title: Comparative proteomic analysis of the contractile-protein-depleted fraction from normal versus dystrophic skeletal muscle.
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 prefractionation step can be employed to eliminate certain aspects of this analytical obstacle. 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 were 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 hindlimb muscles. Interesting new biomarker candidates included Hsp70, transferrin, and ferritin, whereby their altered concentration levels in dystrophin-deficient muscle were confirmed by immunoblotting.
authors: Department of Biology, National University of Ireland, Maynooth, Kildare, Ireland.
authors: Steven
authors: S
authors: Carberry
authors: Department of Physiology II, University of Bonn, D-53115 Bonn, Germany.
authors: Margit
authors: M
authors: Zweyer
authors: Department of Physiology II, University of Bonn, D-53115 Bonn, Germany.
authors: Dieter
authors: D
authors: Swandulla
authors: Department of Biology, National University of Ireland, Maynooth, Kildare, Ireland. Electronic address: kay.ohlendieck@nuim.ie.
authors: Kay
authors: K
authors: Ohlendieck
mesh_terms: D000818:Animals; D002498:Centrifugation; D013058:Mass Spectrometry; D051379:Mice; D008958:Models, Molecular; D009119:Muscle Contraction; D009124:Muscle Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000502:physiology / Q000503:physiopathology; D009136:Muscular Dystrophies / Q000378:metabolism* / Q000503:physiopathology; D011487:Protein Conformation; D040901:Proteomics / Q000379:methods*
chemical_list: D009124:Muscle Proteins
keywords: Dystrophinopathy; Ferritin; Hsp70; Muscle proteomics; Transferrin