Proteomic 2010: Analyseverfahren, Studien und Ergebnisse

Proteomic 2010: Analyseverfahren, Studien und Ergebnisse Dr. Barbara Sitek Medizinisches Proteom-Center Prof. Dr. Helmut E. Meyer PD Dr. Kai Stühler Ruhr-Universität Bochum INSTAND-Symposium Düsseldorf 17.09.2010

Medizinisches Proteom-Center Leiter: Prof. H. E. Meyer Arbeitsgruppen: Administration (Bernd Gröttrup) Bioinformatics (Dr. Christian Stephan) Funktionelle Proteomics (Prof. Dr. Katrin Marcus) Cellular Proteomics (Dr. Katja Kuhlmann) Neuroproteomics (PD Dr. Kai Stühler) Redox Proteomics (Jun.-Prof. Dr. Lars Leichert) Immune Proteomics (Dr. Eckhard Nordhoff) Mitarbeiter: ca. 70 Personen

Vom Genom zum Proteom Genom Gesamtheit aller vererbaren Informationen eiener Zelle Proteom Gesamtheit der Proteine, die von einem Genom unter exakt definierten Bedingungen und zu einem bestimmten Zeitpunk exprimiert werden

Warum Proteine? identisches Genom aber unterschiedliches Proteom! Das Genom sagt, was potentiell in einer Zelle passieren könnte, das Proteom sagt uns, was tatsächlich passiert.

Genom Proteom Mensch: 30,000 Gene entsprechen 200,000 bis zu 2,000,000 unterschiedliche Proteine alternatives splicing (Entfernung der Introne) mrna Editing posttranskriptionalale Modifikationen mißtranslatierte Formen Degradationsprodukte

Proteomics: Erforschung des Proteoms Methoden in Proteomics: 1D Elektrophorese 2D Elektrophorese Westernblotanalytik Immunhistochemie Massenspektrometrie

Probenmaterial für Proteingewinnung Biopsie Transplantation Zellkultur Tiermodelle Proteinaufschluß Proteinlysat

SDS-PAGE (PolyAcrylamid-GelElektropgorese) 1D Gel nach Silberfärbung

Gleichnamige Ladung aller Proteine durch SDS

SDS-PAGE (PolyAcrylamid-GelElektropgorese)

2D Elektrophorese: 1 Dimension Trennung nach dem isoelektrischn Punkt

2D Elektrophorese: 2 Dimension Trennung nach dem Molekulargewicht

2D Elektrophorese Proteinprobe Isoelectric Focussing (20 cm) SDS-PAGE (30 cm) Isoelektrische Fokussierung Klose J & Kobalz U, Electrophoresis. 1995 Jun;16(6):1034-59 SDS-PAGE

2D Gel

Bereich der 2D-PAGE pi: 3-10 MW: 5-150 kda 65.000 Proteinsequenzen aus der NCBInr Datenbank

Identifizierung von Proteinen

Immunhistochemie Gewebe Gewebeschnitt Nachweis eines Proteins im Gewebe mit Hilfe von Antikörpern

Anwendung von Proteomics in der medizinischen Forschung Qualitativer und Quantitativer Vergleich verschiedener Proteome Krankes Gewebe Gesundes Gewebe Proteine, die mit einer Krankheit assoziiert sind

Proteome analysis of hepatic fibrosis at hepatitis C for the detection of new fibrosis biomarkers

Epidemiology of the hepatitis C - infection 170 million people infected worldwide Germany: 500.000 affected persons (prevalence of 0,6%) 5000 new diseases per year Most common reason for liver transplantation Non invasive diagnostic markers are missing! (liver biopsy is still the gold standard)

Different courses of the chronic hepatitis C infection female gender, young people 20-30 years acute HCV-infection chronic course 60% - 80% cirrhosis in 20% HCC 1% - 4% per year < 20 years alcohol, suppression of the immune-system, co-infection

Different stages of liver cirrhosis in laparoscopy cirrhosis = fibrosis grade 4

Hepatic fibrogenesis - hepatic stellate cells

Where Biomarkers can be found? Tissue Concentration of marker Body fluids near to disease place, e. g. urine, pancreatic juice, liquor Serum Spatial distance to the disease place

The Human Plasma Proteome Anderson et al., Mol. Cell. Prot. 2002

Biomarker discovery via tissue profiling Patients N = 5-20 (+) (-) Microdissection 2D-PAGE Serummarker candidate Mass spectrometry Western Blot N = 30-50 ELISA N > 200 Protein I.D. Serum/Plasma Blood Validation by IHC. N = 100-200

Methods for isolation and proteome analysis of cirrhosis relevant cells 1) Microdissection 2) DIGE saturation labelling

Isolation of cirrhotic and non-cirrhotic liver cells using manual microdissection Liver parenchyma: before microdissection needle fibrotic non-fibrotic Liver parenchyma: after microdissection Serial sections of 10 μm 100-120 cells per section manual microdissection is time consuming: 3-5000 cells 4 5 hours low amount of protein

Classical 2-DE of low protein amount Application of silver staining revealed ~ 100 protein spots analysing 2µg protein. Sensitivity of silver staining is not sufficient to detect such low protein amount. 2 µg protein, cell culture

DIGE Saturation labelling dyes Labelling of scarce sample amount (2 µg) 2colors: Cy3and Cy5 Labelling of cysteine thiol group All cysteines in a peptide are labelled (saturation)

DIGE Saturation labelling workflow Sample 1 (Int. Stand.) Labelling with Cy3 2D Electrophoresis Fluoreszence- Scanner Cy3 Overlay Quantification with DeCyder Software Sample 2 Labelling with Cy5 Cy5

