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Proteome Analysis of Cultured Fibroblasts from Type 1 Diabetic Patients and Normal Subjects

Departments of Clinical and Experimental Medicine (L.P., E.I., R.M., M.V., P.T.) and Biological Chemistry (G.A.), University of Padova, 35128 Padova, Italy; and Protein Technology Wallenberglab (G.A., P.J.), SE 22007 Lund, Sweden

Address all correspondence and requests for reprints to: Professor Paolo Tessari, Dipartimento di Medicina Clinica e Sperimentale, Università di Padova, via Giustiniani 2, 35128 Padova, Italy. E-mail: paolo.tessari{at}unipd.it.

Abstract

Context: Protein profiling of diabetic tissues could provide useful biomarkers for early diagnosis, therapeutic targets, and disease response markers. Cultured fibroblasts are a useful in vitro model for proteome analysis and study of the molecular mechanisms involved in diabetes.

Objective: The objective of the study was to isolate and characterize the proteins of cultured fibroblasts, obtained by skin biopsy, from long-term type 1 diabetic patients without complications and age- and sex-matched normal subjects as controls.

Design: Proteins were separated by two-dimensional electrophoresis (2-DE), and the gel images were qualitatively and quantitatively analyzed. Protein identification was performed by matrix-assisted laser desorption/ionization mass spectrometry.

Results: Reproducible protein maps of fibroblasts from diabetic and healthy subjects were obtained. A total of 125 protein spots were isolated and identified, among them 27 proteins not previously reported in published human fibroblast 2-DE maps, including 20 proteins never reported previously in the literature in human skin fibroblasts. Quantitative analyses revealed six protein spots differentially expressed in the fibroblasts from the diabetic vs. the control subjects (P < 0.05), representing glycolytic enzymes and structural proteins. An increase of triosephosphate I isomerase of two splice isoforms of pyruvate kinase and -actinin 4 and a decrease of tubulin-ß2 and splice isoform 2 of tropomyosin ß-chain were detected.

Conclusions: We generated 2-DE reference maps of the proteome of human skin fibroblasts from both normal and uncomplicated type 1 diabetic patients. Differences in glycolytic enzymes and structural proteins were found. The functional implications of the identified proteins are discussed.