Abstracts

Phosphopeptide enrichment by immobilized metal ion affinity chromatography with novel cellulose sorbents

I. Feuerstein ¹ , G. Stecher ¹ , G.K. Bonn ¹, S. Morandell ² , T. Stasyk ² , H. Huang ² , D. Teis ² , L.A. Huber ^2
1 Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innsbruck, Austria
² Dept. of Histology and Molecular Cell Biology, Medical University, Innsbruck, Austria

To perform phosphoproteomics and signal transduction studies gel-independent and non-radioactive techniques are desirable. Immobilized metal ion affinity chromatography (IMAC) is such a method, suitable to selectively isolate and enrich phosphopeptides from a peptide mixture. The aim of this study is to find a fast and reliable technique to analyse phosphorylated targets of the MAPK (Mitogen activated protein kinase) signalling pathway, which is a central player in the differentiation, proliferation and survival of cells and organisms. The complexity of biological samples like a total cell lysate, requires the enrichment of phosphopeptides prior to protein-identification. Therefore, an experimental strategy for the separation of phosphoproteins using two different IMAC sorbents will be presented. One investigated sorbent - POROS 20 MC - was obtained from Perseptive BioSystems. It is based on cross-linked poly-(styrene-divinylbenzene) particles which are coupled to iminodiacetic acid.
Another material, developed in our laboratory, is based on cellulose, which was activated with epichlorhydrine [1], followed by the coupling of iminodiacetic acid to the active surface of the sorbent. The immobilized complex-forming chelating groups were saturated with ferric (III). The selectivity of both materials was demonstrated by separation and enrichment of phosphopeptides from different samples like a digest of horse myoglobin or in vitro phosphorylated MAPK1 mixed with synthetic phosphopeptides. The analysis of the separated phosphopeptides was performed by RP-HPLC-ESI-MS.
Finally, both materials show high selectivity and reproducibility. In addition the cellulose material is rather pressure stable, which makes it excellent suitable for packing in HPLC columns for preparative isolation of analytes. Finally, cellulose can be produced rather easily and at low cost. Therefore, IMAC using IDA - Fe ³+ - cellulose may offer the possibility of a cheep and satisfying method for enrichment of phosphopeptides.

[1] Toomik, R. and Toomik, P. Preparation of ferric adsorbent paper and its interaction with phosphate-containing biomolecules. Preparative Biochemistry 22(3-4), 183-97. 92.

Work in the Bonn and Huber laboratories is funded by the Austrian Genome Programme (GEN-AU) and the ministry of education, science and culture (bm:bwk).

Correspondence:
Isabel Feuerstein
Institute of Analytical Chemistry and Radiochemistry
Leopold Franzens University Innrain 52a, A-6020 Innsbruck, Austria
Tel: +43 (512)-507-5199
Fax: +43 (512)-507-2767
Email: Isabel.Feuerstein@uibk.ac.at

Functional Analysis of Proteins Involved in Lipid Metabolism in Yeast and Mammalian Cells

Olaf Merkel, Gudrun Gann, Harald Scholz, Heimo Wolinski, Klaus Natter, Mathias Kals, Regina Leber, Sepp D. Kohlwein

IMBM, SFB Biomembrane Research Center, Universität Graz, Schubertstrasse 1, 8010 Graz, Austria

Lipid-associated disorders are major health problems in industrialized countries. The approach taken in our lab in the framework of "GOLD - Genomics of Lipid-associated Disorders" makes use of a simple eukaryote, the yeast Saccharomyces cerevisiae , which is a well-established model system for molecular biological research. By comparing pathways and functions of relevant enzyme activities involved in lipid metabolism in yeast (Kohlwein lab) and mouse (Zechner lab) we aim at identifying novel genes based on sequence or functional homologies. Potential targets are analysed using an integrated profile of genomic, proteomic and metabolomic technologies, focused on lipid metabolism. This strategy involves:

1. Purification of affinity-tagged proteins for functional analyses in vitro. Particular efforts focus on enzymes involved in fatty acid transport, fatty acid activation and their incorporation into lipids, as well as on the mobilization of lipids.

2. Investigation of GFP-fusions for localization studies of the lipid proteome, directed towards characterization of the spatial organization of lipid metabolism as well as identifying lipid trafficking routes in the cell, and morphological alterations resulting from lipid/membrane defects. The yeast protein localization database (YPL.db) currently contains some 500 high resolution images of lipid enzyme localization data.

3. Characterization of transcriptional alterations in response to mutations of lipid genes or specific inhibitors (DNA arrays).

4. Development and application of protocols for medium/high-throughput lipid analyses, including automated TLC (neutral lipids), GLC (fatty acids, sterols), HPLC (neutral and phospholipids) and MS techniques (lipid molecular species).

5. Heterologous expression of homologous mammalian genes in yeast, for functional analysis and protein purification.

6. Development of a yeast lipid metabolism database, integrating data from different sources and researchers.

The integration of these experimental strategies has yielded several novel genes of unknown function with relevance to mammalian systems, that may be involved in lipid metabolism. These genes are currently undergoing detailed molecular characterization.

Correspondence:
DI. Dr. Olaf Merkel
IMBM Schubertrasse 1
Karl Franzens Universität Graz
8010 Graz, Austria
Tel: +43-316-380-5489
Email: olaf.merkel@uni-graz.at

Functional proteomic screening for lipases in mouse adipose tissue

Birner-Grünberger R. ¹ , Strauss J. ² , Susani-Etzerodt H. ¹ , Waldhuber M. ¹ , Schmidinger H. ¹ , Riesenhuber G. ¹ , Rechberger G. ² , Kollroser M. ³ , Laas A. ² , Zimmermann R. ² , Zechner R. ² and Hermetter A. ¹

¹ Institut für Biochemie, Technische Universität Graz, Austria; Institut für Molekularbiologie, Biochemie und Mikrobiologie
² , Karl-Franzens-Universität Graz, Austria; Institut für Gerichtsmedizin
³ , Karl-Franzens-Universität Graz, Austria

The goal of our joint project is to discover novel genes, processes and pathways that regulate lipid homeostasis in humans, mice and yeast being a prototype model organism in lipid metabolism. The contribution of our group in the joint project is proteome screening with specific suicide lipase inhibitors which are fluorescently or biotin labelled. Since the inhibitors covalently bind to lipases which are serine hydrolases the enzyme-inhibitor complexes can be isolated by 1D- or 2D-gelelectrophoresis, detected with a laser scanner and analyzed by mass spectrometry (nano-HPLC-iontrap) after tryptic in gel digestion. Screening of the proteome of mouse adipose tissue led to the identification of lipase-, esterase- and (lyso-)phospholipase-type enzymes. Next to known enzymes, also yet uncharacterized proteins were identified. The identification was confirmed by recognition of the transient COS-expressed enzymes by our inhibitors. Thus, our results indicate that our inhibitors specifically bind to lipolytic enzymes and can therefore be used as a powerful tool for screening for lipolytic activities in complex biological samples.

