular proteomes, the respective protein extracts are differentially labeled with light/heavy ICPT, combined in equal ratio, then jointly separated by SDS-PAGE, subjected to in-gel proteolytic digestion followed by peptide analysis using nano-reversed phase chromatography coupled to MS/MS combined with bioinformatics. Proteins or peptides differentially labeled by ICPT exhibit no differences in electrophoretic and chromatographic separation, thereby facilitating accurate MS-based protein quantification. ICPT-coded peptides show a characteristic mass shift of 5 Da in mass spectra; relative quantification of proteins is based on peak areas of the respective peptide pairs. Major advantages of ICPT are that quantitative proteomics experiments can be performed on all common types of MS instrumentation, such as ion traps and quadrupole time-of-flight instruments and that it is compatible with 1-/2-D gel electrophoresis of proteins. Furthermore, ICPT offers the unique ability to uncouple MS-based quantification and MS/MS-based identification of proteins, thereby providing high flexibility in data acquisition and the ability to significantly reduce the amount of MS/MS data. Label free proteomics

focused on the development of a new label free technique allowing fast and high parallel quantification of proteins for comprehensive proteome analysis by targeted Liquid Chromatography Mass Spectrometry (LC-MS(MS/MS)) analysis. We optimized the several steps of the label free approach integrating sample preparation, protein digest, LC-MS measurements, statistical data analysis and protein identification by LC-MSMS (Figure 3).

Figure 3:

Comprehensive quantification of specific changes in biological systems in response of a certain treatment or perturbation is one of the most important tasks in proteomics but also one of the most challenging ones. For this purpose we

Workflow for label free comprehensive proteome analysis including sample preparation, protein digest, LC-MS analyses, statistical quantification, identification by LC-MSMS and database search.

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After evaluation of this new technique in respect of dynamic range, reproducibility, sensitivity and accuracy of quantification, the comprehensive proteome analysis of complex samples has been performed. We analyzed human lung carcinoma cells A549 treated and not treated with TGF-beta for the quantification. For the determination of significant expression changes, 10 biological replicates with 500 ng per sample were analyzed by detecting, matching and quantifying approximately 12.000 peptides per measurement. Statistical analysis revealed differentially regulated peptides which were identified in subsequent targeted MS/ MS runs. Altogether 371 differential peptides were assigned to 117 different protein sequences. These results have been compared with results obtained by the DIGE technique where the same carcinoma cell line A549 was analyzed (see below). The comparison revealed 16 proteins which have been identified in both experiments. However, most of the proteins were found only in the label free approach, indicating that the

performance of this new technique is very promising in the coverage of cellular changes caused by a certain treatment. Moreover, label free proteomics has the potential to become an essential complement to current quantification methods, as its throughput capabilities allow processing large numbers of heterogeneous biological samples and obtaining statistically valid quantification data. The label free technique offers a great applicability for industrial as well as clinical questions. Thousands of proteins can be quantitatively monitored helping to control, to optimize or to understand biotechnological procedures. Industrial processes as e.g. biocatalysis, fermentation or production of chemicals could be run with increased efficiency and cost reduction. Furthermore, our label free proteomics approach is an effective tool for several clinical applications allowing identification of proteins involved in pathological processes. Proteome analyses reveal patient relevant information, e.g. disease staging or treatment progression and success.

Difference in Gel Electrophoresis (DIGE)


General use of DIGE technology in differential proteome analysis During the last years a new 2D-Gel based technology has been developed in order to analyze proteomes of cells, tissues and body fluids. The so called DIGE (Differential in Gel Electrophoresis) technology allows multiplexing of up to 3 samples in one gel by using different fluorescent dyes. This leads to an improved spot detection, quantification and reproducibility. During the project period this method has been established and optimized and can be used for the differential analysis of proteomes. Establishment of the DIGE technology The compatibility of different types of tissues und cells is an important factor for the successful analysis of proteomes. During the project period a total of 23 different cell types and 10 different human tissues were tested for compatibility. Furthermore sample preparation procedures for different samples such as liquor, plasma and muscle tissue could be established. Beside the sample preparation, protein labeling proved to be another critical step in DIGE experiments. Factors influencing the labeling efficiency and behavior have been detected. In addition, different fluorescent dyes from several manufacturers have been checked in IEF/SDS 2D-gel experiments. Furthermore standard operating procedures for DIGE experiment also for small sample amounts (1-5 g of protein) have been set up to ensure the reproducibility of the method.

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Prof. Dr. Helmut E. Meyer et al.

