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

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

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