This work is financed by the GOLD (Genomics of Lipid-associated Disorders) project (http://gold.uni-graz.at/) which is one of four joint GEN-AU (GENome research in AUstria) projects (http://www.gen-au.at/) funded by the Austrian Federal Ministry for Education, Science and Culture.

Correspondence:
Ruth Birner-Grünberger, PhD
Institut für Biochemie
Technische Universitaet Graz
Petersg.12/2 A-8010 Graz
Austria
Tel.:+43316-8736464
FAX:+43316-8736952
Email: ruth.birner@tugraz.at

Quantitative mass spectrometry of histone modifications

Stefan Kubicek, Antoine H. F. M. Peters, Ines Steinmacher, Richard Imre, Susanne Opravil, Thomas Jenuwein, and Karl Mechtler

Research Institute of Molecular Pathology (IMP), The Vienna Biocenter, Dr. Bohrgasse 7, A-1030 Vienna, Austria

Histones are basic proteins that form the nucleosome core structure thereby generating the principal packaging platform for DNA. Their tails, which are extending from this structure, bear different posttranslational modifications (methylation, acetylation, phosphorylation, ubiquitination) at numerous sites. Combinations of these modifications guide the establishment of distinct chromatin domains and the existence of a histone code has been proposed, describing their involvement in the regulation of gene expression.

We developed a novel approach for qualitative and quantitative analysis of posttranslational histone modifications. Whereas routine analysis methods (Western blot, IF, ChIP) are antibody dependent, our mass spectrometry-based technique is not prone to cross-reactivities and furthermore gives quantitative information. Modification levels at different amino acid residues (e.g. H3-K9, H3-K27, H3-K36, H4-K20) and histone isoforms (H3.1/2, H3.3) can be determined, and detection of novel modification sites is possible.

Histones isolated by SDS-PAGE were subjected to a chemical modification of all unmodified and mono-methylated lysines by propionylation. This procedure led to the generation of fragments of equal length - independent of the original lysine-modification status - after a tryptic digest. Generating similar peptides was a prerequisite for the following quantification by nano-HPLC separation of fragments and comparing peak areas in ESI-MS spectra to external standards. In particular, in histones isolated from murine ES cells deficient for the Suv39h HMTases, we observed a significant reduction of the H3-K9 tri-methylation level, that is characteristic for these enzymes as defined by antibody-based approaches. Furthermore, we could quantify combinations of different modifications (K9 and K14, K27 and K36) within the same histone tail and we identified four novel methylation sites on murine histone H3 (K18, K56, K64, K122).

The method described here provides an unbiased approach to investigate genome-wide changes in histone modifications. It will help to define in vivo enzyme target specificities of the numerous histone-modifying enzymes, as well as the nucleosomal changes involved in discriminating different cell states.

Correspondence:
Stefan Kubicek
Research Institute of Molecular Pathology (IMP)
Dr. Bohrgasse 7
A-1030 Wien
AUSTRIA
Tel: +43-1-79730-523
F ax: +43-1-798-7153
email: kubicek@nt.imp.univie.ac.at

SCREEN-OUT - A Method for Selected Mutagenesis

Natalia Khilkevitch, Mathias Müller, Gottfried Brem and Thomas Czerny

Institute of Animal Breeding and Genetics, University for Veterinary Medicine, Vienna, Austria

Recent progress in genome projects has driven the need for functional annotation of genomes in model species. A solution to this problem would be a gene-driven approach based on chemical mutagenesis. Critical for the application of such a method is to find a reliable and inexpensive technique to identify unknown mutations randomly introduced by a mutagen. The aim of this project is to detect mutations in F1 medaka fish, which originate from ENU-treated males, using a novel mutation detection method (Screen-out). The principle of this method is to test a gene fragment in a E. coli system for the integrity of a reading frame. A sequence amplified by PCR (genomic DNA or cDNA) is inserted into a plasmid vector upstream of a toxin gene. A stop-codon in the test reading frame consequently prevents the synthesis of a fusion protein containing a toxic tag. The presence of a nonsense mutation is therefore revealed by growth of colonies, whereas wild type sequences inserted into the vector result in death of the bacteria. We have initiated a pilot screen in medaka F1 fish concentrating on candidate genes involved in early eye development. The Screen-out procedure thus represents a reverse genetics approach, with the potential to analyse gene function in a large scale.

Correspondence:
Natalia Khilkevitch
Institut für Tierzucht und Genetik
Veterinärmedizinische Universität Wien
Josef Baumann Gasse 1
A-1210 Wien
Austria/Europe
Phone: +43-1-25077-5604
Fax: +43-1-25077-5693
Email: natalia.khilkevitch@i122server.vu-wien.ac.at

Automated Nanoelectrospray Mass Spectrometry for the Detection of Noncovalent Protein-Protein Interactions

Sheng Zhang ¹ , Colleen K. Van Pelt ¹ , and Joerg Niebel ²

¹ Advion BioSciences, Ithaca, New York, USA,
² Advion BioSciences Ltd, Wachenheim, Germany

Studies on noncovalent interactions have mainly focused on the binding of relatively small molecules to enzymes, or with protein-protein interactions in natural oligomers. Application of nanoESI/MS to the study of noncovalent protein complexes formed between two different proteins is limited. As progress in the proteomics field is made, protein complex studies are becoming more popular as they are important not only for drug discovery but functional proteomics. A complete understanding of proteome relies on characterizing the interactions of proteins with other macromolecules. Proteins function in cell is most often mediated by protein-protein interactions that are central to many cellular activities.

