new bio analytical techniques and developments

Symposium:New bioanalytical techniques and developments

Heiko Andresen, Kim Zarse, Carsten Grötzinger, Oliver J. Kreuzer, Marc Birringer, Eva Ehrentreich-Förster, Frank F. Bier

Development of Peptide Chips for Biomedical Applications

Jan Barnikow, Ron Tynes, Maik Kindermann, Stefan Steurer, Andreas Brecht

The SNAP-tagTM - a unique tool for covalent labeling and immobilization of proteins

Nadja Bilko, Denys Bilko

Ex vivo proliferation and differentiation of cord blood cells in gel culture system

Matthias Böcker, Pia Heidenreich, Harald Fuchs, Tilman Schäffer

Scanning ion conductance microscope with shear-force control

Andreas Böhm, Albert Sickmann

A Clustering Solution for Distributed Data Analysis in Mass Spectrometry (paOla)


Automatic Synchronizing and self-Updating Protein Sequence Database Management System

Andreas Böhm, Florian Grosse-Coosmann, Albert Sickmann

Calculating theoretical ms spectra for given sequences

Gregor Witte, Claus Urbanke, Ute Curth

Analytical Ultracentrifugation pinpoints the interaction of DNA polymerase III and primase with EcoSSB to its C-terminal region

Andreas Gorschlüter, L.H. Mak *, M. Knoll *, N. Dankbar, C. Sundermeier

Electro-Magnetic Biosensor for the Measurement of Binding Forces between Single Molecules

Sergey V. Tokalov, Herwig O. Gutzeit, Jutta Ludwig-Müller, Barbara Kind, Alexander Franz, Yvonne Henker

Cellular target proteins of quercetin and flavonoid metabolism in human cells

Pia M. Heidenreich, Sebastian Schrot, Hans-Joachim Galla, Harald Fuchs, Tilman E. Schäffer

Scanning Ion Conductance Microscopy - Topographical non-contact imaging of soft surfaces

Robert Henderson, David Dryden, Michael Edwardson, Michael Waring

DNA, Drugs and Proteins: Insights from the Atomic Force Microscope

Alejandro Heredia, C. C. Bui, Ueli Suter, Peter Young, Tilman Schäffer

Mechanical properties of myelinated and de-myelinated peripheral axons with the atomic force microscope

Sonja Hess, Xiaoli Chen, Samuel W. Cushman, Lewis K. Pannell

Quantitative proteomics of the secretory proteome of adipocytes

Peter Hinterdorfer

Topography and Recognition Imaging using AFM

Heinrich Hörber

Single molecule mechanics

Bernhard Andreas Krenig

A new developed biochemical process in purifikation

Stefan Kreusch, Heidrun Rhode, Horst Hoppe, Thomas Moore, Renate Bublitz, Joachim Misselwitz, Margarete Schulze, Gerd Cumme

Separation and analysis of native human proteomes using parallel chromatography with microplates: Alport syndrome versus healthy serum

Ziv Reich

Exploring Protein-Binding Mechanisms and Energy Landscapes by Single-Molecule Mechanical Unbinding Experiments

Nicole Reifschneider, S. Goto, Norbert A. Dencher, Frank Krause

Tissue diversity of the mitochondrial membrane proteome of Rattus norvegicus studied by peptide mass fingerprinting

Sascha Rexroth, Jürgen Meyer zu Tittingdorf, Frank Krause, Holger Seelert, Norbert Dencher

Identification of integral thylakoid membrane proteins from Chlamydomonas reinhardtii by peptide mass fingerprinting and MALDI-MS

Heidrun Rhode, M. Schulze, E. Mitre, G.A. Cumme, T. Rausch, D. Philipp, A. Horn

Turbo-mixing in microplates

Thomas Schulenborg, Heike Schäfer, Christian Bunse, Helmut E. Meyer, Katrin Marcus

Detection of phosphorylation sites using a hybrid triple quadrupole/linear ion trap mass spectrometer

Barbara Sitek, Ognjan Apostolov, Kathy Pfeiffer, Kai Stühler, Helmut E. Meyer, Angelika Eggert, Alexander Schramm

Identification of proteins differentially expressed upon neurotrophin receptor activation using DIGE and MALDI-MS

Manfred Wuhrer, Carolien A. M. Koeleman, André M. Deelder, Cornelis H. Hokke

Analysis of protein glycosylation using normal-phase nano-scale liquid chromatography-mass spectrometry of glycopeptides and oligosaccharides

Heiko Andresen, Kim Zarse, Carsten Grötzinger, Oliver J. Kreuzer, Marc Birringer, Eva Ehrentreich-Förster, Frank F. Bier

Development of Peptide Chips for Biomedical Applications

Miniaturisation of standard analytical techniques is a prominent task of biomedical research and development. Special attention is given to the microarray technology whose diagnostic potential enhanced and complemented genome and transcriptome analysis in the human genome project. The human proteome, however, is a multiple more complex than the human genome, emphasizing the need of an analytic tool on protein level that is comparable in efficiency and performance to the DNA microarray technology. Synthetic low-molecular weight peptides have outstanding purposes for use as variable probes in chip-based analysis, since they can be assembled in defined orientation and high density. Furthermore, fully automated synthesis makes them an economically attractive alternative to recombinant proteins. We developed a peptide chip for assaying the binding reaction of specific monoclonal antibodies to linear peptide epitopes. For the prototypic version, 13mer peptides mimicking linear epitopes of model proteins were synthesized by standard Fmoc-synthesis, equipped with a biotin linker and printed on NeutrAvidin-coated glass slides. The peptide probes were used to capture specific monoclonal antibodies with known linear epitopes. A fluorescently labelled secondary antibody was used for array readout. The peptide chip assay showed highly specific signals for all tested antibodies and sensitivity in the range of ng•ml-1.

contact:

Dipl.-Ing. Heiko AndresenFraunhofer Institut f. Biomedizinische Technik Molekulare Bioanalytik und Bioelektronik [email protected] Arthur-Scheunert-Allee 14558 Nuthetal (Deutschland)

