Identification of Fibrosis-Relevant Proteins Using DIGE (Difference in Gel Electrophoresis) in Different Models of Hepatic Fibrosis

C. Henkel; M. Roderfeld; R. Weiskirchen; B. Scheibe; S. Matern; E. Roeb

doi:10.1055/s-2004-813911

Zeitschrift für Gastroenterologie, Table of Contents

Z Gastroenterol 2005; 43(1): 23-29
DOI: 10.1055/s-2004-813911

Übersicht

Identification of Fibrosis-Relevant Proteins Using DIGE (Difference in Gel Electrophoresis) in Different Models of Hepatic Fibrosis

Identifizierung fibroserelevanter Proteine mittels DIGE (Difference in Gel Electrophoresis) in verschiedenen FibrosemodellenC. Henkel¹ , M. Roderfeld¹ , R. Weiskirchen² , B. Scheibe³ , S. Matern¹ , E. Roeb¹

¹Medizinische Klinik III Universitätsklinikum RWTH, Aachen
²Institut für Klinische Chemie und Pathochemie, Universitätsklinikum RWTH, Aachen
³GE Healthcare Bio-Sciences, Discovery Systems, Freiburg

The authors thank Udo Roth, Stefan Müller and Franz Georg Harnisch for protein identification via MALDI-TOF MS (Center for Molecular Medicine, University of Cologne (CMMC). We gratefully acknowledge the technical assistance of Bettina Jansen, Judith Dahmen, and Karin Mascke-Neuss. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 542, TP C3 and RO 957/6-1), from the Federal Ministry of Education and Reseach of Germany (Network of Competence in Medicine, Hep-Net), and Aachen University (START project Identification of Molecular Markers and Gene Therapy of Fibrosis and Wound Healing).

Abstract

Zusammenfassung

In den letzten Jahren gewann Proteomics mehr und mehr an Bedeutung. Die zweidimensionale (2D-)Gel-Elektrophorese ist eine weit verbreitete Methode, um das Proteinmuster zweier biologischer Stadien miteinander zu vergleichen (z. B. gesundes und krankes Gewebe) und Unterschiede im Proteinmuster zu ermitteln. Bisher stellen Gel-zu-Gel-Variationen und daraus resultierende Artefakte bei der Detektion von Expressionsunterschieden ein wesentliches Problem im Bereich Proteomics dar. Die „Difference in Gel Electrophoresis”-Methodik (DIGE) ermöglicht das Auftrennen zweier Proteome in einem Gel. Die zu vergleichenden Proteinlysate werden mit unterschiedlichen Fluoreszenzfarbstoffen markiert (Cy3 und Cy5) und in einem Gel aufgetrennt. Ein weiterer Vorteil von DIGE ist die Möglichkeit, einen internen Standard, mit einem dritten Fluoreszenzfarbstoff (Cy2) markiert, ebenfalls im selben Gel aufzutrennen, um eine quantitative Expressionsanalyse zu ermöglichen. Drei verschiedene Fibrosemodelle wurden mit der jeweiligen Kontrolle verglichen („Tissue Inhibitor of Metalloproteinases-1”(TIMP-1)-überexprimierende HepG2-Zellen im Vergleich zu einer HepG2-Kontrolle, frisch isolierte HSC im Vergleich zu aktivierten HSC und gesunde Mausleber im Vergleich zu fibrotischer Mausleber). Unter den differenziell exprimierten Proteinen konnten einige als bereits fibroserelevant beschriebene Proteine bestätigt werden. Zusätzlich konnten aber auch neue, für die hepatische Fibrose bisher nicht als relevant bekannte, Proteine identifiziert werden.

Abstract

Proteomics became a more and more important technique for the large-scale analysis of proteins during the last years. Two-dimensional (2D) electrophoresis as a major tool of proteomics is a powerful method to compare two different biological stages (e. g. healthy and diseased tissue) and to find differences in their protein pattern. One major problem in proteomics is the gel to gel variation of two-dimensional gel electrophoresis, which could cause artefacts in the detection of expression differences. The “difference in gel electrophoresis” (DIGE) technique allows the separation of two proteomes in the same gel. The protein pools were labelled with different fluorescent dyes and equal amounts of protein were separated in the same gel. Another advantage of DIGE is the possibility to separate an internal standard labelled with a third dye in the same gel to allow quantitative expression analysis. We compared proteomes of three different fibrosis models with the appropriate control (tissue inhibitor of metalloproteinases-1 (TIMP-1) overexpressing HepG2 cells in comparison to a HepG2 control, freshly isolated HSC in comparison to activated HSC and healthy mouse liver in comparison to fibrotic mouse liver). Among the differentially expressed proteins several were already found to be relevant for fibrosis but we also detected some proteins like the selenium binding protein 2 which might be relevant for hepatic fibrosis.