The advantages of ESI/MS over other techniques in protein complex studies include high sensitivity, speed of analysis and the ability to identify unknown proteins. In analysis of quantity-limited protein samples for noncovalent binding studies, nanoelectrospray MS is highly desirable. The purpose of this work was to demonstrate if an automated nanoelectrospray system coupled to a mass spectrometer could reliably be used to study noncovalent protein- protein interactions

The interaction of trypsin inhibitors to their target enzymes has been extensively studied and provides an ideal model for studying protein-protein interactions by nanoESI/MS. Two target proteins (bovine trypsin and trypsinogen) were used for determination of noncovalent binding interactions with bovine pancreatic trypsin inhibitor (BPTI) and soy bean trypsin inhibitor (SPTI) respectively. BPTI or SPTI was mixed with trypsin and trypsinogen respectively in 10 mM ammonium acetate solution prior to automated nanoESI/MS analysis using a NanoMate coupled with a Q-TOF micro MS.

The experimental results demonstrate that automated nanoESI/MS offers an important new platform for detecting noncovalent interactions between proteins. This automated chip-based nanoelectrospray system used for studying protein complex not only reduces sample consumption, increases analysis speed, but also dramatically improves the efficiency and quality of the ESI/MS method.

Correspondence:
Linda Allen
Marketing Specialist
Advion BioSciences Ltd
Rowan House
28 Queens Road
Hethersett Norwich
NR9 3DB
UK
Tel; +44 (0)1603 813970
Fax: +44 (0)1603 813971
Email: allenl@advion.com

Large Scale Expression Profiling and Functional Annotation of Adipocyte Differentiation

H Hackl ¹, T Burkard ¹, C Paar ¹, R Fiedler ¹, A Sturn ¹, G Stocker ¹, RM Rubio ², J Quackenbush ², A Schleiffer ³, F Eisenhaber ³, and Z Trajanoski ¹

¹ Institute of Biomedical Engineering and Christian Doppler Laboratory for Genomics and Bioinformatics, Graz University of Technology, 8010 Graz, Austria
² The Institute for Genomic Research, Rockville, MD 20850, USA
³ Research Institute of Molecular Pathology, 1030 Vienna, Austria

Distinct profiles of gene expression mirror the complex molecular mechanisms that regulate development during cellular differentiation and throughout life. To study the key events and processes in adipogenesis we analyzed the gene expression of 3T3-L1 cell line during differentiation from fibroblast like preadipocytes to mature adipocytes with a 27.648 element focused murine cDNA microarray, comprising adipose specific genes and ESTs from early embryonic stages (NIA 15k clone set). Three independent time series experiments of 3T3-L1 adipocyte differentiation were performed in reference design. RNA from 8 time points (0d, 6h, 12h, 24h, 2d, 3d, 7d, 14d) was hybridized against RNA from the preconfluent stage to study the gene expression profile over the whole differentiation process. 780 genes found to be more than twofold up- or downregulated in at least 4 timepoints in comparison to the preconfluent stage were selected for further analysis. Clustering algorithms were performed (k-means, principal component analysis) to categorize these genes by their expression course. Additional, functional annotation of the identified ESTs was performed using a novel system for computational analyses. Many known and unknown differentially expressed genes in the mitotic clonal expansion phase and the late terminal adipocyte differentiation could be identified and confirmed by Real Time PCR. Due to the focused approach and a novel thorough functional annotation process new promising targets were revealed. Further computational analysis of the promoter sequences and experiments with RNAi of the selected targets are currently under way to provide novel insights into the regulatory mechanisms of adipogenesis.

Correspondence:
Hubert Hackl
Institute of Biomedical Engineering
Graz University of Technology
Krenngasse 37, 8010 Graz, Austria
Tel +43-316-873-5345
Fax +43-316-873-5340
Email hubert.hackl@tugraz.at

The inflammatory Glycosylaminoglycan-Binding Proteome

B. Gesslbauer, M. Kollroser ¹ , G. Chiarandini, A.J. Kungl

Institute of Pharmaceutical Chemistry and Pharmaceutical Technology, University of Graz, Universitätsplatz 1, 8010 Graz and
¹ Institute of Forensic Medicine, University of Graz, Universitätsplatz 3, 8010 Graz

Glycosylaminoglycans (GAGs) such as heparan sulfate are complex and highly charged polysaccharides which were found to participate in several (patho-) physiological processes by specifically interacting with proteins. These proteins represent therefore potential targets for a manifold of therapeutic interventions. In a first step we have analysed the entire GAG binding proteome of endothelial cells by 2-D chromatography (ion exchange and reversed phase) followed by nanospray-MS/MS peptide analysis. By this means, 344 proteins were reliably identified in one experiment. In order to identify glycosylaminoglycan-binding proteins that are involved in inflammatory processes we have optimised a proteomics protocol which is based on 2D-gel-electrophoresis followed by tryptic in-gel digestion and peptide sequencing performed with nano-HPLC ESI-MS/MS. For this purpose, proteins from TNF- a stimulated and unstimulated human umbilical vein endothelial cells were pre-fractionated by affinity chromatography using heparin-agarose. The resulting proteins were separated in the first dimension by isoelectric focusing followed by SDS-PAGE in the second dimension. After image analysis, upregulated protein spots were excised and digested with trypsin. Extracted peptides were concentrated on a reversed phase pre-column. After a washing step, in order to remove contaminants and salts, the pre-column was brought in-line with a nano-column and the mass spectrometer. By this means, the peptides were eluted from the pre-column onto the analytical column using a step gradient for separation and ion trap detection. MS/MS spectra were recorded in the full scan mode followed by database search for matching peptide sequences using Mascot. The sensitivity of this method was found to be in the low femtomol range. Several TNF- α upregulated glycosylaminoglycan-binding proteins have been identified by this approach. Our results represent a further step towards the molecular characterisation of primary inflammatory responses yielding a yet to be extended number of potential therapeutic targets. (This work was supported by the European Commission, grant no. QLK3-CT-2001-01976 and by the GEN-AU programme of the Austrian Federal Ministry for Education, Science and Culture).