Jan Barnikow, Ron Tynes, Maik Kindermann, Stefan Steurer, Andreas Brecht

The SNAP-tagTM - a unique tool for covalent labeling and immobilization of proteins

We present a novel protein tag and its applications which range from protein labeling over immobilization to purification. The SNAP-tag is based on the DNA-repair enzyme alkylguanine-DNA-alkyltransferase.1 It reacts with O6–alkylguanine derivatives forming a thioether bond between the protein and the alkylgroup of its substrate.2 The selectivity of the reaction in combination with the formation of a stable linkage between the protein-tag and its substrate offers attractive possibilities for handling and labeling of recombinant fusion proteins even in complex samples.3 The in vivo labeling of SNAP-tag fusion proteins has been tested with a range of fluorescent dye substrates in several mammalian cell lines. The nontoxic compounds allow live cell imaging. Cellular localization experiments with various fusion proteins targeting the label to cytosceleton, plasmamembrane, nucleus or cytoplasm resulted in the respective staining in combination with very low background signals. In vitro, the labeling proceeds with more than 90% labeling achieved within 10 min (RT, 10 µM substrate, up to 5 µM fusion protein). Fluorescence polarization allows to follow the reaction in realtime with dyes that cover a broad range of the spectrum. Labeled SNAP-tag fusion proteins can be used in fluorescent interaction assays. Another attractive application is the detection and quantitation of labeled fusion proteins on SDS-gels without further staining or blotting and immunodetection steps. The technology is currently tested and further applications are developed in a number of joint research projects with academical and industrial partners.

Literature1) A. Juillerat, T. Gronemeyer, A. Keppler, S. Gendreizig, H. Pick, H. Vogel, K. Johnsson, 2003, Chemistry&Biology 10: 313-7. 2) A. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel, K. Johnsson, 2003, Nature Biotechnology 21: 86 - 89. 3) A. Keppler, K. Kindermann, S. Gendreizig, H. Pick, H. Vogel, K. Johnsson, 2004, Methods 32: 437-44.

contact:

Dr. Jan BarnikowCovalys Biosciences AG

[email protected] Benkenstr. 254 4108 Witterswil (Switzerland)

Nadja Bilko, Denys Bilko

Ex vivo proliferation and differentiation of cord blood cells in gel culture system

Cord blood (CB) cells are being used increasingly as a source of hematopoietic stem cells (HSC) for transplantation. It is known that ex vivo expansion of HSC depends on many factors and is not achievable, because addition of the major growth factors to the growth media in vitro does not fulfill all requirements for successful long-term cell maintenance. Often cultures are supplied with factor-producing feeder layers. It has been suggested, that stromal presence is important for hematopoiesis in vitro and in vivo, but the question remains: whether diffusible factors produced by stromal cells are sufficient for the regeneration of primitive haematopoietic cells, or whether direct cell-to-cell contacts would be required. Meantime, the culture of complex undefined cell populations makes the implementation of some molecular diagnostic methods non-consistent, and at times impossible. As for the potential use of cultured haematopoietic cells for cytotherapeutic purposes, contamination of the transplant material with stromal cells of the donor should be minimised, or eliminated. During present studies, influence of various feeder layers and feeder layer condition media on AC133+ cells derived from human umbilical cord blood was investigated and their proliferative, differentiative and clonogenic activity was studied. Original patented model of gel diffusion capsules (DC) was implemented. Purified cells were injected in to the inner cavity of the DC in DMEM culture media with 15% FCS, that were cultured in 6-well plates supplemented with 7ml of DMEM growth media in absolute humidity at 370C and 5% CO2 for the duration of two weeks. Feeder layers were prepared from 4-6 weeks old human embryos (FLh) and co-cultured feeder cells (FCh). Effects of the condition media (FL-CMh) were also determined. Culture and feeder layer media was at first supplemented with LIF and later with IL-3 and GM-CSF. Estimation of functional properties of AC133+ suspension cell cultures were performed in secondary subculture experiments using semisolid culture media. After 14 days subcultured material was analysed and its clonogenic activity was determined by direct and indirect analysis and quantification of the colony forming units. Multipotential CFU-MIX (CFU-GEMM) and unipotential progenitor cells CFU-GM, BFU-E and CFU-E were observed and analysed using morphological and cytochemical methods. Our data indicated deffinite ex vivo expansion and myeloid differentiation of cord blood AC 133+ cells in the presence of FCh or FL-CMh feeder layer supported culture models. Prolonged support of primitive haematopoietic cells (undifferentiated cells such as promyelocytes, myelocytes and metamyelocytes) and their clonogenic capacity and functional characteristics in feeder layer positive cultures, indicates that diffusible factors are sufficient for haematopoiesis and that direct cell-to-cell contacts may not be exclusively required for successful long term in vitro haematopoiesis.

contact:

PhD, DSci Nadja BilkoNational University \ Centre of Molecular and cell investigations [email protected] G.Skovoroda 2 04070 Kiev (Ukraine)

Matthias Böcker, Pia Heidenreich, Harald Fuchs, Tilman Schäffer

Scanning ion conductance microscope with shear-force control

We have built a scanning ion conductance microscope (SICM) that measures the local ion conductance of surfaces. A pulled micropipette, having an opening diameter of less than 100nm and filled with electrolyte, acts as local ion conductance probe and is scanned over a porous sample surface. To obtain a meaningful interpretation of the measured ion conductance, the pipette-sample distance needs to be kept constant. This is achieved by implementing a complementary shear-force distance control: The micropipette is mechanically oscillated at a fixed frequency (3-10kHz), in a direction parallel to the surface. When the pipette is approached to the surface, the oscillation amplitude decreases due to arising shear forces. This amplitude decrease is detected optically by focusing a laser beam onto the thin end of the micropipette and measuring the modulation of the transmitted or reflected light. A feedback loop uses this signal to keep the pipette at constant distance to the surface while scanning, thereby simultaneously measuring two complementary surface properties: topography and ion conductance. We have used this microscope for imaging gratings, CDs and cells.

contact:

Dr. Tilman SchäfferUniversität Münster Center for Nanotechnology [email protected] Gievenbecker Weg 11 48149 Münster (Germany)

additional information

http://bioforce.centech.de


A Clustering Solution for Distributed Data Analysis in Mass Spectrometry (paOla)

