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: Ca 2+ -binding proteins play a key role in many regulatory processes in skeletal muscles, including excitation-contraction coupling and fiber relaxation. Recent advances in the large-scale separation and high-throughput screening of entire protein populations enabled the systematic analysis of the luminal Ca 2+ -binding protein calsequestrin in pathological muscle. In this review, we briefly outline the physiological functions of calsequestrin and related ion-regulatory proteins in skeletal muscle and then describe the application of mass spectrometry-based proteomics for studying calsequestrin and related Ca 2+ -handling proteins such as sarcalumenin, parvalbumin and regucalcin. The proteomic profiling of animal models of muscular dystrophy has revealed distinct secondary changes in these Ca 2+ -binding proteins. Dystrophinopathy was shown to exhibit a drastic reduction in luminal and cytosolic Ca 2+ -binding proteins and an impaired luminal calcium buffering capacity. This diminished ability to sequester excess calcium in the sarcoplasmic reticulum might represent a key pathophysiological step in the damage pathway of X-linked muscular dystrophy. In conjunction with Ca 2+ -leakage through the sarcolemma resulting in chronically increased cytosolic Ca 2+ -levels and an enhanced proteolytic degradation of contractile proteins, abnormal Ca 2+ -storage may exacerbate the dystrophic phenotype. In the long-term, the proteomic characterization of calsequestrin might be useful for the establishment of novel biomarkers of secondary changes in muscular dystrophy.Calcium Signaling. 08/2014; 1(2):2373-1168.
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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.BioMed research international. 01/2014; 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; · 2.58 Impact Factor