Optimization of large gel 2D electrophoresis for proteomic studies of skeletal muscle

The Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, Department of Physiology, University of Maryland School of Medicine University of Maryland, Baltimore, MD 21201, USA.
Electrophoresis (Impact Factor: 3.03). 04/2012; 33(8):1263-70. DOI: 10.1002/elps.201100642
Source: PubMed


We describe improved methods for large format, two-dimensional gel electrophoresis (2DE) that improve protein solubility and recovery, minimize proteolysis, and reduce the loss of resolution due to contaminants and manipulations of the gels, and thus enhance quantitative analysis of protein spots. Key modifications are: (i) the use of 7 M urea and 2 M thiourea, instead of 9 M urea, in sample preparation and in the tops of the gel tubes; (ii) standardized deionization of all solutions containing urea with a mixed bed ion exchange resin and removal of urea from the electrode solutions; and (iii) use of a new gel tank and cooling device that eliminate the need to run two separating gels in the SDS dimension. These changes make 2DE analysis more reproducible and sensitive, with minimal artifacts. Application of this method to the soluble fraction of muscle tissues reliably resolves ~1800 protein spots in adult human skeletal muscle and over 2800 spots in myotubes.

  • [Show abstract] [Hide abstract]
    ABSTRACT: In skeletal muscle fibers, the excitation-contraction-relaxation cycle is a highly evolved process that is mediated by the contractile proteins - myosin and actin - and the regulatory elements - troponin and tropomyosin. Contractile fibers exhibit enormous complexity and heterogeneity on the molecular level, which is reflected by the diversity of protein isoforms that constitute the actomyosin apparatus. The main components of the contractile apparatus exist in high abundance and are relatively soluble, making them ideal candidates for a systematic analysis by liquid chromatography or gel electrophoresis-based proteomics. This review discusses the proteomic profiling of contractile components in adapting, degenerating and aging skeletal muscle tissues. The proteomic identification of altered contractile proteins may be useful for the establishment of biomarker signatures that can be applied in the examination of the physiological adaptability, cellular plasticity and pathological susceptibility of the neuromuscular system.
    No preview · Article · Jun 2013 · Expert Review of Proteomics
  • [Show abstract] [Hide abstract]
    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.
    No preview · Article · Aug 2013 · Analytical Biochemistry

We use cookies to give you the best possible experience on ResearchGate. Read our cookies policy to learn more.