Aims and Perspectives of ZAP Subprojects


Software analysis of image data and statistical analysis An important factor concerning DIGE studies is the image analysis of the acquired gel images. For this purpose several commercial available software solutions are available. Four different programs have been compared within the project. The application of more than one image analysis software leads to partially complementary results based on the used algorithms for spot detection, matching and volume quantitation. A large study including 75 gel pictures has been set up comparing two different states of the cell line A459. This data set has been used for extensive statistical analysis. It could be shown that the spot volume data is normal distributed, which is the requirement for the application of several advanced statistical methods. Confidence intervals have been introduced as a criterion that a protein spot is differential between two groups of samples. In addition several methods to handle missing values have been compared. The acquired results and established statistical methods could be incorporated in the software Statistical DIGE Analyzer. This platform independent JAVA applet has been developed for the statistical analysis of DIGE data subsequent to the gel-image analysis. Proteome studies In order to show the feasibility of using DIGE for a wide range of applications, several proteome studies have been carried out. Different clinical samples such as microparticles isolated from plasma of patients suffering from heart attack, liquor from huntington patients and colon-cancer tissue could be analyzed successfully. DIGE performed also very well on model systems: a mouse model of leukemia and a cell model of lung fibrosis were successfully analyzed. Conclusion Reproducible DIGE experiments can be used as a valuable platform technology. This technology can be applied for several applications like biomarker discovery and proteome wide monitoring of drug effects. During the ZAP project more than hundred biomarker candidate proteins for different diseases have been identified. These valuable pools of proteins are now available for further evaluation experiments. The discovered proteins might be used for clinical applications like early detection of cancer or the monitoring of disease states.

Glycoproteomics
Project 3 evolves methods for the quantitative and qualitative analysis of glycosylated proteins. The applied techniques were designed for the characterization of recombinant proteins like therapeutic antibodies, but they are also applicable for differential analysis in order to identify glycan biomarkers for a specific disease. The precise analysis of glycosylated proteins includes the application of versatile methods. Whereas below two of the most important strategies attractive for the economic and scientific use are described: the determination of the composition and structure of the glycan as well as its localisation in the amino acid sequence of the protein (figure 4). The former

Figure 4: Workflow for the analysis of glycosylated proteins. We established two different strategies for (A) the identification of the glycan structure and composition of a single glycoprotein or a glycoprotein mixture and for (B) a direct identification of the glycosylation sites.

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is achieved by the release of the sugar moiety by PNGase F treatment in the case of N-glycosylation. Furthermore, glycans were labeled with 2-amino benzamide (5AB) to allow for a sensitive detection by nanoLC-ESI-MS/MS. This mass spectrometry based strategy enabled a detailed characterisation of glycans differing in structure and origin, like e.g. biantennary sugars of the complex type from human serum transferrin (figure 5A). The compositional information about the glycans was the basis for a second strategy the localization of the glycosylation site. Therefore, a characteristic of the mass spectrometric fragmentation by collision induced dissociation (CID) was used, namely that glycosidic bondages are more fragile than peptide bondages. This phenomenon leads to the predominance of fragment ion masses explaining neutral losses of the sugar moiety of the glycopeptide. Some of these masses were prominent in fragment ion spectra of glycans as well as glycopeptides and therefore can act as reporter ions for the detection of peptides containing a glycan moiety (figure 5B). Furthermore, the established methods were successfully applied for the identification of phosphorylation sites in several proteins, e.g. in the protein fetuin which was also used for the identification of biantennary and triantennary complex type sugars in glycopeptides.

Figure 5: (A) CID-MS/MS spectrum of the precursor ion m/z = 1172,4 Da of a human serum transferrin glycan after PNGase F release and 2-amino benzamide (2-AB) labeling. All signals could be allocated to parts of the biantennary sugars of the complex type in human serum transferrin. (B) CID-MS/MS spectrum of the precursor ion m/z = 1152,3 Da of a trypsin released human serum transferrin glycopeptide (AA 622 to 642). The fragment ions m/z = 366.1 Da, m/z = 528.1 Da and m/z = 657.2 Da are typical protonated fragments of this complex type glycan. They were used as reporter ions to extract glycopeptides from nanoLC-ESI-MS/MS data.

Abstracting the described results ZAP was able to develop easy to use and highly accurate methods for the comprehensive characterisation of recombinant and tissue extracted glycoproteins.

More information can be found in Klare N, Hamacher M, Marcus K, Grnewald P, van Hall A. und Meyer HE. Die komplexe Welt der Zuckerreste Glykoproteomics im stlichen Ruhr gebiet BioTec. 2007 Nr. 3, Mai/Juni , 1213. Klare N, Grnewald P, Marcus K, Meyer HE, van Hall A. und Hamacher M. Zucker nutzbar machen moderne Proteomforschung im Fokus Laborwelt. Nr. 2/2007, 2929.

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Prof. Dr. Helmut E. Meyer et al.

Aims and Perspectives of ZAP Subprojects Protein Biochips


Chip Technologies Microarray technologies are very useful to analyse genomes and gene expression profiles. However, recently protein microarrays are gaining in importance, since the function of genes is finally realized throughout proteins. For the production of valuable protein biochips it is necessary to have a rich diversity of proteins in large quantities. To generate such proteins frequently cDNA expression libraries are used that allow the expression and purification of a large number of different proteins. For this project we used the hEX1 library, which is constructed from human fetal brain mRNAs and contains the largest diversity of recombinant human proteins currently available for binding assays. During the period of this project we applied the protein technology to two typical applications, namely the characterization of purified antibodies and disease diagnosis based on autoimmune antibody profiles. Characterization of Antibodies using Protein Microarrays
It is of great importance that specificity and reactivity of antibodies used for diagnosis or therapy are well characterized. Thus, the determination of these parameters is of considerable scientific interest as well as economically important. The binding profile of several antibodies from the Swedish human protein atlas project has been investigated using protein biochips, which contained approx. 400 different proteins from the hEX1 library and in addition different amounts of the primary antigen.