In mass spectrometry huge amounts of data are acquired each day. This data must be analyzed in less or maximal equal time it is recorded. Otherwise the volume of data will grow over time will need more and more time to be processed. Reducing the processing time can be achieved by three ways: By applying high-performance hardware or by increasing the degree of parallelism. The third possibility is tuning of the used algorithms, but this can seldom be done in case of commercial software. We decided to combine the first two possibilities and designed a clustered computer environment named "protein analysis on linux architecture" (paOla). This design implements a queuing architecture with a task submitting interface. The queue is implemented in a way ensuring every submitted task will have its turn. Using paOla, the analyzing tasks can be distributed easily and in a transparent way over independent CPUs and machines, so these tasks do not interfere each other. Actually in our lab the cluster consists of four computers with two CPUs each, allowing a maximum of eight analyzing tasks running at the same time. The job types are configurable, so adding new analyzing algorithms or even new task types can be done with nearly no effort. The key administration features of the queue like task submission, killing, stopping, viewing progress and status are all implemented in an easy-to-use web-based service that can be used even by inexperienced users.

Literature[1] Boehm, A.M., F. Grosse-Coosmann, and A. Sickmann. Bioinformatics, 2004. in press. [2] Perkins, D.N., et al. Electrophoresis, 1999. 20(18): p. 3551-3567. [3] Eng, J.K., A.L. McCormack, and I. Yates, John R. Journal of the American Society for Mass Spectrometry, 1994. 5(11): p. 976-989. [4] Wall, L., T. Christiansen, and R.L. Schwartz, Programming Perl. 1996: O´Reilly. [5] Pedrioli, P., The SASHIMI Project. http://sashimi.sourceforge.net/ 2003.

contact:

Andreas BöhmUniversität Würzburg Rudolf-Virchow-Center for Experimental Biomedicine [email protected] Versbacher Str. 9 97078 Würzburg (D)


Automatic Synchronizing and self-Updating Protein Sequence Database Management System

A common problem in proteomics is the deficient quality of some available sequence databases, especially concerning accessibility and redundancy, for example the non-redundant sequence database (nrdb), which is not truly non-redundant. Often they are available online only for a short period of time, then disappear from the internet and cannot be obtained in other ways. Common proteomic or genomic "database" formats (omic-databases) like FASTA or geneDB are neither designed for efficient maintenance nor practical for data management purposes in environments where millions of sequences must be handled. Therefore we decided to develop our own sequence database system and to keep it up to date by integrating a practicable automatism for maintenance purposes. This system queries each online database for available updates and merges the differences in our system called the Automatic Syncing and Updating Protein Sequence Database (ASUP-SDB). Making the database non-redundant by applying known and our own algorithms, the key feature is, that the classes of mistakes which may emerge while making the database non-redundant are known and thus we can react appropriately to these circumstances. ASUP-SDB is capable of storing sequence data as well as the appropriate information concerning splice-variants, post-translational modifications, isoforms, localization and taxonomy. Of course it allows precise retrieval of this information and exports in common omic formats, too. As a remarkable feature ASUP-SDB is capable of downloading external online databases by looking for updates and synchronizes with these data automatically using several algorithms. The ASUP-SDB supports common import and export omic formats such as FASTA or swissprot, for instance. Other importing or exporting formats can be added easily. As ASUP-SDB uses an underlying relational database management system (RDBMS), maintenance of sequence information is centralized and thus omic-databases used for sequence analysis such as mass spectrometry are kept up to date simply by exporting them from the central ASUP-SDB on a regularly basis.

Literature[1] Pearson, W.R. and D.J. Lipman. PNAS, 1988. 85(8): p. 2444-2448. [2] Altschul, S.F., et al. JMB, 1990. 215(3): p. 403-410. [3] http://www.ncbi.nih.gov/, 1988. [4] http://us.expasy.org/sprot/, 2004

contact:


Andreas Böhm, Florian Grosse-Coosmann, Albert Sickmann

Calculating theoretical ms spectra for given sequences

Scientists usually want to verify the ion matching process of algorithms that look up peptide sequences in DNA or protein databases. The verification step is often done numerically or visually. Not all search algorithms present the appropriate theoretical spectrum information within their results. Thus, the theoretical spectrum for each result should be calculated from the sequence of the matched peptide. We present an operating-system-independent command line tool for this purpose that can be integrated easily into complex as well as existing environments, and can be used to present the theoretical spectrum to the user in either graphical or tabular format by third party products. The source code and binaries for windows and linux can be downloaded from http://www.protein-ms.de

LiteratureKernighan, B. W. and Ritchie, D. M. (1990) The C Programming Language National Institute for Standards and Technology (2004) The Nist Reference on Constants, Units and Uncertainty, http://physics.nist.gov/cuu/Constants Perkins, D. N., Pappin, D. J. C., Creasy, D. M., Cottrell, J. S. (1999) Probability-Based Protein Identification by Searching Sequence Databases Using Mass Spectrometry Data, Electrophoresis, 20, 3551-3567 Yates, J. R., Eng, J. K., Clauser, K. R. and Burlingame, A. L. (1996) Search of Sequence Databases with Uninterpreted High-Energy Collision-Induces Dissociation Spectra of Peptides, J Am Soc Mass Spectom, 7, 1089-1098

contact:


Gregor Witte, Claus Urbanke, Ute Curth

Analytical Ultracentrifugation pinpoints the interaction of DNA polymerase III and primase with EcoSSB to its C-terminal region