Correspondence:
Andreas J. Kungl, PhD
Professor for Biophysical Chemistry
Institute of Pharmaceutical Chemistry and Pharmaceutical Technology
University of Graz
Universitätsplatz 1
A-8010 Graz, Austria
Tel: +43 316 380 5373
Fax: +43 316 382541
E-Mail: andreas.kungl@kfunigraz.ac.at

Platelet proteomics: a tool to analyse age-related changes in the protein expression profile

Zellner M. ¹ ,Winkler W. ¹, Miller I. ², Chang M ³, Grillari J ³, and Oehler R. ^1,4

¹ Surgical Research Laboratories, University of Vienna,
² Austrian Proteomics Platform -Institute of Medical Chemistry, University of Veterinary Medicine Vienna, and
³ University of Natural Resources and Applied Life Science Vienna, Austria

Increased reactivity of blood platelets can be observed in a number of different disease states including arteriosclerosis, diabetes, obesity, septic shock as well as in aged persons. This effect is mediated by a decrease in the activation threshold of these cells due to an imbalance between reactive oxygen species and the cellular anti-oxidative capacity. Oxidative stress in platelets is therefore assumed to be a major contributor to the high risk of thrombotic complications in these patients. The presented study characterises modifications of platelet protein expression profile during ageing. We isolated platelets from peripheral venous blood of 15 aged and 15 young subjects (mean age 85 and 25 respectively). Blood sampling under well defined clinical conditions and immediate cell preparation turned out to be crucial for the success of the entire study. To analyse proteome modifications we applied the novel fluorescence difference gel electrophoresis system DIGE. The fluorescence images of the gels were analysed by the DeCyder software package of Amersham. The 2D-gel electrophoresis was highly reproducible (cv = 4.7%) and 1400 spots were detected repeatedly in every gel of the same group. Comparison of the two groups revealed several distinct statistically significant changes (p < 0.05): 7 spots were increased (1.5 to 2.5-fold) and 4 spots were decreased (1.4 to 1.6-fold) in aged subjects. The study shows that platelets from aged subjects have a different protein expression and modification profile in comparison to young control. The identity of the changed proteins will be determined soon by mass spectrometry. The relationship of these proteins to oxidative stress will be investigated in future studies by comparison with proteome changes in other disease states and in in-vitro oxidative stress experiments.

Correspondence:
Rudolf Oehler, Ph. D.
Associate Professor of Medical Chemistry
Surgical Research Laboratories, University of Vienna
Allgemeines Krankenhaus der Stadt Wien
Level 8, Leitstelle 8H, door 8G9-13
Waehringer Guertel 18-20
A-1090 Vienna, Austria
Tel.:+43-1-40400-6979
Fax:+43-1-40400-6782
E-Mail: rudolf.oehler@akh-wien.ac.at

Characterization of the Biological Effects of IGF1/2 Overexpression in Human Breast Cancer Cells: A Link from Transcription Profiles to Functional Biology

Pacher M, Mogg M, Schweifer N, Mikula M, Eger A, Mikulits W, Kubista E, and Schreiber M

Dept. of Obstetrics & Gynecology, University of Vienna and Ludwig Boltzmann Institute of Clinical and Experimental Oncology

Insulin-like growth factor (IGF)-mediated proliferation and survival are essential for normal development and function in the mammary gland. Hyperactivation of IGF signaling is known to play an important role in human breast cancer. To clarify the molecular mechanism by which IGF signaling promotes breast cancer, we generated stable breast cancer cell lines (MCF-7 derivatives) overexpressing IGF1 or IGF2 more than forty-fold at the protein level. Total RNA from these cell lines was analyzed using Affymetrix U133A+B-GeneChips. Among the genes upregulated are known target genes of the IGF system (e.g. VEGF), as well as unknown or unexpected genes (amino acid transporters, genes involved in aminoacid biosynthesis). IGF1/2 overexpressing cells exhibit accelerated, serum independent proliferation, increased cell size, and increased malignancy. Western blot analysis with phosphospecific antibodies and kinase assays show a strong activation of the PI3K-Akt pathway, but only a moderate constitutive activation of the MAPK-pathway. Inhibition of the Akt-pathway with the PI3K inhibitor LY294002 neutralizes the growth advantage of IGF overexpresing cells, demonstrating that the Akt-pathway mediates the growth-promoting function of IGFs. Biochemical studies to unravel the signaling pathways downstream of Akt have identified 4E-BP1, a regulator of translational initiation, as the major Akt target responsible for enhanced growth of IGF1/2 overexpressing cell lines. Thus, deregulation of translational initiation is the cellular event leading to transformation and enhanced malignancy.

Correspondence:
Mag. Margit Pacher-Zavisin
Univ. Klinik fuer Frauenheilkunde
AKH Wien
Waehringer Guertel 18-20
A-1090 Vienna, Austria
Tel.: ++43/ +1/ 40400-7822
E-Mail: margit.pacher-zavisin@akh-wien.ac.at

Are telomerase overexpressing endothelial cells young?

Chang M^*, Grillari J^*, Mayrhofer C⁺, Fortschegger K^*, Allmaier G⁺, Katinger H^* and Voglauer R^*

^*Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna
⁺Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, A-1060 Vienna

Serial passaging of human primary cells in vitro ends up in an irreversible growth arrest, called replicative senescence.The main cause for the limited lifespan of normal cells is supposed to be shortening of telomeres, since overexpression of the catalytic subunit of human telomerase (hTERT), that prevents this telomere erosion, leads to immortalization of a variety of primary cells. Additionally, ectopic telomerase expression maintains the non-tumorigenic and highly differentiated phenotype of the normal counterparts. However, changes of the whole protein profile associated with telomerase overexpression have not been under investigation yet. Therefore, we have transfected human umbilical vein endothelial cells (HUVECs) with plasmids containing hTERT and a vector control. While the telomerase negative control cells reached replicative senescence 15 population doublings after transfection (PDpT), hTERT overexpressing cells have reached 100 PDpT so far and are still growing. For the determination of the differentiation grade, we performed a phenotypic characterization including staining for endothelial cell markers and simulation of the inflammation process by TNF-α. Additionally we analyzed the cells for their potential for neoangiogenesis and for transformation. Together with young subconfluent, young quiescent and senescent HUVECs, our newly established hTERT overexpressing cell line was investigated using 2-dimensional-fluorescence-difference-gel-electrophoresis. We identified proteins showing similar expression levels in hTERT and young cells as opposed to senescent cells like cytokeratin 7, reticulocalbin 1, calumenin and glutathion S-transferase. However, we also found proteins changed equally in immortalized and in senescent HUVECs compared to early passage cells (carbohydrate (chondroitin 6) sulfotransferase 3, F-box only protein 21, Era G-protein-like 1). Additionally, our characterization of the hTERT cell line showed an overall young phenotype, but in the case of simulation of an inflammation we observed an intermediate stage. This leads us to the conclusion that hTERT overexpression extends the lifespan of HUVECs, sustains an endothelial cell specific differentiated phenotype, but does not maintain an early passage nature.