The following diagram shows that binding intensity of two antibodies increases almost linearly with the amount of spotted antigen. All antibodies exhibited a high specificity, since they only displayed weak cross reactivities with a small number (<5) of the other 400 antigens on the protein biochip (data not shown).

Figure 6 Reactivities of two different antibodies against Vimentin and PDIA3 with their primary antigens, depending on the amount of spotted antigen.

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Parkinsons Disease Diagnosis based on Protein Microarrays Antibodies against human proteins (autoantibodies) occur naturally in the serum of healthy individuals without causing pathological problems. During autoimmune diseases and various types of cancer the autoantibody profile changes as a consequence of alterations of the immune system. Even for neurodegenerative diseases, such as Alzheimer or Parkinson, the generation of disease specific autoantibodies has been discussed. This opens the way to use protein biochips as diagnostic tool for different diseases. We used 20 sera of Parkinson patients and 16 sera of healthy individuals to test whether differences of the autoimmune repertoire exist between the two groups. Using supervised learning algorithms (support vector machines) we were able to successful recognise patients suffering from Parkinsons disease. This preliminary study shows that the achieved sensitivity and specificity of over 90% (Fig.7) is very promising. We propose that our results are suitable to generate a diagnostic chip for Parkinsons disease.

Figure 7 Performance of the established support vector machine (SVM). SVM were used to classify data sets of 20 Parkinsons disease patients and 16 healthy controls. A high sensitivity and specificity of over 90 % was obtained indicating that Parkinson disease is well suited for Biochip based diagnosis.

Conclusions Protein biochips are an emerging technology with great potential for the characterization of therapeutic and diagnostic antibodies as well as for diagnosis of different diseases based on serum samples.

Development of a homologous Expression System The proteins of the hEX1 expression library are currently produced in a heterologous prokaryotic system (E. coli). This system does not support posttranslational modifications of proteins which are normally found in eukaryotes and are essential for biological function. To further improve the quality of our proteins, we have established an expression system using human cell lines. As first step towards such a system we tested the properties of forty proteins. We have successfully expressed these proteins in human embryonic kidney cells (HEK293A) and optimized culture conditions as to maximize protein synthesis.

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Prof. Dr. Petra Mutzel, Prof. Dr. Katja Ickstadt, Prof. Dr. Bernhard Steffen, Prof. Dr. Wolfgang Urfer (em.), Prof. Dr. Jrg Rahnenfhrer et al.

Aims and Perspectives of ZAP Subprojects Biostatistics and Bioinformatics


Overview Profound and optimized research in proteomics relies on adapting methods from biostatistics and bioinformatics. Subproject 5 was the connecting element of the ZAP concept, supporting the other subprojects. Researchers from biostatistics and bioinformatics developed models and algorithms for quantitative proteomics and for glycoanalytics, elaborated statistical validation methods for the DIGE system, generated software tools for analyzing protein biochips and optimized the Data Collection Center of the HUPO BPP (see http:www.hbpp.org). Cluster Analysis and Classification for LC/MS Data and Protein- Protein Interactions Different pre-processing algorithms for LC/MS Data were evaluated and incorporated into the Bio-jETI platform of subproject 5.4. Cluster analysis was applied to LC/MS experiments for identifying clusters of differentially expressed peptides. A retention time alignment algorithm for LC/MS Data based on cluster analysis and nonlinear regression was developed in cooperation with subprojects 5.1 and 5.3, Protagen AG and Bruker GmbH. Cluster analysis and different multiple testing procedures were applied to protein biochip data.

Statistical Analysis of data from LC/MS, DIGE and Protein Biochips We performed various statistical analyses for proteomic data, with a focus on the development and evaluation of preprocessing methods for MS and DIGE data and the development and application of classification algorithms for MS and biochip data. A major achievement is a new algorithm for alignment, binning and normalization of LC/MS data that was devolped in cooperation with the other subprojects. Moreover, a new genetic algorithm for classification was proven to be competitive to existing methods. Currently, a method for combining peptide information for the whole protein is being developed.

Algorithm Engineering for LC based Proteomics approaches Our primary goal was to validate and extend existing as well as to develop new algorithms for the application in quantitative proteomics and to protein biochips. We developed and implemented high-throughput quantitative data analysis algorithms for a chemical labeling as well as a label-free approach to high-throughput quantitative proteomics without the need for proteolysis. In addition, statistical preprocessing as well as clustering algorithms for the identification of protein-protein interactions were developed.

