Proteomic Analysis of Dystrophic Muscle

Department of Biology, National University of Ireland Maynooth, Maynooth, Co. Kildare, Ireland.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2012; 798(1):357-69. DOI: 10.1007/978-1-61779-343-1_20
Source: PubMed


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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    • "By using MudPIT it is possible to overcome several drawbacks associated with two-dimensional gel electrophoresis (2D-PAGE), especially under-representation of extreme acid/basic proteins [19] and the poor sensitivity for lowly expressed proteins. Moreover the MudPIT method simplifies sample handling, avoids sample loss in gel matrix and increases throughput and data acquisition [20], [21]. "
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    ABSTRACT: Duchenne muscular dystrophy (DMD) is the most common childhood myopathy, characterized by muscle loss and cardiorespiratory failure. While the genetic basis of DMD is well established, secondary mechanisms associated with dystrophic pathophysiology are not fully clarified yet. In order to obtain new insights into the molecular mechanisms of muscle dystrophy during earlier stages of the disease, we performed a comparative proteomic profile of the spared extraocular muscles (EOM) vs. affected diaphragm from the mdx mice, using a label based shotgun proteomic approach. Out of the 857 identified proteins, 42 to 62 proteins had differential abundance of peptide ions. The calcium-handling proteins sarcalumenin and calsequestrin-1 were increased in control EOM compared with control DIA, reinforcing the view that constitutional properties of EOM are important for their protection against myonecrosis. The finding that galectin-1 (muscle regeneration), annexin A1 (anti-inflammatory) and HSP 47 (fibrosis) were increased in dystrophic diaphragm provides novel insights into the mechanisms through which mdx affected muscles are able to counteract dystrophy, during the early stage of the disease. Overall, the shotgun technique proved to be suitable to perform quantitative comparisons between distinct dystrophic muscles and allowed the suggestion of new potential biomarkers and drug targets for dystrophinopaties.
    PLoS ONE 06/2013; 8(6):e65831. DOI:10.1371/journal.pone.0065831 · 3.23 Impact Factor
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    • "However, it is not well understood how many molecular and cellular factors are involved in the overall process leading to the highly complex pathology of dystrophinopathy. Thus, in order to determine the hierarchy of secondary pathobiochemical effects that render a dystrophic muscle more susceptible to necrosis, it is crucial to elucidate global alterations due to the disintegration of the dystrophin-glycoprotein complex [18]. Mass-spectrometrybased proteomics suggests itself as a suitable analytical tool for such large-scale and high-throughput approaches to study the effects of dystrophin deficiency. "
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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: The age-related loss of skeletal muscle mass and associated progressive decline in contractile strength is a serious pathophysiological issue in the elderly. In order to investigate global changes in the skeletal muscle proteome after the fifth decade of life, this study analysed total extracts from human vastus lateralis muscle by fluorescence difference in-gel electrophoresis. Tissue specimens were derived from middle-aged (47-62 years) vs. aged (76-82 years) individuals and potential changes in the protein expression profiles were compared between these two age groups by a comprehensive gel electrophoresis-based survey. Age-dependent alterations in the concentration of 19 protein spots were revealed and mass spectrometry identified these components as being involved in the excitation-contraction-relaxation cycle, muscle metabolism, ion handling and the cellular stress response. This indicates a generally perturbed protein expression pattern in senescent human muscle. Increased levels of mitochondrial enzymes and isoform switching of the key contractile protein, actin, support the idea of glycolytic-to-oxidative and fast-to-slow transition processes during muscle aging. Importantly, the carbonic anhydrase (CA)3 isoform displayed an increased abundance during muscle aging, which was independently verified by immunoblotting of differently aged human skeletal muscle samples. Since the CA3 isoform is relatively muscle-specific and exhibits a fibre type-specific expression pattern, this enzyme may represent an interesting new biomarker of sarcopenia. Increased levels of CA are indicative of an increased demand of CO2-removal in senescent muscle, and also suggest age-related fibre type shifting to slower-contracting muscles during human aging.
    International Journal of Molecular Medicine 07/2012; 30(4). DOI:10.3892/ijmm.2012.1056 · 2.09 Impact Factor
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