Correspondence:
DI Martina Chang
Institute of Applied Microbiology
Muthgasse 18
A-1190 Vienna
AUSTRIA
Tel: +43 1 36006 6805
Fax: +43 1 3697615
E-mail: h9240248@edv2.boku.ac.at

Interaction of Two Parkinson-Linked Gene Products: O- Glycosylation of the Human Brain-Derived Substrate a-Synuclein p22 Is Required for Ubiquitination by Parkin.

Michael Schlossmacher ¹, Hideki Shimura ¹ , Mark Sutton-Smith ², Jennifer Chan ³, Howard Morris ², Anne Dell ², Kenneth Kosik¹ and Dennis Selkoe ¹.

¹ Center for Neurologic Diseases, Department of Neurology and
³ Department of Pathology, Brigham & Women's Hospital, Harvard Medical School, Boston, USA; and the
² Division of Glycobiology, Department of Biochemistry, Imperial College, London, UK.

We examined the modification of an a-synuclein (aS) variant, aSp22, and the relation to its cognate E3 ubiquitin ligase, parkin. Both proteins are linked to heritable forms of Parkinson disease. aSp22 was present in primate (but not rodent) brain, occurring as 21-22 kDa isoforms. Lectin-binding analyses and enzymatic digestions revealed a complex glycosylation of human aSp22: b (1-3)-N-acetylgalactosamine disaccharide with terminal neuraminic acid sugar(s). Intact sialylation was required for the binding to and poly-ubiquitination of aSp22 by wild-type (but not mutant) parkin proteins in vitro . Accordingly, the homozygous inheritance of parkin deletions led to the accumulation of non-ubiquitinated forms of aSp22 in 4 / 4 human brains and the development of young-onset Parkinson disease (Shimura et al., Science 2001). Surprisingly, parkin gene inactivation in mouse brain did not result in a parkinsonian phenotype (Goldberg et al., J. Biol. Chem., 2003). Purification of human aSp22 to apparent homogeneity and comprehensive mass spectrometry confirmed the presence of full-length, human aS and revealed variable Met-oxidation events. Extracts of Substantia nigra from human control brain, the site of greatest pathology in Parkinson disease, contained the highest amount of detectable aSp22. Our data identify in the O- glycosylation of aSp22 a primate brain modification that is required for binding to parkin. Therefore, aSp22 may represent a neuroanatomic, biochemical and functional link between two Parkinson disease genes in the human (but not) rodent brain.

Correspondence:
Michael Schlossmacher, M.D.
Center for Neurologic Diseases
Brigham & Women's Hospital
Harvard Institutes of Medicine #764
77, Avenue Louis Pasteur
Boston, MA. 02115   USA
Tel.: (617).525-523
Fax: (617).525-5252
E-mail: mschlossmacher@rics.bwh.harvard.edu

Parasitic infection in horses: Serum protein pattern changes during infestation and treatment

Miller, I. ¹ , Duvigneau, C. ¹, Hartl, R. ¹, Dobretsberger, M. ,sup>2, Sieber, K. ³, Prosl, H. ³, Gemeiner, M. ¹

¹ Institut für Medizinische Chemie, Veterinärmedizinische Universität Wien
² Lehr- und Forschungsgut, Veterinärmedizinische Universität Wien
³ Institut für Parasitologie und Zoologie, Veterinärmedizinische Universität Wien

Intestinal parasitic infection may have considerable to major impact on the health of horses: clinical symptoms range from mild inflammatory enteropathy to decreased levels of performance or growth as well as to severe weight loss and debilitation. Regular anthelmintic treatment is therefore known to contribute to the animals' well-being. One of the drugs used is ivermectin, which is highly effective against a broad spectrum of equine intestinal parasites in different developmental stages. It is also discussed whether or not it has immunomodulatory properties, which could facilitate the defence mechanisms against nematodes. A group of six horses was subjected to oral treatment with ivermectin, blood samples were collected in regular intervals from day 0 to 14. Effectivity of the treatment was checked by parasitological investigations (nematode egg counts from feces). Besides determining haematological values as well as cytokine and IgE levels, serum protein patterns were studied as a function of time. Method of choice for the proteomic approach was differential gel electrophoresis using pre-electrophoretic labelling of the samples with CyDyes. Special interest was paid to changes in acute phase proteins (e.g. SAA, haptoglobin) and immunoglobulin patterns (e.g. IgG(T), a subclass unique in horses).

Correspondence:
Ing. Ingrid Miller
Institut für Med. Chemie
Veterinärmedizinische Universität Wien
Veterinärplatz 1 A-1210 Vienna Austria
Tel. +431-25077-4224
Fax +431-25077-4290
E-Mail: ingrid.miller@vu-wien.ac.at

Poster Presentation

Characterisation and isolation of lipolytic enzymes using a fluorescent activity tag

Waldhuber M, Schmidinger H, Susani-Etzerodt H, Birner-Grünberger R, Hermetter A

Institut für Biochemie, Technische Universität Graz, Austria

In Western Europe, more than 50% of the population is overweight, and throughout the world about twice as many people - approximately 15 million per year - die from cardiovascular diseases, such as heart attack and stroke, than die from cancer. Dysregulation of the vascular and cellular metabolism of lipids and the excessive deposition of neutral lipids in adipocytes and the arterial wall are causally involved in the pathogenesis of atherosclerosis and obesity. The goal of our joint project is to discover novel genes, processes and pathways, which regulate lipid homeostasis in humans, mice and yeast being a prototype model organism in lipid metabolism. The contribution of our group in the joint project is proteome screening with specific suicide lipase inhibitors, which are fluorescently labelled. Since the inhibitors covalently bind to lipases, which are serine hydrolases, the enzyme-inhibitor complexes can be resolved by 1D- or 2D-gelelectrophoresis, and detected with a laser scanner. One inhibitor, namely O-((6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl) aminoethyl- O-( p -nitrophenyl) n -hexylphosphonate (NBD-HE-HP), is able to label isolated lipases and esterases. Moreover it inhibits lipolytic activity of mouse adipose tissue and labels the known lipase in adipose tissue, hormone sensitive lipase. Thus NBD-HE-HP can be used as a powerful tool for screening for lipolytic activities in complex biological samples. This work is financed by the GOLD (Genomics of Lipid-associated Disorders) project (http://gold.uni-graz.at/), which is one of four joint GEN-AU (GENome research in AUstria) projects (http://www.gen-au.at/) funded by the Austrian Federal Ministry for Education, Science and Culture.