Software Engineering for LC based Proteomics approaches We developed Bio-jETI, a graphical and service-oriented framework for orchestrating biological processes. The technology was used to integrate LC/MS analysis algorithms based on R in cooperation with subprojects 5.1 and 5.2 and to postprocess data earned from the Decyder M/S analysis software. These approaches were presented at different international fairs and conferences leading to publications and partnerships in both industry and research.

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Zentrum fr Systembiologie
The Zentrum fr Systembiologie (ZfS) was established in 2006 as an interdivisional project at the Max-Planck-Institut fr molekulare Physiologie. It is located at and coordinated by the Max-Planck-Institut fr molekulare Physiologie. The scientific partners of the ZfS are the departments of Professors Alfred Wittinghofer, Philippe Bastiaens, Roger S. Goody und Herbert Waldmann. The Zentrum fr Systembiologie (ZfS) at the Max-Planck-Institut fr molekulare Physiologie is working on the analysis of biological networks and the development of methods aiding in this research. The resulting technological and scientific innovations are of major interest for companies of the biomedical or biotechnological sector and will therefore strengthen the local economy. Aim is also to distinguish Dortmund as a centre for biomedical sciences and development in Northrhine-Westfalia. The ZfS focuses on interdisciplinary research on complex biological systems. Modern technologies are used to investigate how cells or organisms function as holistic networks and why and how molecular defects can unbalance the systems equilibrium. These findings are prerequisites for understanding the formation of complex diseases (e.g. cancer) and of vital importance for drug development. The purpose of the centre is to generate new scientific findings and to promote efficient transfer of the scientific research into industry. The objective is to strengthen the regional life science cluster, to improve the general conditions for knowledge transfer and to advance interdisciplinary exchange. The increase in scientific excellence together with our focus on the applicability of the research raises the level of innovation. The focus of the centre lies in research with the capability of advancing new leading edge technologies and new products. The funding is cofinanced by the European Union (European Fonds for regional development) and the state of Northrhine-Westfalia. Apart from the two sister centres ZACG and ZAP, the ZfS is also strongly cooperating with several other local research centres and institutions and is well embedded into local and international networks of economic and scientific partners. The Zentrum fr Systembiologie is using state of the art technologies like space and time resolved fluorescence microscopy, fluorescence correlation spectroscopy, electron microscopy, X-ray structure analysis, optical tomography, mass spectrometry and bioinformatics in its research projects. It is organised into a total of ten independent equipment platforms for the analysis and automation of systems biology research.

Zentrum fr Systembiologie

Zentrum fr Systembiologie: Equipment platforms


Equipment platform 1: Fluorescence Life Time Imaging Microscopy for Cell Array Screening To understand how cells function and are able to communicate with their environment, the classical approach of focussing on a protein function or an interaction is no longer sufficient. To obtain all data necessary, high throughput screening methods are required. The ZfS is developing and establishing cell array screening procedures in which mammalian cells are cultured on glass slide arrays. The cells are either treated with different chemical agents (substance search) or are driven out of equilibrium by targeting and disturbing cellular switch points of signal transduction (for instance through RNAi). This technology is combined with fluorescence lifetime imaging microscopy. The lifetime of a fluorophore describes how rapidly the fluorescence emission decays after an exciting light pulse. The decay rate is a specific characteristic of the fluorophore and its microenvironment (binding state, pH-value, temperature, solvent and many other factors). Because of its sensitivity to the local environment, FLIM technology is an important method for exploring the spatial and temporal parameters of cellular structures and function. FLIM screens are therefore able to determine how cells react to various internal or external cues. Amongst other projects we apply this method to study the influence of growth factors on cellular signal transduction. Growth factors are extracellular messengers that regulate cell growth and are of special significance for understanding pathological changes like cancer, which result in uninhibited cell growth. Equipment platform 2: Polychromatic Flow Cytometry A first step in the analysis of holistic networks is the description of the components. We analyse growth factor induced posttranslational modifications like phosphorylations in mammalian cells transfected with various fluorescent fusion proteins. Following expression the cells are exposed to varying stimuli. Localisation und extent of phosphorylations can be determined using fluorescence energy transfer (FRET) and fluorescence lifetime imaging (FLIM). Activation of GTPases following exchange of GDP to GTP can be analysed in a comparable approach using GTP-specific binding domains (GBDs). Quantification of these interactions and sorting of the cells for further analysis is done in vivo via fluorescence activated cell sorting analysis. This approach is extendable to the analysis of other modifications like ubiquitinylation, sumoylation or methylation.

