Abstract
In this article, we illustrate the application of difference in-gel electrophoresis for the proteomic analysis of dystrophic skeletal muscle. The mdx diaphragm was used as a tissue model of dystrophinopathy. Two-dimensional gel electrophoresis is a widely employed protein separation method in proteomic investigations. Although two-dimensional gels usually underestimate the cellular presence of very high molecular mass proteins, integral membrane proteins and low copy number proteins, this method is extremely powerful in the comprehensive analysis of contractile proteins, metabolic enzymes, structural proteins and molecular chaperones. This gives rise to two-dimensional gel electrophoretic separation as the method of choice for studying contractile tissues in health and disease. For comparative studies, fluorescence difference in-gel electrophoresis has been shown to provide an excellent biomarker discovery tool. Since aged diaphragm fibres from the mdx mouse model of Duchenne muscular dystrophy closely resemble the human pathology, we have carried out a mass spectrometry-based comparison of the naturally aged diaphragm versus the senescent dystrophic diaphragm. The proteomic comparison of wild type versus mdx diaphragm resulted in the identification of 84 altered protein species. Novel molecular insights into dystrophic changes suggest increased cellular stress, impaired calcium buffering, cytostructural alterations and disturbances of mitochondrial metabolism in dystrophin-deficient muscle tissue.
Highlights
Skeletal muscle proteomics is concerned with the global analysis of protein populations from voluntary contractile tissues
The optimization of animal models is a crucial part of biomedical proteomics and proteomic biomarker discovery
In the field of muscular dystrophy research, the mdx mouse is a widely used model system to study the molecular pathogenesis of dystrophinopathy and the suitability of novel experimental therapies to counteract dystrophic symptoms
Summary
Skeletal muscle proteomics is concerned with the global analysis of protein populations from voluntary contractile tissues. These types of muscle-associated proteins include especially integral membrane proteins, very high molecular mass proteins and low-abundance proteins To overcome this technical problem, other than using sophisticated liquid chromatography approaches [14], alternative electrophoretic methods can be employed to supplement routine 2D-gel based investigations. In the field of experimental muscle pathology, a large number of animal genocopies are available to study the basic mechanisms of major neuromuscular diseases [40] These genetic disease models are often not perfect phenocopies of a highly complex human etiology, proteomic screening of pathobiochemical changes in their expression profiles can be helpful for the initial identification of new biomarker candidates [41]. Altered proteins are associated with the contractile apparatus, the extracellular matrix, the cellular stress response, metabolite transport and mitochondrial energy metabolism
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