Correspondence:
Markus Waldhuber
Department of Biochemistry
University of Technology Graz
Petersgasse 12/2 8010 Graz
Phone: 0043-316-873/6465
Fax: 0043-316-873/6952
E-Mail: wood@sbox.tugraz.at

Poster Presentation

Initial proteomic investigations on Coxiella burnetii , the causative agent of Q fever

L. Skultety ¹, L. Hernychova ², J. Stulik ², M. Kroca ³, A. Macela ², R.Toman ¹

¹ Institute of Virology, Slovak Academy of Sciences, 845 05 Bratislava, Slovak Republic,
² Purkyne Military Medical Academy, 500 01 Hradec Kralove, Czech Republic,
³ Central Military Medical Institute, 561 66 Techonin, Czech Republic

Coxiella burnetii is the etiological agent of Q fever, which belongs to the most frequent diseases of rickettsial origin in the world. The most common, acute form of Q fever, is characterized as a flue-like illness or atypical pneumonia, or less frequently as granulomatous hepatitis, with a significant incidence of neurologic complications. Persistent infections in humans may lead to chronic form of Q fever, which may be associated with endocarditis, and consequently, C. burnetii -induced endocarditis is often fatal. Q fever is a zoonosis which affects feral and domestic large and small animals. The microbe is transmitted by contact with animal faeces, urine, blood and pregnancy fluids or by inhaling dust from infected premises. It is extremely resistant to harsh environmental conditions due to spore formation, it readily becomes airborne, and it is highly infectious for humans. Because of these properties, C. burnetii is currently at the list of biological warfare (BW) agents in "Category B". There have been ongoing studies on the chemical composition and structure of the C. burnetii lipopolysaccharide. However, there is a lack of information on the proteins that represent the major immunoreactive antigens in the serological diagnosis of Q fever. Proteomic studies could bring new data in this respect, which could be applied to detection/diagnosis and prophylaxis of the agent/disease. Our initial investigations have focused on mapping of the C. burnetii proteins. Thus, the whole cell lyzate of the bacterium was run on 2D SDS-PAGE electrophoresis and eight major spots were selected for MS analyses. Based on MS data and subsequent search in the protein database, the following proteins were fully identified: chaperone protein DnaK, chaperonin 60 kDa, chaperonin 10 kDa, ribosomal protein L7/L12, and DnaJ-like protein dj1A (mucoidy activation protein mucZ).

Correspondence
Dr. Rudolf Toman
Head of Department of Rickettsiology and Chlamydiology
Institute of Virology, Slovak Academy of Sciences
Dubravska cesta 9
845 05 Bratislava
Slovak Republic
Tel: +421-2-59302418
Fax: +421-2-54774284
E-Mail: virutoma@savba.sk

Microwave-assisted protein hydrolysis with formic acid - a rapid and selective alternative to trypsin as the cleaving reagent.

Rechberger G.N. ¹, Desai B. ² , Kappe C.O. ² , Kollroser M. ³

¹ Institute of Molecular Biology, Biochemistry and Microbiology, Karl-Franzens-University Graz, Heinrichstrasse 31a, A-8010 Graz
² Institute of Chemistry, Karl-Franzens- University Graz, Heinrichstrasse 28, A-8010 Graz
³ Institute of Forensic Medicine, Karl-Franzens- University Graz, Universitätsplatz 4, A-8010 Graz

The microwave enhanced tryptic digestion of proteins both in-gel and in-solution has recently been published [Prot. Sci. (2002), 11:2676-2687]. We were able to reproduce the significant acceleration of the enzymatic proteolysis process. In order to provide a constant microwave irradiation to the sample the applied power of the microwave reactor was fixed while the temperature was raised up to 60°C. A nano-HPLC-system and a LCQ™ Deca XP ion trap mass spectrometer for peptide analysis were used. After irradiation at the BSA/trypsin solution with 20W for 1 minute we obtained a sequence coverage of 18% of albumin.

In the next step, we combined the advantages of microwave with formic acid as a selective cleavage agent. Li et al. [Anal. Chem. (2001), 73:5395-5402] hydrolysed BSA with 2% formic acid in solution for 2 hours at 108°C and achieved a sequence coverage of 30%. In preliminary experiments, we obtained a sequence coverage of 19% out of an in-solution digest after 10 minutes of microwave irradiation at 140°C.

Correspondence:
Dr. Gerald N. Rechberger
Institute of Molecular Biology, Biochemistry & Microbiology (IMBM)
Karl-Franzens University Graz
Heinrichstraße 31a
A8010 Graz
Austria
Tel. : +43/(0)316/380-1933
Fax   :+43/(0)316/380-9016
Email: gerald.rechberger@uni-graz.at

Cap-independent Translation of Tumor-specific Transcripts

Daniela Kozina, Christian Haslinger and Wolfgang Sommergruber

Boehringer Ingelheim Austria GmbH, A-1121 Wien, Austria

Translation in eukaryotic cells is normally initiated by ribosome scanning from the 5' end of the capped mRNA. However, under stress conditions such as heat shock or in highly proliferating (tumor) cells initiation of translation may also occur via a mechanism which is independent of the cap structure - in this case ribosomes are directed to the start codon via an internal ribosome entry site (IRES).
In many viruses cap-independent translation is facilitated by such an IRES-motif containing a poly-pyrimidine stretch and exhibiting a pronounced secondary structure in the 5'-UTR (untranslated region), which enables a direct interaction of RNA with ribosomes in the absence of cap-recognition (cleavage of eIF-4G). This allows fast recruitment of transcripts for an enhanced translation. For instance, c-myc, a proto-oncogene, is regulated by deadenylation and IRES-regions. C-myc mRNA contains two initiation codons (GUG and AUG), 4 alternative promoters (P0-P3) leading to different 5'-UTRs with or without IRES-regions. The long-size 5'-UTRs (P0 and P3) are used for cap-independent translation as detected in Burkitt's lymphoma.
Additional trans-acting factors support the shift from cap-dependent towards the cap-independent translation in virally infected cells and this very likely holds true for transformed cells.