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Zentrum fr Systembiologie: Equipment platforms


Equipment platform 3: High Resolution Mass Spectrometry Mass spectrometry is an extremely sensitive method for determining molecular mass. The molecules under analysis are first ionised in an ion source, transferred via an electric field into an analyser where they are separated according to their mass/charge relationship and then detected. To be able to calculate the mass, the ion charge must be known, which, however, is always a whole-number multiple of the elementary charge. Mass spectrometry has a multitude of applications in systems biology research. For instance, it allows the analysis of the composition of cellular protein complexes and thus has an important function for the identification of network components. This technology is also often used to investigate inducible protein modifications such as phosphorylation, which has great significance for the regulation of cellular signalling pathways. With high resolution mass spectrometry, a resolution can be achieved that allows the determination of the sum formula for small molecules. Equipment platform 4: Fluorescence (Cross) Correlation Microscopy This method uses fluctuations of the fluorescence intensity over time to gain information on concentrations, diffusion constants and interactions. It is therefore a powerful quantitative technique for monitoring molecular interactions. Using this method we could for example in cooperation with partners from the EMBL, Heidelberg, investigate signalling complexes at endogenous expression levels in the cytoplasm of single yeast cells. We could show that signal transduction in the pheromone pathway is not dependent on the formation of new complexes but rather on the spatial regulation of Fus3 MAP kinase activity. This is an important new insight into the regulation of cellular responses and shows that interfering with the formation of protein complexes is not always the optimal way of modulating cellular signalling.

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Zentrum fr Systembiologie

Equipment platform 5: Confocal and Widefield Microscopy This platform encompasses a whole range of fluorescence techniques. Apart from standard wide-field and confocal fluorescence microscopy, the platform enables us to use FRAP, FRET and TIRF in order to investigate how signalling networks process extracellular information thereby determining cellular phenotype. FRAP (Fluorescence Recovery After Photobleaching) is a method for the determination of molecule mobilities in living cells. The technique relies on fluorescently labelled molecules being bleached within a defined area of the cell (photo bleaching). Subsequently, it is observed how fluorescently labelled molecules from the surrounding areas enter the bleached region. The manner and speed with which this is occurring give important information about the mobility of the labelled molecules. From this data conclusions can be drawn about the function and regulation of cellular components within the biological system. TIRF stands for total internal reflection fluorescence and is a method suited for the analysis of processes in close proximity to the plasma membrane of cells. The basic principle is based on the fact that when laser light is totally reflected at the interface between coverslip and cell, the effects of the electromagnetic field reach several hundred nanometers deep into the cell. This electromagnetic field is able to excite those fluorophores lingering within its sphere of action. As a consequence of the limitation of this sphere to the proximity of the totally reflecting interface, a selective illumination of the sample in the vicinity of the plasma membrane results. In this way membrane-associated processes can be observed in every detail without interference from fluorescence out of the inner reaches of the cell. The method is of high impact because of the multitude of signalling processes occurring at membranes. FRET (fluorescence resonance energy transfer) is a highly sensitive method for measuring distances and interactions. The method is based on the principle that excitation of a fluorophore (donor) can non-radiatively induce the fluorescence activity of another fluorophore (acceptor) if donor and acceptor are in close vicinity of each other (Frster radius). The high sensitivity of the method combined with the spatial resolution provided by the high-end microscopes shows where within a cell the FRET-biosensor is active and gives valuable information about location, activity and dynamics of network components.

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Zentrum fr Systembiologie: Equipment platforms


Equipment platform 6: Cryo Electron Tomography Light microscopy can permit optical resolution of structures down to about 1 m. Smaller objects (e.g. internal structures of cell organelles, viruses, membranes, two-dimensional crystals, macromolecular complexes and individual molecules) can be investigated structurally with electron microscopy. One of the most precise techniques to correlate light and electron microscopy is the photooxidation of diaminobenzidine (DAB) through bleaching of fluorescent dyes. Radicals formed during bleaching of fluorophores oxidize DAB molecules to a specific electron dense precipitation, directly visible at electron microscopic level. The protocol was evolved to a very high sensitivity to detect even the low amounts of radicals formed during bleaching of green fluorescent protein (GFP). This novel method, originally considered as technological caveat, bridges directly the gap between light and electron microscopy for GFP. The fine DAB precipitation is sufficient for detection in electron tomograms allowing the analysis of pleomorphic objects and membrane systems in 3D. Usually, electron microscopic images are two-dimensional renderings of three-dimensional objects. This means that important information about the structure of a sample is missing. In cryo-electron tomography the sample is tilted stepwise. The EM images taken from different angles are then reconstructed into a 3D structure. Cryo-electron tomography of vitrified sections combines the potential of 3D imaging at molecular resolution with unperturbed structural preservation. The specimens are immobilized in vitreous ice by rapid freezing without use of any chemical fixatives or staining procedures. The thickness of mammalian/human cells prevents from direct analysis under electron beam, therefore 5070 nm sections are cut for cryoelectron observation, or 150250 nm sections for 3D analysis by cryo-electron tomography. With this approach, we studied microtubule substructures in mammalian cells. Equipment platform 7: Bioinformatics The complexity of biological systems and the quantity of data generated on our platforms make the bioinformatics platform a central element of the Centre of Systems Biology. Here the large quantities of data that are produced in laboratory experiments are analysed and archived. Computer-based models of the system under investigation are generated from the experimental data. Based on these models, new hypotheses about the behaviour and characteristics of the system can be formulated and tested anew in practical experiments. This iterative cycle of virtual and real experiments leads to a constant optimisation and expansion of the model, permitting a deeper understanding of the investigated system.

