Proteomic analysis of dystrophic muscle.
ABSTRACT Mass spectrometry-based proteomics had a major impact on the global characterization of skeletal muscles and has decisively enhanced the field of neuromuscular pathology. Proteomic profiling of x-linked muscular dystrophy has identified a large number of new signature molecules involved in fiber degeneration. Here, we describe the difference in-gel electrophoretic analysis of the dystrophic diaphragm muscle from the MDX mouse model of Duchenne muscular dystrophy. This chapter summarizes the various experimental steps involved in muscle proteomics, such as sample preparation, fluorescence labeling, isoelectric focusing, second-dimension slab gel electrophoresis, image analysis, in-gel digestion and electrospray ionization mass spectrometry.
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ABSTRACT: X-linked muscular dystrophy is a highly progressive disease of childhood and characterized by primary genetic abnormalities in the dystrophin gene. Senescent mdx specimens were used for a large-scale survey of potential age-related alterations in the dystrophic phenotype, because the established mdx animal model of dystrophinopathy exhibits progressive deterioration of muscle tissue with age. Since the mdx tibialis anterior muscle is a frequently used model system in muscular dystrophy research, we employed this particular muscle to determine global changes in the dystrophic skeletal muscle proteome. The comparison of mdx mice aged 8 weeks versus 22 months by mass-spectrometry-based proteomics revealed altered expression levels in 8 distinct protein species. Increased levels were shown for carbonic anhydrase, aldolase, and electron transferring flavoprotein, while the expressions of pyruvate kinase, myosin, tropomyosin, and the small heat shock protein Hsp27 were found to be reduced in aged muscle. Immunoblotting confirmed age-dependent changes in the density of key muscle proteins in mdx muscle. Thus, segmental necrosis in mdx tibialis anterior muscle appears to trigger age-related protein perturbations due to dystrophin deficiency. The identification of novel indicators of progressive muscular dystrophy might be useful for the establishment of a muscle subtype-specific biomarker signature of dystrophinopathy.BioMed Research International 10/2012; 2012:691641. DOI:10.1155/2012/691641 · 2.71 Impact Factor
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ABSTRACT: Cardiorespiratory complications are frequent symptoms of Duchenne muscular dystrophy, a neuromuscular disorder caused by primary abnormalities in the dystrophin gene. Loss of cardiac dystrophin initially leads to changes in dystrophin-associated glycoproteins and subsequently triggers secondarily sarcolemmal disintegration, fibre necrosis, fibrosis, fatty tissue replacement, and interstitial inflammation. This results in progressive cardiac disease, which is the cause of death in a considerable number of patients afflicted with X-linked muscular dystrophy. In order to better define the molecular pathogenesis of this type of cardiomyopathy, several studies have applied mass spectrometry-based proteomics to determine proteome-wide alterations in dystrophinopathy-associated cardiomyopathy. Proteomic studies included both gel-based and label-free mass spectrometric surveys of dystrophin-deficient heart muscle from the established mdx animal model of dystrophinopathy. Comparative cardiac proteomics revealed novel changes in proteins associated with mitochondrial energy metabolism, glycolysis, signaling, iron binding, antibody response, fibre contraction, basal lamina stabilisation, and cytoskeletal organisation. This review summarizes the importance of studying cardiomyopathy within the field of muscular dystrophy research, outlines key features of the mdx heart and its suitability as a model system for studying cardiac pathogenesis, and discusses the impact of recent proteomic findings for exploring molecular and cellular aspects of cardiac abnormalities in inherited muscular dystrophies.03/2014; 2014:246195. DOI:10.1155/2014/246195
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ABSTRACT: In basic and applied myology, gel-based proteomics is routinely used for studying global changes in the protein constellation of contractile fibers during myogenesis, physiological adaptations, neuromuscular degeneration and the natural aging process. Since the main proteins of the actomyosin apparatus and its auxiliary sarcomeric components often negate weak signals from minor muscle proteins during proteomic investigations, we have here evaluated whether a simple pre-fractionation step can be employed to eliminate certain aspects of this analytical obstacle. In order to remove a large portion of highly abundant contractile proteins from skeletal muscle homogenates without the usage of major manipulative steps, differential centrifugation was used to decisively reduce the sample complexity of crude muscle tissue extracts. The resulting protein fraction was separated by two-dimensional gel electrophoresis and 2D-landmark proteins identified by mass spectrometry. To evaluate the suitability of the contractile protein-depleted fraction for comparative proteomics, normal versus dystrophic muscle preparations were examined. The mass spectrometric analysis of differentially expressed proteins, as determined by fluorescence difference in-gel electrophoresis, identified 10 protein species in dystrophic mdx hind limb muscles. Interesting new biomarker candidates included Hsp70, transferrin and ferritin, whereby their altered concentration levels in dystrophin-deficient muscle was confirmed by immunoblotting.Analytical Biochemistry 08/2013; DOI:10.1016/j.ab.2013.08.004 · 2.31 Impact Factor