To date only a few examples are known where genes become cap-independently translated in eukaryotic cells e.g. VEGF (Vascular endothelial growth factor), Apaf-1 (Apoptotic protease activating factor) and FGF2 (Fibroblast growth factor 2).
Very little is known about utilisation of this mechanism in tumor cells.

Correspondence:
Daniela Kozina
Boehringer Ingelheim Austria GmbH
Dr. Boehringergasse 5-11
A-1121 Wien
Austria
Tel: +43-1-80105-2474
Email: daniela.kozina@vie.boehringer-ingelheim.com

Characterization of E2-family enzymes UBCH10 and UBCH5

Sebastian Glatt, Wolfgang Sommergruber and Michael Gmachl

Boehringer Ingelheim Austria, Dr. Boehringer Gasse 5-11, A-1121 Vienna, Austria

The Anaphase Promoting Complex (APC) is a multi-protein complex displaying an E3 protein ubiquitin ligase activity essential for mitotic entry and exit. Critical target proteins, among others, are cyclin A, cyclin B and securin, which are crucial for the cell to pass through mitosis. APC performs its activity (ubiquitination of target proteins) with the aid of a ubiquitin activating enzyme (UBA1) and one of two different ubiquitin conjugating enzymes (UBCH5 and/or UBCH10). Recently, reconstitution experiments using individual APC subunits identified one subunit (Apc11) as the catalytically active one. This subunit harbors the ability to form long polyubiquitin chains. However, this core activity differs markedly from the reaction catalyzed by the whole APC (holoAPC). One of these differences observed is the failure of UBCH10, but not UBCH5, to support the APC11 catalyzed formation of polyubiquitin chains.
We could show, using an siRNA approach, that the APC degrades crucial mitotic target proteins exclusively with the aid of UBCH10 in vivo. In contrast to previous studies, using a dominate negative form of UBCH10, we could outline the importance of the mitosis-specific E2 enzyme UbcH10 for cell survival, progression through mitosis, and the inability of UBCH5 to complement the mitotic functions of UbcH10 in vivo.
The intrinsic functions of the N-terminal extension (Nxt) of UbcH10 were characterized in vitro, by using UbcH5-Nxt fusion proteins and UbcH10 lacking the extension. In contrast to previous studies we proofed the interaction of both E2s with the two APC-subunits Apc2 and Apc11, which supports the idea of a Apc2/11 ligase core module.
I could show that the N-terminal extension has no effect on these interactions, but the UbcH5-Nxt fusion proteins produced polyubiquitin chains of higher molecular mass, allowing the conclusion, that the N-terminal extension itself has an effect on processivity and ubiquitin chain assembly.

Correspondence:
Sebastian Glatt
Boehringer Ingelheim Austria GmbH
Dr. Boehringergasse 5-11
A-1121 Wien
Austri
Email: sebastian.glatt@vie.boehringer-ingelheim.com

The use of monolithic capillary columns in 2D-HPLC for prostate cancer biomarker discovery

Wolfgang Walcher ^*, Kurt Kofler^**, Christian W. Huck^*, Helmut Klocker^**, Georg Bartsch^**, Günther K. Bonn^*

* Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens-University Innsbruck, A-6020 Innsbruck, Austria
** Department of Urology, University Hospital Innsbruck, A-6020 Innsbruck, Austria

A 2D-HPLC approach is tested for its capability of prostate cancer biomarker discovery in a collection of 200,000 blood sera, for which detailed patient information and long-term follow-up are available. The 2D-HPLC system consists of a strong cation exchange column (100 x 0.30 mm i.d., Poros 10S), a 200 µm i.d. PS-DVB Monolith (20 x 0.20 mm i.d., trap column) and a 100-µm i.d. PS-DVB Monolith (60 x 0.10 mm i.d., separation column). The use of a cation exchange column enables the fractionation of the sample. The 200-µm i.d. reversed-phase column allows concentration and desalting and the 100-µm i.d. RP column establishes a high efficiency separation of peptides and proteins. The 100-µm i.d. PS-DVB monolithic capillary columns generate peak capacities of 84 and 137 for proteins and peptides, respectively. Additionally to the unique separation efficiency, peak volumes of about 50 nanoliter (100-µm i.d. PS-DVB Monolith) facilitate outstanding detection limits. For further investigation of peptides and proteins the HPLC peaks were fractionated onto a MALDI target by the use of a robot (PROBOT, LC-Packings a Dionex company) and transferred to a MALDI-TOF-TOF mass spectrometer.

Correspondence:
Univ.-Prof. Dr. Mag. Günther Bonn
Chair
Institute of Analytical Chemistry and Radiochemistry
Leopold-Franzens-University Innrain 52a, A-6020 Innsbruck, Austria
Tel: +43 (512)-507- 5171
Fax: +43 (512)-507-580519
Email: guenther.bonn@uibk.ac.at

Protein phosphorylation

Karl Mechtler

Institute of Molecular Pathology, Dr. Bohr-Gasse 7, 1030 Vienna, Austria

Proteins communicate with each other through post-translational modifications, and locating modified sites is a key challenge in proteomics. Phosphorylation, the most common modification by far, is central the cell signaling, and knowledge of where and when proteins are phosphorylated could help researchers decipher disease mechanisms, including those for cancer and diabetes. The most commonly used method for mapping such sites is tryptic digestion of protein, followed by tandem mass spectrometry of the resulting fragments. This technique can fail, however, when used to analyze low-abundance proteins or proteins with substoichiometric phosphorylation (that is, not all of the molecules are phosphorylated).
With a combination of "shotgun 2 D separation" and special digest methods we are able to find about more than 150 phosphorylation sites in a protein complex.