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Zentrum fr Systembiologie

Equipment platform 8: Protein Production The reconstruction of isolated biological function units to molecular networks is an important aspect of systems biology. This approach allows us to compare the structural, biochemical and biophysical characteristics of single components with those of the assembled networks. This way, we gain a deep insight into the regulation of self-organising processes. For the reconstruction of molecular networks, pure proteins have to be available in sufficient quantity. This is achieved by extracting the proteins from cells und purifying them. Since most cellular proteins exist only in relatively small numbers per cell, proteins are often produced in host cells, enabling more efficient and increased production. To accomplish this, first the respective gene sequence of the protein must be unravelled. Next, this is inserted into a vector which enables the transmission of the genetic information into new host cells. The host cells are often bacteria, but can also be yeast, insect or mammalian cells that are capable of producing large quantities of the protein. Afterwards, the cells are broken up and the protein under investigation is purified using different chromatographic and precipitation methods. The isolated proteins as well as reconstructed networks can then be analysed in detail. Here a number of methods (x-ray structure analysis, high resolution electron microscopy, nuclear magnetic resonance spectroscopy, and many others) can be used. Our protein production platform uses state-of-the-art technology in order to accelerate and automate the laborious and time-consuming methods of protein production and purification and can process up to 1000 cloning projects per year.

Equipment platform 9: Fluorescence Life Time Imaging Tomography for the Detection of Biosensors in Living Mice In the Centre of Systems Biology optical sensors are being developed which allow the tracing of enzymatic reactions and signalling activities in tissues and organisms. Such fluorescent biosensors can be expressed in whole organisms or expressed selectively in special tissues. We use these to study how signal transduction processes take place in multicellular specimen. For this, new tomographic imaging and processing techniques are being developed which enable the observation and 3D representation of how signals are transmitted in the body. With these new techniques, it is also possible to observe which cells or tissues are targeted by specific signals and how they react. For these investigations, measurement of the lifetime of the biosensor is particularly suited, since it is especially sensitive to different environments. Equipment platform 10: Fluorescence Life Time Imaging Microscopy (FLIM) for the analysis of clinical tissue samples Early detection of malignant tissue changes is of crucial importance for clinical diagnostics. The more differentiated the analysis of the tissue samples is, the earlier reliable conclusions can be drawn about pathologically changed tissue structures. Important parameters are e.g. fluorescence intensity of reliable biomarkers and their spatial distribution. In addition, measuring the fluorescence lifetime (mean residence time of the fluorophore in an excited state) provides crucial information. Since the fluorescence lifetime is very sensitive to changes in the micro-environment, we can directly monitor the activity of various oncogenes. This has the potential to be developed into methods for early and reliable diagnoses. The results of the FLIM analysis can be correlated with histological experiments. Besides improved early detection, this method also allows deeper insight into the origin and development of pathological conditions.

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Partners in Lebenswissenschaftliche Innovationsplattform Dortmund

TechnologieZentrumDortmund Management GmbH

Partners in Lebenswissenschaftliche Innovationsplattform Dortmund

Max-Planck-Institut fr molekulare Physiologie


Max-Planck-Institut fr molekulare Physiologie is one of the leading German research institutes in the field of basic biomedical research. The aim of the scientists is to elucidate the function of the building blocks which regulate all processes of life the genes as carriers of genetic information and the proteins as structural materials and catalysts of the cells. With the aid of molecular biology and organic chemistry, laser scanning microscopy and structural analytics, researchers here in Dortmund have discovered numerous mechanisms active in the development of tumours, neurological development disorders and inherited blindness. In the past years, Institute scientists have gained a multitude of valuable insights that already enable the development of innovative methods for the diagnosis of cancer and a more efficient search for new anti-tumour drugs. Max-Planck-Institut fr molekulare Physiologie owes this success last but not least to a still unusual liaison: the close interweaving of genome and proteome research with the analytical and synthetic methods of organic chemistry. At many universities and institutions these disciplines have been separate worlds until now. However, one aspect is clear: Although the decoding of the genome and the proteome of man, mouse and many other organisms have revolutionised the life sciences in recent years, knowledge of genes and proteins alone will not suffice to understand the manifold phenomena of life. It first requires interactions of the molecules with each other always a domain of chemistry to decide the fate of cells, organs and entire organisms. That is why chemists and biologists in Dortmund have been working hand in hand for years both within the institute and also with external partners. The intensive, long-standing cooperation between the MPI and the chemistry department of the Technische Universitt Dortmund and the biochemistry department and medical school of the University of Bochum provides vivid evidence of this, as do the innovative projects which very recently have evolved from this unusual liaison:

The International Max Planck Research School in Chemical Biology offers an interdisciplinary special study course programme in English to both German and international students, enabling them to complete their PhD degrees at a faster-than-usual pace. The Chemical Genomics Centre (CGC), in which chemists, biochemists and biologists of six Max Planck Institutes work together on the development of specific molecules to influence disease-relevant proteins and plant proteins. Zentrum fr Angewandte Chemische Genomik is a joint project with the Technische Universitt Dortmund and coordinated by the BioMedizinZentrum Dortmund. The Zentrum fr Systembiologie (ZfS), which pursues research on diseases and complex biological networks with an interdisciplinary approach. The Institute employs a staff of about 450 people and is organised in four departments as well as several independent teams. Each department is headed by a director who is autonomous in his scientific activity. All directors are scientific members of the Max Planck Society and jointly decide on the orientation and research activities of the institute. The position of managing director rotates every two years to each of the directors. Department of Structural Biology (Prof. Dr. A. Wittinghofer) Department of Systemic Cell Biology (Prof. Dr. P. Bastiaens) Department of Physical Biochemistry (Prof. Dr. R. S. Goody) Department of Chemical Biology (Prof. Dr. H. Waldmann)

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Partners in Lebenswissenschaftliche Innovationsplattform Dortmund

Technische Universitt Dortmund


Technische Universitt Dortmund includes 189 chairs, institutes and central science institutions. In the winter term 2006/07 22,000 students were registered. Currently they can choose from among 128 study courses. About 4,000 freshers start their studies at Technische Universitt Dortmund each year. Approximately 1,800 graduates leave the university with diploma, M. A., state examination or BA/MA, 220 with PhD every year. 14.4 percent of the students come from abroad. The highest number of students is registered for language and cultural studies (30.4 percent), for engineering sciences (27.3 percent) and mathematics and natural sciences (25.4 percent). About 27 percent of them are studying to become a teacher. The university has 2,800 employees: 300 professors, 900 academic and 1,050 non-academic employees, 450 employees financed with third-party funds and approximately

100 trainees. That means the university is one of the biggest employers and training companies in the city of Dortmund. It has an annual budget of over 200 million Euros, 37 of which are third-party funds financial contributions for research projects from public and private-sector clients and sponsors. Five collaborative research centres and three participations, one DFG transregios and one participation, three research groups and seven participations, four graduate colleges and one participation, for NRW Graduate School a participation in a MPI Research School as well as close connections with one institute of the Max Planck and two institutes of the Leibniz Society and two Fraunhofer institutes, clearly show the universitys high research capacity.

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Partners in Lebenswissenschaftliche Innovationsplattform Dortmund

Medizinisches Proteom-Center

The Medizinisches Proteom-Center (MPC) is one of the leading institutes in the field of protein analysis in Germany. The MPC is part of the medical faculty of the Ruhr-Universitt Bochum. In order to analyze and identify proteins the institute is equipped with state-of-the-art techniques for mass spectrometry combined with methods for multi-dimensional protein separation. The MPC is divided into the several working groups Redox Proteomics, Functional Proteomics, Cellular Proteomics, Neuroproteomics, Bioinformatics and Administration. The MPC maintains a multiplicity of cooperations. Various national, European and international projects are coordinated here, like e.g. the HUPO Brain Proteome Project (HUPO BPP) under the patronage of the Human Proteome Organisation (HUPO). The aim is the characterization of all proteins of a tissue at a certain time, the so-called proteome. Differential analysis of the protein samples of healthy and diseased or juvenile and old tissues uncovers differences between these conditions and makes an improved diagnosis of the disease possible as well as the development of therapeutic procedures. The MPC possesses an excellent and modern infrastructure for Proteomics with methods such as MALDI-MS, LC-ESI-MS/MS, 2D-PAGE, nano- HPLC and multidimensional HPLC.

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Partners in Lebenswissenschaftliche Innovationsplattform Dortmund

TechnologieZentrumDortmund GmbH/ TechnologieZentrumDortmund Management GmbH/ BioMedizinZentrum Dortmund


Dortmund and its surrounding areas offer ideal sites for technology companies. The combination of a landscape teaming with innovative research centres and an industrial city at the heart of Germany containing all modern aspects create complex synergetic effects and impulses for these companies. In addition, universities and colleges engineering and natural science departments cooperate with 24 other different institutes and research facilities to create a considerable potential for research and development. Imbedded in this area of research and development, the TechnologieZentrumDortmund (TZDO) has become a leader in innovative ideas and a source of modern technologies since its opening in 1985. The close proximity to the university, polytechnic and renowned research institutes is a major factor for its success and promotes the technology transfer between economy and science. More than 280 companies inside the Centre and the surrounding Technology Park profit from this practical relationship between research, development, the industry and the service sector. The Centre and the Park have developed into important location factors for the regional economic structure. 110 technology companies and their 1,300 employees work in eight building complexes in the TZDO that have an overall floor area of 80,000 m. More than 16,000 employment opportunities were created, including those at the Park, of which 8,448 are direct and 4,224 are indirect additional employment opportunities. Approximately 9,504 employees are residents of Dortmund. The average per capital turnover of EURO 90,000 indicates that the companies generate an overall turnover of EURO 800 million. Extensive Service Management The TechnologieZentrumDortmund offers companies a complete service package. The offer ranges from communication devices such as telephone, fax, e-mail, internet and includes, for example reception and telephone services, participation at trade fairs and events, national as well as international contacts and cooperative aid and is completed by extensive consulting services provided by qualified specialists. The TZDO is designed to function as a development centre and a laboratory for testing prototypes and pre-production batches in selected technological fields, which are based upon the technologies pooled from Dortmunds existing scientific and economic potential. Micro-systems technology Software/telecommunications/multimedia Electronics/electromagnetic compatibility (EMC) Quality assurance Logistics/material flow/packaging Environmental technology Automation/robotics Biomedicine, Proteomics, Bio-IT