Single-stranded DNA binding (SSB) proteins are involved in DNA replication, repair and recombination. They bind without sequence specificity to single-stranded DNA (ssDNA) but not to double-stranded DNA [1]. This results in stabilizing the ssDNA, preventing hairpin formation and holding it in a suitable conformation for the action of other proteins involved in e.g. DNA replication. The E.coli SSB protein (EcoSSB) is composed of three regions. The DNA-binding region comprises the N-terminal 2/3 of the protein and is succeeded by a flexible, glycine-rich linker. The negatively charged region of the last 10 amino acids of EcoSSB is highly conserved among bacterial SSB proteins. Though this highly conserved C-terminus is not involved in DNA binding it is essential for the survival of the bacterial cell [2]. It has previously been shown that a mutation in this C-terminus weakens the interaction between EcoSSB and the χ subunit of DNA-polymerase III, the main replication enzyme in E.coli [3]. Using sedimentation velocity experiments we show that a mutant protein lacking the last 26 amino acids of EcoSSB does not interact with χ. Therefore the C-terminus of EcoSSB is essential for the interaction with χ. At low salt concentrations we demonstrate by analytical ultracentrifugation that the affinity of χ towards EcoSSB is enhanced approximately 20-fold in the presence of ssDNA. DNA melting experiments show that the affinity of EcoSSB towards ssDNA is enhanced specifically in the presence of χ. Thus the interaction of EcoSSB and χ prevents premature dissociation of EcoSSB at the lagging strand of the replication fork, thereby enhancing the processivity of DNA polymerase III [4]. At the lagging strand of the replication fork EcoSSB also interacts with primase, thereby preventing a premature dissociation of the RNA primer synthesized by the primase [5]. While the sedimentation constant of a complex of wild-type EcoSSB and ssDNA is shifted from 27 S to 39 S by the presence of primase, a complex of C-terminally shortened EcoSSB and ssDNA is barely affected. Thus, we can demonstrate for the first time that the interaction of EcoSSB and primase is also mediated by the C-terminus of EcoSSB.

Literature1. Greipel, J., C. Urbanke, and G. Maass. in Protein-Nucleic Acid Interaction, W. Saenger and U. Heinemann, Editors. 1989, Macmillan: London. p. 61-86. 2. Curth, U., J. Genschel, C. Urbanke, and J. Greipel. Nucleic Acids Res, 1996. 24(14): p. 2706-11. 3. Kelman, Z., A. Yuzhakov, J. Andjelkovic, and M. O'Donnell. Embo J, 1998. 17(8): p. 2436-49. 4. Witte, G., C. Urbanke, and U. Curth. Nucleic Acids Res, 2003. 31(15): p. 4434-40. 5. Yuzhakov, A., Z. Kelman, and M. O'Donnell. Cell, 1999. 96(1): p. 153-63.

contact:

Dr. Ute CurthMedizinische Hochschule Hannover Institut für Biophysikalische Chemie [email protected] Carl-Neuberg-Str. 1 30625 Hannover (Germany)

Andreas Gorschlüter, L.H. Mak *, M. Knoll *, N. Dankbar, C. Sundermeier

Electro-Magnetic Biosensor for the Measurement of Binding Forces between Single Molecules

We report on the development of a high-sensitive biosensor combining an electrical detection of microbead labelled analytes with a magnetical system for binding strength control. The new sensor technology allows the determination of specific binding forces between analyte and the corresponding capture molecules (e.g. proteins, antibodies, DNA). The sensor chip comprises many biochemically functionalised ultramicroelectrodes to which one end of the ligand-receptor pairs is attached. By labelling the other end of these molecular pairs with superparamagnetic microparticles a magnetically generated force can be applied to the molecular bonds and an unbinding can be induced. This unbinding event is electrochemically detected. With knowledge of the applied force, the binding force can be determined. We present such binding force measurements on the system neutravidin-biotin and on the antibody-antigen system BSA and monoclonal anti-BSA. Because the sensor system allows a binding strength determination at many individual molecular pairs simultaneously, it seems promising that a fast investigation of environmental conditions on ligand-receptor interaction will be possible in the future.

contact:

Dr. Andreas GorschlüterUniversität Münster Physikalisches Institut [email protected] Wilhelm Klemm-Str. 10 48149 Münster (Deutschland)


*) Institut für Physikalische Chemie, Münster A.G., L.M., N.D. & C.S.: former address: ICB GmbH, Münster

Sergey V. Tokalov, Herwig O. Gutzeit, Jutta Ludwig-Müller, Barbara Kind, Alexander Franz, Yvonne Henker

Cellular target proteins of quercetin and flavonoid metabolism in human cells

Despite the wealth of information concerning biological effects of flavonoids a systematic approach to analyse the molecular targets is still lacking and, for this reason, a rational evaluation of the risks or benefits of flavonoid- containing food or of possible pharmaceutical applications has not been possible. We have exploited the property of quercetin to elicit fluorescence when bound to specific target proteins. The effect was studied in detail using BSA and insulin as model proteins. Furthermore, cellular target proteins in the polyploid nuclei and in cytoplasmic structures of Drosophila follicles were visualised in living cells by fluorescence microscopy. Nuclear target proteins were also present in human leukaemia (HL-60) cell cultures. However, vital HL-60 cells quickly lost their fluorescence while apoptotic cells were characterised by a strong and persisting signal (analysis by microscopy and flow cytometry). This observation indicated rapid metabolic conversion of quercetin in vital human cells. The observed dynamics of decreasing fluorescence was confirmed and quantified by HPLC analysis. While apoptotic cells still contained considerable amounts of quercetin, vital cells rapidly metabolised the flavonoid (e.g. methylation or glycosylation). Of several known metabolites which were tested with BSA and insulin for their fluorogenic properties, only isorhamnetin but no other metabolite elicited strong fluorescence.

contact:

Prof. Dr. Herwig O. GutzeitTU Dresden Zoologie [email protected] Helmholtzstr. 10 01069 Dresden (Deutschland)

Pia M. Heidenreich, Sebastian Schrot, Hans-Joachim Galla, Harald Fuchs, Tilman E. Schäffer

Scanning Ion Conductance Microscopy - Topographical non-contact imaging of soft surfaces

We have developed a scanning ion conductance microscope (SICM) that can obtain topographical images of soft and biological surfaces such as cells, with a lateral resolution of 50 nm. SICM is a non-contact scanning probe method which is based on the measurement of an ion current that flows through the tip opening of a micropipette. The ion current generally decreases when the tip approaches the surface. This “current squeezing” effect is used as a contrast mechanism in SICM. It is possible to control the tip-sample distance with this effect while scanning across the sample surface. In this case, the topography of the sample is imaged. Our experimental setup also can be operated in a spectroscopic mode. The measured current-distance curves do not only show the current squeezing when the tip is approached to a sample. They contain a lot of information about sample and tip properties, but not all effects are fully understood yet.