Fluorescence labelling of low protein amount Fluorescence saturation labelling allows to detect proteins (1 µg) from microdissection. Application of DIGE saturation labelling revealed ~ 2500 protein spots analysing 1 µg protein. 1 µg protein, cell culture

Reproducibility of microdissection and saturation labelling, 1000 cells 2.500 spots 2.400 spots 2.420 spots 2.400 spots

Work flow Liver transplantation from 7 hepathetis C patiants Microdissection of fibrotic and non-fibrotic cells Protein labelling => DIGE Fluor saturation dyes Differential 2D gel electrophoresis => Analysis using DeCyder software Validation of the proteins using IHC Biomarker detection in serum

Experimental design Microdissected cells Mixture of 7 liver tissues Fibrotic Cy5 n=7 5000 cells Non-fibrotic Cy5 internal standard 3 µg CY3 protein identification 400 µg n=7 3500 cells

Comparison between liver parenchyma (green) as standard and microdissected cells of fibrotic / non-fibrotic liver tissue (red) red: 2500 cells, fibrotic green: 3µg liver tissue yellow: congruence red: 2500 cells, non-fibrotic green: 3µg liver tissue yellow: congruence

Representative 2 DE gels for fibrotic and non-fibrotic cells fibrotic non-fibrotic

Identification of differentially expressed spots Microdissected cells Internal standard 400 µg, Cy3 Fibrotic 35.000, Cy3 Non-fibrotic 40.500, Cy3

Summary of differentially expressed spots Number of spots analysed in all gels 2500 Number of high abundant spots in fibrotic cells (fold change >10) Number of up-regulated spots in fibrotic cells (fold change <10 and >3) Number of high abundant spots in non fibrotic cells (fold change >10) Number of up-regulated spots in non fibrotic cells (fold change <10 and >3) 11 16 15 17 49 regulated protein spots 27 non-redundant proteins

Proteomic analysis in cirrhotic and non-cirrhotic liver parenchyma cirrhotic non cirrhotic cirrhotic, 5000 Zellen non cirrhotic, 3500 Zellen

Proteomic analysis in fibrotic and non-fibrotic liver parenchyma Fibrotic Non-fibrotic Proteins associated to hepatic fibrosis?

Biomarker candidate: Tropomyosin t-test: 2.50 x 10-08 Fold change: 56 Non cirrhotic cirrhotic cirrhotic non cirrhotic

Validation of Tropomyosin using Immunhistochemie non fibrotic fibrotic

Tropomyosin western blot of different Hepatitis C /alcohol cirrhosis serum samples vs. control samples Hep C cirrhosis alcohol induced cirrhosis controls positive control 170 Hep B fibrosis 3ng 5ng 10 ng 130 100 70 55 40 35 25

Tropomyosin a typical tissue leakage protein Tropomyosin control 0,0005µg 0,001µg 0,005µg 0,01µg 0,05µg Patient 5 control Patient 6 control Anderson et al., Mol Cell Proteomics. 2002 Nov;1(11):845-67. Patient 4 HCV- type 1 Patient 3 HCV- type 1 Patient 2 HCV- type 1 2 ng/ µl ~ 0,003µg Patient 1 HCV- type 1

Biomarker candidate: Microfibril associated glycoprotein 4 t-test: 4.80 x 10-10 Fold change: 45 Non cirrhotic cirrhotic cirrhotic non cirrhotic

Immunohistochemistry of Microfibril associated glycoprotein 4 Non cirrhotic Cirrhotic

100 70 55 40 35 MFAP 4 western blot of different Hepatitis C /alcohol cirrhosis serum samples vs. control samples MFAP 4 HCV- cirr Child C HCV- cirr Child C HCV- cirr Child C HCV- cirr Child C HCV- cirr Child C HBV- fibrosis HAI 4 Alc cirr Child C Alc cirr Child C Alc cirr Child B Alc cirr Child B Control Control Control Control Control Neg. control (cell culture) Pos. control (cell culture) Alc cirr Child B

ELISA of MFAP-4 Serum concentration in controls and patients with HCV or alcoholic cirrhosis Statistical tools: ANOVA and Tukey s post-hoc test

ELISA of MFAP-4 Serum concentration in patients with different fibrosis stage Statistical tools: ANOVA and Tukey s post-hoc test

Summary 2-DE-based differential proteome analysis of 3.500 microdissected cells is feasible using saturation fluorescence dyes. Analysis of cirrhotic versus non-cirrhotic cells revealed 27 nonredundant proteins. Identification and validation of the first candidate progression markers with a very high success rate of 5 out of 7 candidate proteins. Validated proteins are good candidates for targeted approach (ELISA, MRM, IHC etc.) in serum/plasma or tissues.

Acknowledgment Medizinisches Proteom-Center Ruhr-Universität Bochum Prof. Dr. Helmut E. Meyer Dr. Kai Stühler Dr. Gereon Poschmann Dr. Corinna Henkel Eva Hawranke Kathy Pfeiffer Sebastian Wiese Jun.-Prof. Dr. Bettina Warscheid Department of Internal Medicine Knappschaftskrankenhaus, Bochum Prof. Wolf Schmiegel Dr. Christian Mölleken Department of Pathology Christian Albrechts University, Kiel PD Dr. Bence Sipos Dr. Ibrahim Alkatout Dr. Jutta Lüttges Sabine Roggenbrodt Birgit Streletzki Prof. Dr. Günter Klöppel Department of Medical Biology, University of Southern Denmark, Odense Prof. Dr. Uffe Holmskov Ida Tornøe Anders Schlosser Department of General Surgery and Transplantation University Hospital, Essen Prof. Dr. C. Broelsch