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New technological fields such as photonics, nanotechnology or molecular electronics are added in the next years. BioMedizinZentrumDortmund (BMZ) is designed to house not only the field of biomedicine but also the fields of bioinformatics and proteomics. Dortmund is the nationwide leader in tomorrows market of microstructure technology. Visible evidence of this is the expanded Microstructure Centre (MSC) and the Modular Interface Technology Centre (MITC). Additional uses for miniaturized components are being made available to users in the industry from these centres. Dortmund, as a part of the New Economys international competition, will be expanded into one of Europes leading sites within the scope of the Dortmund Project. Six main objectives are being focused on: Setting up new leading industries: information technology, micro-systems technology and e-commerce/e-logistics Strengthening of the resident companies Expanding training, qualification, science and research The expansion of Dortmund as a location into a modern business metropolis with a high quality of life and leisure time Time saving methods from a one-stop shop for those starting up a new business or settling in Dortmund A clear increase in the level of employment

Contact Persons
TechnologieZentrumDortmund Management GmbH BioMedizinZentrumDortmund www.tzdo-do.de Andr van Hall Projectmanagement BioMedizinZentrumDortmund

Zentrum fr Angewandte Chemische Genomik


Technische Universitt Dortmund www.tu-dortmund.de Prof. Dr. Christof M. Niemeyer Dr. Udo Feldkamp Department of biological-chemical Microtechnology Prof. Dr. Andreas Schmid Lars M. Blank, PhD, Bruno Bhler, PhD; Katja Bhler, PhD Department of Biotechnology Prof. Dr. Roland Winter Dr. Werner Horstman, PD Dr. Claus Czeslik Department of physical Chemistry I

Max-Planck-Institut fr molekulare Physiologie www.mpi-dortmund.mpg.de Prof. Dr. Herbert Waldmann Department of chemical Biology

TechnologieZentrumDortmund Management GmbH BioMedizinZentrumDortmund www.tzdo-do.de Dr. Klaus F. Rammert, Sandra Citrich, Evgenia Hill Projectmanagement and Assistence ZACG

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Zentrum fr Systembiologie

Zentrum fr Angewandte Proteomik


Ruhr-Universitt Bochum Medizinisches Proteom-Center www.medizinisches-proteom-center.de Prof. Dr. Helmut E. Meyer Dr. Michael Hamacher Dr. Katja Kuhlmann, Jun.-Prof. Bettina Warscheid, Dr. Barbara Sitek, Dr, Kai Sthler, Dr. Gereon Poschmann, Dr. Stefan Helling, Prof. Dr. Katrin Marcus, Dr. Heike Ghler, PD Dr. Axel Kowald, Dr. Nicola Klare, Dr. Christian Stephan

Zentrum fr Systembiologie
Max-Planck-Institut fr molekulare Physiologie www.mpi-dortmund.mpg.de Prof. Dr. Alfred Wittinghofer Department of structural Biology Prof. Philippe Bastiaens Department of systemic Cell Biology Prof. Dr. Roger S. Goody Department of physical Biochemistry Prof. Dr. Herbert Waldmann Department of chemical Biology Dr. Astrid Krmer Projectmanagement ZfS

Technische Universitt Dortmund www.tu-dortmund.de Prof. Dr. Katja Ickstadt Arno Fritsch, Evgenia Saveleva Department of Statistics Prof. Dr. Rahnenfhrer; Prof. Wolgang Urfer (Prof. em.) Katharina Podwojski Department of Statistics Prof. Dr. Petra Mutzel Wolfgang Paul Department of Informatics Prof. Dr. Bernhard Steffen Christian Kubzcak Department of Informatics

TechnologieZentrumDortmund Management GmbH BioMedizinZentrumDortmund www.tzdo-do.de Dr. Petra Grnewald, Elisabeth Marten Projectmanagement and Assistence ZAP

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Supported by Ministry of Innovation, Science, Research and Technology of Northrhine-Westphalia

Co-financed by European Union Fonds for Regional Development (EFRE)

TechnologieZentrumDortmund Management GmbH

BioMedizinZentrumDortmund Otto-Hahn-Strae 15 44227 Dortmund Germany Contact: Phone Fax E-Mail Internet

+49-231-9742-130 +49-231-9742-159 info@bmz-do.de www.bmz-do.de www.zap-do.de www.zacg-do.de

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