contact:

Pia HeidenreichUniversität Münster Center for NanoTechnology & Physikalisches Institut [email protected] Gievenbecker Weg 11 48149 Münster (Germany)

Robert Henderson, David Dryden, Michael Edwardson, Michael Waring

DNA, Drugs and Proteins: Insights from the Atomic Force Microscope

Since its adoption by the biomedical community in the early 1990s, the atomic force microscope has been widely used to study structure of and interaction between biological macromolecules. It has the advantage over other techniques that images can be obtained at a high resolution of molecules under fluid and in real time. One of the most fruitful areas of study has been in the field of DNA. To demonstrate the breadth of information that can be obtained from this technique, this presentation will focus on the use in our laboratory of atomic force microscopy to investigate: (1) the mechanism of action of at type I restriction enzyme, EcoKI, that binds to and cleaves DNA, and (2) the structural effects upon DNA of anti-cancer drugs that intercalate into the nucleic acid molecule.

contact:

Dr Robert HendersonUniversity of Cambridge Department of Pharmacology [email protected] Tennis Court Road CB2 1PD Cambridge (United Kingdom)

Alejandro Heredia, C. C. Bui, Ueli Suter, Peter Young, Tilman Schäffer

Mechanical properties of myelinated and de-myelinated peripheral axons with the atomic force microscope

Schwann cells are the principal glial cells of the peripheral nervous system (PNS). They envelop the axons in the PNS of vertebrates by forming insulating myelin sheaths, thereby providing saltatory conduction and axonal regeneration following nerve injury. Saltatory conduction is achieved by reducing the depolarization regions to only the Ranvier nodes, which are the small gaps that are directly exposed to the extracellular environment between adjacent Schwann cells. Conversely, axons regulate the differentiation, survival and/or proliferation of Schwann cells at several stages during their development. Several human diseases (multiple sclerosis, Pelizeaus-Merzbacher disease, etc) result from a deficiency or disruption of the myelin sheaths. We used the atomic force microscopy (AFM) to image myelinated and de-myelinated mouse peripheral nerve axons. We furthermore established a method of measuring locally resolved, quantitative elastic properties of axons with the AFM, and acquired two-dimensional images of their local elastic modulus. Typical elastic moduli of re-hydrated axons at physiological conditions were in the 100-900 kPa range. We further present initial results of investigations linking the mechanical properties of the axons to the ionic concentration of the solution.

contact:

Alejandro HerediaUniversität Münster Center for Nanotechnology [email protected] Gievenbecker Weg 11 48149 Münster (Germany)


A.H.: Universität Münster and Instituto de Ciencias Nucleares, UNAM, México. C.C.B. & P.Y.: Klinik und Poliklinik für Neurologie, Universitätsklinikum Münster U.S.: Institute of Cell Biology, ETH Hönggerberg Zürich, Switzerland.

Sonja Hess, Xiaoli Chen, Samuel W. Cushman, Lewis K. Pannell

Quantitative proteomics of the secretory proteome of adipocytes

In the past adipose cells have generally been regarded as energy storage sites. Only recently, the critical endocrine function of adipocytes to release signaling molecules has been recognized. It is now known that adipose cells and their regulation play a central role in the pathogenesis of obesity and related diseases. However, the signaling cascades are not well understood. Therefore, we have developed a 2D-LC-MS/MS and 18O proteolytic labeling strategy to identify and compare levels of secretory proteins with low abundance in the conditioned medium of adipose cells w/o or w ith insulin stimulation. This approach allowed us to catalog a large number of secretory proteins and to detect the different levels of many secreted proteins in the basal versus insulin treatment. Adipose cells were isolated from rats and cultured in a serum free DMEM medium for 48h. Culture medium was concentrated and separated on a Zorbax C3 column using a gradient from an aqueous solution of 0.05%TFA to acetonitrile. Eight combined fractions were collected. Each fraction was reduced, alkylated, digested with Lys-C and trypsin. The samples were separated on a C18 column. The peptides were characterized by LC-MS/MS using a QTOF2 mass spectrometer. Data were analyzed using a MASCOT search engine. For 18O proteolytic labeling, 16O-to-18O exchange in the digested peptides from eight individual fractions was carried out in parallel in H216O and H218O with immobilized trypsin and the ratios of isotopically distinct peptides were measured by LC-MS. We have set up proteomics experiments using a 2D LC-MS/MS and 18O proteolytic labeling strategy to identify and quantify secretory proteins of adipose cells without or with insulin treatment. Proteins were identified by using MASCOT searches of the NCBInr database. A minimum criterion of one positive peptide identification was set. In addition, all secretory proteins were checked by SignalP predictions for their identity as secretory proteins. From comparison of stro mal-vascular cells that are also known as preadiopcytes, we could clearly show that mature adipocytes function as secretory cells. A total of 70 secreted proteins have been identified using this approach. When comparing the proteins identified in this study with those found in earlier studies, it is evident that our approach is extremely powerful in identifying secretory proteins of adipose cells since the number of secretory proteins identified has been considerably increased in our study. To those few proteins that are already known to be secreted by adipose cells, we have compiled a group of known secretory proteins previously not associated with adipose cells and a few unknown proteins. Comparative proteomics of 18O proteolytic labeling allowed us to detect the different levels of many secreted proteins in the basal versus insulin treatment. 18O proteolytic labeling was chosen because of its accuracy, versatility and applicability in animal and human studies. Taken together, our proteomic approach is able to identify and quantify the secretory proteome of adipose cells that is applicable to animal and human studies. This will certainly lead to an improved understanding of the signaling cascades of adipose cells and thus, pathogenesis of obesity and its related diseases.