Correspondence:
Karl Mechtler
Research Institute of Molecular Pathology
Dr. Bohr-Gasse 7 A-1030 Vienna Austria
Tel: +43 (1) 79730
Fax: +43 (1) 7987153
Email: mechtler@imp.univie.ac.at

Approaches towards the stem cell proteome

Andreas J. Kungl ¹, Christoph Zenzmaier ², Bernd Gesslbauer ¹, Nina Grobuschek ¹, Karl-Heinz Preisegger ², Anita Jandrositz ²

¹ Institute for Pharmaceutical Chemistry and Pharmaceutical Technology, University of Graz, Austria
² Lifecord Inc., Graz, Austria

The proteome of stem cells is a challenging target for various reasons: (i) to find new surface (protein) markers which characterise stem cells and which can be used for stem cell identification/isolation (in addition to known proteins such as CD34); (ii) to identify new (protein) expansion factors in the serum of stem cells which enable for multiplying stem cells without loosing their pluripotent character; (iii) to unravel the protein interaction network of stem cells which is responsible for pluripotency. We have therefore investigated the proteome of human umbilical cord blood stem cells (UCBSC) using two different approaches, namely 2-D gel electrophoresis and 2-D chromatography [1]. Out of 51 matching spots obtained from five different UCBSC samples on 2-D gels (pH 4 -7), 20 proteins were identifed by trypsin in-gel digestion followed by nano-rpHPLC and MS/MS detection. Using cation exchange and reversed phase HPLC for separating peptides obtained from the trypsin solution digest of a UCBSC raw lysate, 215 proteins were identified by MS/MS detection. The details of the methods and the biological context of the proteins will be discussed.

References

Bioinformatic Solutions for High-Throughput Proteomics

Magnetic-Bead based Sample Fractionation and Purification prior to MALDI-TOF Analysis for Clinical Proteomic Approaches

Markus Kostrzewa*, Wolfgang Pusch, Dirk Peters*, Arnd Ingendoh, Arndt Asperger*

*Bruker Daltonik GmbH, Permoserstrasse 15, 04318 Leipzig, Germany
Bruker Daltonik GmbH, Fahrenheitstrasse 4, 28359 Bremen, Germany

Proteomic profiling by MALDI-TOF has been shown to be a promising tool for the identification of disease markers. For the analysis of the enormous diversity of peptides and proteins in crude human samples by mass spectrometry, an appropriate sample conditioning is necessary to reduce the sample complexity and to remove contaminants, which would interfere with the MS analysis.
Described here is a protocol for sample preparation based on magnetic particles. Proteins and peptides are bound to the functionalized surface of super-paramagnetic microparticles in the presence of appropriate binding solutions. Non-bound components are removed by subsequent wash steps, before the analytes are eluted and prepared on a MALDI sample target.
The major strengths of the method are speed, sensitivity, ease of use, adaptability to any desired sample volume and a high flexibility (e.g., different steps of fractionation can be easily coupled to each other). The availability of magnetic beads of various types of surface functionality, including C ₃ , C ₈ , C ₁₈ and IMAC , allows further optimisation of the technique with respect to the nature of the protein investigated and the scope of analysis.
Using this method, more than 200 peptides/proteins in the mass range between 1 and 10 kDa could be observed from biological samples. Specific procedures have been developed, e.g. for the enrichment of peptides/proteins a one-step MB-CX procedure, for specific capture of negatively charged peptides the MB-AX procedure. Examples will be given for biological samples of different origin.

Correspondence:
Dr. Arnd Ingendoh
Bruker Daltonik GmbH
Fahrenheitstr. 4
D-28359 Bremen
Tel.: +49-421-2205-244
Fax: +49-421-2205 104
Email: ai@bdal.de

Improvements in Sensitivity and Speed by a High Capacity Ion Trap for Proteomic Analyses

Markus Lubeck, Ulrike Schweiger-Hufnagel, Gabriela Zurek, Andreas Brekenfeld, Carsten Baessmann, Michael Schubert, Arnd Ingendoh

Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany

Three-dimensional ion traps are widely used as workhorses for proteomics applications due to their high sensitivity, fast MS to MS/MS switching and high scan speeds.
A novel 3D ion trap MS with a new trap geometry has been developed, which shows a 10-20 fold higher ion storage capacity than previously possible with 3D traps. The resulting improved ion statistics have benefits for proteomics in terms of a higher sensitivity, a higher dynamic range and lower numbers of averaged scans for a single spectrum needed. These improvement results in both a higher analysis speed as well as dynamic range, providing an increased sequence coverage of complex samples and/or the possibility to look for details like post-translational modifications or point mutations in a much better way, even in those quite complex mixtures.
New monolithic capillary columns enable separations of digested proteins within a few minutes with peak widths of only a few seconds.   The MS(n) duty cycle has to be as short as possible, especially for complex mixtures when a high number of   precursor ions has to be fragmented or when a more in depth analysis via   MS3   is required, e. g. for de novo sequencing or fragmentation of phosphorylated peptides.
Here, we present examples for the detailed analysis of mixtures of digested proteins with sensitivity levels in the low attomol range.

Correspondence:
Dr. Arnd Ingendoh
Bruker Daltonik GmbH
Fahrenheitstr. 4
D-28359 Bremen
Tel.: +49-421-2205-244
Fax: +49-421-2205 104
Email: ai@bdal.de

Interfacing HPLC to MALDI- and ESI-MS/MS Analysis for High-Throughput and High-Content Proteomics

Stephanie Hahner, Markus Lubeck, Anja Resemann, Wolfgang Jabs, Detlev Suckau, Arnd Ingendoh

Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany

The coupling of LC with mass spectrometry has proven to be a powerful tool for comprehensive proteome analysis. Until recently, ESI has been used online coupled with LC for the analysis of complex protein mixtures. With the introduction of a MALDI-TOF/TOF mass spectrometer with MS/MS capability, LC-MALDI becomes a promising option.
In an even more advanced approach the two MS techniques were combined by post column split of the LC flow providing online and offline MS and MS/MS analysis of a single LC run.
For the online-ESI analysis a new high-capacity ion trap has been used for high MS/MS throughput by acquisition of 1 MS + 4 MS/MS spectra in less than three seconds. This increased duty cycle of the ESI-analysis enables fast LC separation techniques using monolithic capillary columns (gradient < 30min).
The simultaneous deposition of 50% of the eluent onto a MALDI target allow for a non-redundant selection of precursor ions for MS/MS analysis without any temporal constraints. Thus, the combined LC-ESI-MALDIMS/MS approach yields increased information readout from a single LC run within relatively short time due to the complementary MS/MS performance when the two MS-techniques are linked to LC separation.
For complex protein mixtures (> 10 proteins) superior sequence coverage could be obtained for each identified protein when using the combined LC-ESI-MALDI-MS/MS compared to the use of each MS-technology alone.

Correspondence:
Dr. Arnd Ingendoh
Bruker Daltonik GmbH
Fahrenheitstr. 4
D-28359 Bremen
Tel.: +49-421-2205-244
Fax: +49-421-2205 104
Email: ai@bdal.de