Schlüsselwörter

Proteomics - DIGE - Leberfibrose - Proteom - zweidimensionale Gel-Elektrophorese

Key words

Proteomics - DIGE - liver fibrosis - proteome - two dimensional gel electrophoresis

Full Text

References

1 http://us.expasy.org/proteomics_def.html.
2 O’Farrell P H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975; 250 4007-4021
3 Klose J. Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals. Humangenetik. 1975; 26 231-243
4 Unlu M, Morgan M E, Minden J S. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis. 1997; 18 2071-2077
5 Zhou G, Li H, DeCamp D. et al . 2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers. Mol Cell Proteomics. 2002; 1 117-124
6 Yang J W, Czech T, Lubec G. Proteomic profiling of human hippocampus. Electrophoresis. 2004; 25 1169-1174
7 Hiramatsu S, Kojima J, Okada T T. et al . The serum protein profile in chronic hepatitis, cirrhosis and liver cancer. Acta Hepatogastroenterol (Stuttg). 1976; 23 177-182
8 Lanfear J, Fleming J, Walker M. et al . Different patterns of regulation of the genes encoding the closely related 56 kDa selenium- and acetaminophen-binding proteins in normal tissues and during carcinogenesis. Carcinogenesis. 1993; 14 335-340
9 Weiskirchen R, Kneifel J, Weiskirchen S. Comparative evaluation of gene delivery devices in primary cultures of rat hepatic stellate cells and rat myofibroblasts. BMC Cell Biol. 2000; 1 4
10 McGrory W J, Bautista D S, Graham F L. A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5. Virology. 1988; 163 614-617
11 Barnard J A, Snyder P N, Werner M J. et al . Protein synthesis by HepG2 cells infected with influenza B virus. Pediatr Res. 1988; 23 334-337
12 Roeb E, Purucker E, Breuer B. et al . TIMP expression in toxic and cholestatic liver injury in rat. J Hepatol. 1997; 27 836-847
13 Seglen P O. Preparation of isolated rat liver cells. Methods Cell Biol. 1976; 13 29-83
14 Schafer S, Zerbe O, Gressner A M. The synthesis of proteoglycans in fat-storing cells of rat liver. Hepatology. 1987; 7 680-687
15 Tonge R. et al . Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics. 2001; 1 377-396
16 Goerg A, Boguth G, Obermaier C. et al . Two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimension (IPG-Dalt): the state of the art and the controversy of vertical versus horizontal systems. Electrophoresis. 1995; 16 1079-1086
17 Lang A, Schrum L W, Schoonhoven R. Expression of small heat shock protein alphaB-crystallin is induced after hepatic stellate cell activation. Am J Physiol Gastrointest Liver Physiol. 2000; 279 G1333-1342
18 Rossi E, Adams L, Prins A. et al . Validation of the FibroTest biochemical markers score in assessing liver fibrosis in hepatitis C patients. Clin Chem. 2003; 49 450-454
19 Gressner A M. The cell biology of liver fibrogenesis - an imbalance of proliferation, growth arrest and apoptosis of myofibroblasts. Cell Tissue. 1998; 292 447-452
20 Olaso E, Friedman S L. Molecular regulation of hepatic fibrogenesis. J Hepatol. 1998; 29 836-847
21 Siegers C P, Bossen K H, Younes M. et al . Glutathione and glutathione-S-transferases in the normal and diseased human liver. Pharmacol Res Commun. 1982; 14 61-72
22 Vidal F, Toda R, Gutierrez C. et al . Influence of chronic alcohol abuse and liver disease on hepatic aldehyde dehydrogenase activity. Alcohol. 1998; 15 3-8
23 Lee H C, Lee H S, Jung S H. et al . Association between polymorphisms of ethanol-metabolizing enzymes and susceptibility to alcoholic cirrhosis in a Korean male population. J Korean Med Sci. 2001; 16 745-750
24 Okuno M, Akita K Moriwaki H. et al . Prevention of rat hepatic fibrosis by the protease inhibitor, camostat mesilate, via reduced generation of active TGF-beta. Gastroenterology. 2001; 120 1784-1800
25 Fouad F M, Mamer O A, Shahidi F. Comparative evaluation of gene delivery devices in primary cultures of rat hepatic stellate cells and rat myofibroblasts. J Toxicol Environ Health. 1996; 47 601-615
26 Chu R, Lim H, Brumfield L. et al . Protein profiling of mouse livers with peroxisome proliferator-activated receptor alpha activation. Mol Cell Biol. 2004; 24 6288-6297
27 Pandey A, Mann M. Proteomics to study genes and genomes. Nature. 2000; 405 837-846
28 Kashino Y. Separation methods in the analysis of protein membrane complexes. J Chromatogr B Analyt Technol Biomed Life Sci. 2003; 25 191-216
29 Fountoulakis M, Juranville J F, Tsangaris G. et al . Fractionation of liver proteins by preparative electrophoresis. Amino Acids. 26. Epub 2003; 2004; 26 27-36
30 Gygi S P, Rochon Y, Franza B R. et al . Correlation between protein and mRNA abundance in yeast. Mol Cell Biol. 1999; 19 1720-1730

Prof. Dr. Elke Roeb

Department of Internal Medicine III, University Hospital RWTH Aachen

Pauwelsstraße 30

52074 Aachen, Germany

Phone: ++ 49/2 41/80-8 92 00

Fax: ++ 49/2 41/8 08 24 55

Email: eroeb@ukaachen.de