Literature1. Ahima, R.S., & Flier, J.S. (2000) Trends Endocrinol. Metab. 11, 327-332. 2. Kratchmarova, I. et al. (2002) Mol. Cell. Proteomics 1, 213-222. 3. Washburn, M.P., Wolters, D., & Yates, J.R. 3rd (2001) Nat. Biotechnol. 19, 242-247. 4. Hess, S., van Beek, J., & Pannell, L.K. (2002) Anal Biochem. 311, 19-26.

contact:

Dr. Sonja HessNational Institutes of Health NIDDK [email protected] 9000 Rockville Pike 20892 Bethesda, MD (USA)

Peter Hinterdorfer

Topography and Recognition Imaging using AFM

We have developed a method for the localization specific binding sites and epitopes with nm positional accuracy by combining dynamic force microscopy with single molecule recognition force spectroscopy. A magnetically driven AFM tip containing a ligand covalently bound via a tether molecule was oscillated at 5 nm amplitude while scanning along the surface. Since the tether had a length of 8 nm, the ligand on the tip was always kept in close proximity to the surface and showed a high probabilty of binding when a receptor site was passed. We showed that for cantilevers with low Q-factor driven at frequencies below resonance the surface contact only influenced the downward deflections of the oscillations, while binding of the ligand on the tip to the receptors on the surface reduced only the upwards deflections of the oscillations. The recognition signals were well separated from the topographic signals arising from the surface. In this way, topography and recognition image were gained simultaneously and independently with nm lateral resolution. This work was supported by the Austrian Science Foundation Project P-14549 and the GEN-AU initiative.

contact:

Ass. Prof. Dr. Peter HinterdorferJohannes Kepler University of Linz Institute for Biophysics [email protected] Altenbergerstr. 69 A-4040 Linz (Austria)

Heinrich Hörber

Single molecule mechanics

In the context of evolution, nature has found many elegant nano-technological concepts. In the cells of our body innumerable examples of multi-functional, self-organizing and self-reproducing molecular structures are found. All cellular functions are performed by groups of highly specialized molecules carrying out exact programs. These components possess an extremely high efficiency level and their functions are regulated stably and precisely, both by networking with other functional groups as well as by reacting to changing environmental conditions. Thus biologists talk about proteins like machines for good reason: proteins function like machines with specific tasks equipped with built-in programming and often with a chemical on/off switch. It is possible to use and in some cases change the functions of proteins for technological applications. Such modifications need to be monitored carefully and for certain molecules their mechanical properties are the most relevant features. The three-dimensional analysis of thermal position fluctuations can reveal mechanical properties, for instance of molecular motor proteins like Kinesin and Myosin. This became possible by a novel three-dimensional scanning probe microscope, the Photonic Force Microscope (PFM). Such measurements can complement AFM studies of protein unfolding revealing at higher forces information about internal forces determining the three-dimensional structure of these molecules. A double detection scheme has been developed to provide the necessary stability of the AFM doing experiments on short polymer chains and to work in a “force-clamp” mode.

LiteratureJ. K. H. Horber and Mervin Miles, 2003 Science 302, 1002-1005 Hörber J.K.H. 2002 Methods in Cell Biology, Vol. 68, 1-31 Altmann, S. M., R. G. Grunberg, P. F. Lenne, J. Ylanne, A. Raae, K. Herbert, M. Saraste, M. Nilges, and J. K. H. Horber, 2002 Structure 10, 1085-1096. Pralle, A., P. Keller, E. L. Florin, K. Simons, and J. K. H. Horber, 2000 Journal of Cell Biology 148, 997-1007

contact:

Prof. Dr. Heinrich HörberWayne State University Physiology [email protected] 540 E. Canfield Ave. 48201 Detroit (USA)

Bernhard Andreas Krenig

A new developed biochemical process in purifikation

Purification of monoclonal antibodies is a key process in biomedical research. One of the most common used techniques to separate proteins is ion exchange chromatography, but this typically involves time consuming procedures and expensive equipment. The rapid purification protocol is based on electrodialysis. We developed a microelectrodyalisis (MED)process to desalt proteins. Usage of this tool resulted in very high purity of the proteins as shown by subsequent mass spectrometry. So establisched MED process with special ion exchange membranes is more efficient than standard dialysis. Rapid purification periods are needed for handling unstable proteins. Very fast purification of electrolyte ions up to 95% in a volume of 10µl is possible with MED. Purification results are strongly dependend on membranes quality. We developed membranes with permselectivities up to 95%. The MED membranes can be used topurify concentrations >1µmol/10µl. So MED is the preferred method for protein purification. In case of too strong membrane fouling processes, the transport depletion has to be preferred. Both techniques are predominant compared to the standard dialysis procedure and therefore should be the future for purification and concentration of recombinant proteins.

contact:

Dipl. chem, Bernhard Andreas KrenigUniversitätsklinikum Frankfurt Virologie [email protected] Theodor-Stern Kai 7 60950 Frankfurt a.M. (Deutschland)

Stefan Kreusch, Heidrun Rhode, Horst Hoppe, Thomas Moore, Renate Bublitz, Joachim Misselwitz, Margarete Schulze, Gerd Cumme

Separation and analysis of native human proteomes using parallel chromatography with microplates: Alport syndrome versus healthy serum

The Alport syndrome is a hereditary nephropathy caused by mutations in type IV collagen genes. It results in hematuria and/or proteinuria and, therewith, alteration of serum proteins. We analyzed human serum proteomes of 3 Alport syndrome patients versus 3 healthy probands. 0.5 ml serum (about 30 mg protein) were separated by size exclusion chromatography and subsequent anion exchange chromatography. A parallel chromatography device in the 8*12 microplate format was used together with the liquid handling device CyBiä-Well. Protein concentrations within the resulting 2400 fractions of each serum were quantified by UV-absorbance measurements at 205, 215, and 280 nm. Normalized protein concentration differences, syndrome minus healthy, are shown as two-dimensional pattern according to both the separations performed. In liquid aliquots of the native serum fractions obtained, activities of alkaline phosphatase, acetylcholine esterase, and GPI-phospholipase D were determined. Triglycerides and cholesterol as lipoprotein markers and the immunoreactivity of C-reactive protein were determined. Semiquantitative MALDI-MS using mean peak intensity sums of tryptic peptides normalized by internal standard peptide peak heights was applied to validate alterations of selected protein levels. It turned out that Alport syndrome patients partially compensate for loss of lower molecular weight proteins by a higher serum concentration of alpha-2-macroglobulin.

contact:

Dr. Stefan KreuschKlinikum der FSU Jena Inst. f. Biochemie I [email protected] Nonnenplan 2 07743 Jena (D)

Ziv Reich

Exploring Protein-Binding Mechanisms and Energy Landscapes by Single-Molecule Mechanical Unbinding Experiments

Single-molecule experiments provide fascinating possibilities for studying systems in which molecular individuality matters. Here, I describe two applications of single-molecule dynamic force spectroscopy (DFS) to study mechanisms and energy landscapes of protein binding. In the first example, DFS is used to discriminate between alternative modes of protein binding and activation – namely induced-fit and population-shift (1). In the second application, we used DFS to derive the energy landscape roughness of a bi-molecular protein complex (2). This work represents the first measurement of energy landscape roughness of biomolecules.

Literature1. Nevo, R., Brumfeld, V., Elbaum, M., Hinterdorfer, P. & Reich Z. Direct discrimination between models of protein activation by single-molecule force measurements. Biophys. J. In press. 2. Nevo, R., Brumfeld, V., Hinterdorfer, P, & Reich, Z. Direct measurement of protein energy landscape roughness. Manuscript submitted.

contact:

Associate Prof. Ziv Reich

Weizmann Institute of Science [email protected] Ha'nasi Ha'rishon 76100 Rehovot (Israel)

Nicole Reifschneider, S. Goto, Norbert A. Dencher, Frank Krause

Tissue diversity of the mitochondrial membrane proteome of Rattus norvegicus studied by peptide mass fingerprinting

Understanding the role of mitochondria not only in normal cell physiology but also in cellular dysfunction and cellular death is one of the main topics in current proteomic research. Especially blue-native polyacrylamide gel electrophoresis (BN-PAGE) combined with subsequent 2D SDS-PAGE is an ideal tool for this purpose as it is a simple method providing high throughput of various soluble and/or membrane proteins in their native state including protein-protein interactions. Mitochondria of different tissues (liver, kidney, brain, muscle) isolated from Rattus norvegicus were solubilized with the mild detergent digitonin under conditions maintaining respiratory supercomplexes and ATP synthase oligomers. The digitonin-extracts were analysed using BN-PAGE and the more gentle CN (colourless-native)-PAGE. Many mitochondrial integral membrane proteins, besides oxidative phosphorylation complexes, as well as matrix proteins were identified by trypsinization and subsequent matrix assisted laser desorption/ionisation mass spectrometry (MALDI-MS) in one gel. Using this technique a partial mitochondrial proteome map could be assembled providing physiological significant information about protein-protein interactions. Also tissue specific proteins could be observed and will be discussed.

contact:

Nicole ReifschneiderTu-Darmstadt Physikalische Biochemie [email protected] Petersenstr. 22 64287 Darmstadt (Germany)

Sascha Rexroth, Jürgen Meyer zu Tittingdorf, Frank Krause, Holger Seelert, Norbert Dencher

Identification of integral thylakoid membrane proteins from Chlamydomonas reinhardtii by peptide mass fingerprinting and MALDI-MS

Analysis of the membrane integral proteome is mainly dependent in the ability of protein separation. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a technique, so far mainly applied to mitochondrial respiratory chain, capable of efficient membrane protein separation. Applying BN-PAGE to thylakoid membranes after mild solubilisation with digitonin, we identified extremely hydrophobic subunits of the photosystem complexes with 5 - 11 transmembrane helices by trypsination and subsequent matrix assisted laser desorption / ionization - mass spectronomy (MALDI-MS). All photophosphorylation complexes, as well as their supercomplexes, from thylakoids of C. reinhardtii were resolved in a single gel, providing an analytical tool suitable to characterize composition of membrane protein supercomplexes in different physiological states. Using this technique significant differences in supercomplex composition from C. reinhardtii grown under photoautotrophic and photomixothrophic conditions can be observed. While under photoautotrophic conditions the organism is dependent on chloroplastidic energy production, under photomixotrophic growth the relevance of the mitochondrial oxidative phosphorylation is increased noticeably. In response to the physiological state the stability of thylakoid membrane protein complexes changes significantly, in addition to changes in protein expression level.

LiteratureRexroth, S., Meyer zu Tittingdorf, J.M.W., Krause, F., Dencher, N.A. and Seelert, H. Thylakoid membrane proteome at altered metabolic state: Challenging the forgotten realms of the proteome. Electrophoresis 24, 2814-2823 (2003).

contact:

Sascha RexrothTU Darmstadt Physikalische Biochemie [email protected] Petersenstr. 22 64287 Darmstadt (Deutschland)

Heidrun Rhode, M. Schulze, E. Mitre, G.A. Cumme, T. Rausch, D. Philipp, A. Horn

Turbo-mixing in microplates

Currently microplate technology has occupied all fields of rapid analysis and combinatorial chemistry, including high-throughput schedules with rapid reactions. However, sufficiently rapid mixing in microplates still represents an unsolved and underestimated or neglected problem. E.g., a microplate shaker on its highest speed takes about 50 min to produce homogeneous mixtures of samples and reagent solutions in 384-well plates. Here we introduce a mixing method using the phenomenon of Marangoni convection. After deliverance of the watery solutions to be mixed into microplate wells, complete mixing is brought about simply by spotting liquid droplets to the liquid surfaces. These drops contain water miscible organic solvents or detergents which may be selected for compatibility with sample components. In order to check method performance, the microplate wells were filled successively with layers of liquids differing markedly in pH and density, the upmost layer containing an indicator with pH dependent optical absorbance or fluorescence. Upon spotting liquid droplets to the surfaces, mixing was followed-up by optical signal change. Within wells of 96- and 384-well microplates, mixing by Marangoni convection took about one second. The amount of organic solvents necessary for instantaneous production of an ideally homogeneous mixture is small enough not to alter bioactive molecules, neither by heat of solution nor by the resulting concentration of solvent spotted. Moreover, solvents may rapidly evaporate from the mixture. If necessary, mixing may be achieved also by spotting solvent repeatedly into the same well. Applications to enzyme-activity determination and evaluation of liquid handling devices are presented.

contact:

PD Dr. med. habil Heidrun RhodeFriedrich-Schiller-Universität Institut für Biochemie I [email protected] Klinikum 07740 Jena (Deutschland)

Thomas Schulenborg, Heike Schäfer, Christian Bunse, Helmut E. Meyer, Katrin Marcus

Detection of phosphorylation sites using a hybrid triple quadrupole/linear ion trap mass spectrometer

Phosphorylation is a very prominent post-translational modification (PTM) of proteins. PTM, especially phosphorylation, plays an important role in regulation of many cellular processes, folding and localisation of proteins and the interaction of proteins (Kurosawa, 1994). One expects, that every second protein has modifications like phosphorylation or glycosylation (Sickmann et al., 2003). In general phosphorylations in signalling cascades are accompanied by dephosphorylation. Hence, the absolute quantity of phosphorylated protein species is less than 1% of the whole protein amount. Assuming one 2D-gel spot contains about 1-10 pmol protein just 10-100 fmol of the phosphorylated species is available for the proteomic analysis, what means, that identification and localisation of phosphorylation sites is still a challenge in proteome analysis today. In the presented work the localisation of phosphorylation sites of different proteins, e.g. αA-Crystallin, PKA, β-casein is shown using the 4000 Q Trap® – a new hybrid triple quadrupole/linear ion trap mass spectrometer. For analysis, the proteins were separated by 2D-PAGE or 1D-PAGE, respectively. Protein spots were excised and tryptically digested. Resulting peptides were separated by nanoRP-HPLC and the phosphorylation sites were localised after a database search using Proteinscape™ and the PTM-Explorer (both Protagen AG, Dortmund).

LiteratureKurosawa, M: Phosphorylation and dephosphorylation of protein in regulating cellular function, J Pharmacol Toxicol Methods, 1994, 31, 135-9 Sickmann, A, Mreyen, M, Meyer, HE: Mass spectrometry - a key technology in proteome research, Adv Biochem Eng Biotechnol, 2003, 83, 141-76

contact:

Dipl.-Biochem. Thomas SchulenborgRuhr-Universität Bochum Medizinisches Proteom-Center [email protected] Universitätsstr. 150 44801 Bochum (Deutschland)

Barbara Sitek, Ognjan Apostolov, Kathy Pfeiffer, Kai Stühler, Helmut E. Meyer, Angelika Eggert, Alexander Schramm

Identification of proteins differentially expressed upon neurotrophin receptor activation using DIGE and MALDI-MS

The tyrosine kinases TrkA and TrkB are members of the family of neurotrophin receptors, which mediate survival, differentiation, growth and apoptosis of neurons in response to stimulation by the neurotrophins NGF (nerve growth factor) and BDNF (brain derived neurotrophic factor), respectively. Activation of TrkA by NGF causes differentiation of neurons and neuroblastoma cells, whereas it induces proliferation of fibroblasts and apoptosis of medulloblastoma cells. Similarly, TrkB is associated with a favorable biology and good prognosis in medullary thyroid carcinoma, which is in contrast to the situation found in neuroblastoma. Since TrkA/TrkB exhibit high level of sequence similarity and use overlapping pathways for signal transduction there have to be specific effectors critical for receptor and cell-type specific response. In order to analyze biological effects of TrkA und TrkB in a defined model, we have chosen neuroblastoma cell line SY5Y stably transfected with TrkA or TrkB, respectively. We performed a proteome analysis of these cell lines in the absence or presence of their respective ligands in a time course from 0 to 24 h. The use of the recently introduced DIGE (fluorescence 2-D difference gel electrophoresis) system (Amersham Biosciences) allowed us to monitor differences in protein expression between samples in one gel. Following image analysis we have detected 22 BDNF-dependent regulated proteins (expression change > 1,5 fold compared to unstimulated cells, p < 0,05) in TrkB SY5Y cells. The analysis of TrkA SY5Y cells resulted in 9 NGF-dependent regulated proteins (ratio > 1,3 compared to unstimulated cells, p < 0,05). Differentially expressed proteins were subsequently identified by MALDI-PMF/PFF-MS analysis. Functional assignment revealed, that the majority of these proteins is involved in organisation and maintenance of cellular structures (e.g. lamin A/C).

contact:

Barbara SitekRuhr-Universität Bochum Medizinisches Proteom-Center [email protected] Universitätsstr. 150 44801 Bochum (Deutschland)

Manfred Wuhrer, Carolien A. M. Koeleman, André M. Deelder, Cornelis H. Hokke

Analysis of protein glycosylation using normal-phase nano-scale liquid chromatography-mass spectrometry of glycopeptides and oligosaccharides

Reverse-phase nano-scale online-liquid chromatography (LC) electrospray ionization mass spectrometry (MS) is widely used for the analysis of enzymatic digests in proteomics. We here show that in the normal-phase mode using an amide-column, nano-LC-MS has a big potential in glycoproteomics. Firstly, oligosaccharides released from glycoproteins can be analyzed at low femtomole sensitivity with [1] or without [2] derivatization. Oligosaccharide mixtures are separated over a one-hour range which permits the detailed characterization of the different species by MS-MS. Secondly, (glyco-)peptides derived from glycoproteins by enzymtic digestion can likewise be analysed by normal-phase nano-LC-MS, allowing the separation of various glycoforms and the characterization of both glycan and peptide moiety by multiple ion selection and fragmentation steps using ion trap MS. Taken together, normal-phase nano-LC-MS is useful for the sensitive and quick analysis of glycosylation patterns from various biological samples.

Literature[1] Wuhrer, M., Koeleman, C. A. M., Hokke, C. H., Deelder, A. M., (2004) Int. J. Mass Spec., 232, 51-57. [2] Wuhrer, M., Koeleman, C. A. M., Deelder, A. M., Hokke, C. H. (2004) Anal. Chem., 76, 873-878.

contact:

Dr Manfred WuhrerLeiden University Medical Center Biomol. Mass Spectrometry Unit [email protected] P. O. Box 9600 2300RC Leiden (The Netherlands)

new bio analytical techniques and developments

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