Abstract
In skeletal muscle, the dystrophin-glycoprotein complex forms a membrane-associated assembly of relatively low abundance, making its detailed proteomic characterization in normal versus dystrophic tissues technically challenging. To overcome this analytical problem, we have enriched the muscle membrane fraction by a minimal differential centrifugation step followed by the comprehensive label-free mass spectrometric analysis of microsomal membrane preparations. This organelle proteomic approach successfully identified dystrophin and its binding partners in normal versus dystrophic hind limb muscles. The introduction of a simple pre-fractionation step enabled the simultaneous proteomic comparison of the reduction in the dystrophin-glycoprotein complex and secondary changes in the mdx-4cv mouse model of dystrophinopathy in a single analytical run. The proteomic screening of the microsomal fraction from dystrophic hind limb muscle identified the full-length dystrophin isoform Dp427 as the most drastically reduced protein in dystrophinopathy, demonstrating the remarkable analytical power of comparative muscle proteomics. Secondary pathoproteomic expression patterns were established for 281 proteins, including dystrophin-associated proteins and components involved in metabolism, signalling, contraction, ion-regulation, protein folding, the extracellular matrix and the cytoskeleton. Key findings were verified by immunoblotting. Increased levels of the sarcolemmal Na+/K+-ATPase in dystrophic leg muscles were also confirmed by immunofluorescence microscopy. Thus, the reduction of sample complexity in organelle-focused proteomics can be advantageous for the profiling of supramolecular protein complexes in highly intricate systems, such as skeletal muscle tissue.
Highlights
The total number of proteins in contractile tissues is not precisely known [1], the findings from systematic cataloguing studies suggest that several thousand protein species constitute the protein repertoire of the skeletal muscle proteome [2,3,4,5,6]
Not surprising that shotgun proteomics of various skeletal muscles has not succeeded in the systematic cataloguing of all members of the dystrophin complex [2,3,4,5] and that comparative studies using total extracts from dystrophic fibres have been unsuccessful in the comprehensive identification of dystrophin and its associated glycoproteins [41,42,43,44,45,62,63]
The proteomic quantitation of select peptides representing dystrophin within a complex protein mixture has been carried out by combining stable isotope labelled dystrophin as a spike-in standard, one-dimensional gel electrophoretic separation, and mass spectrometry [67]
Summary
The total number of proteins in contractile tissues is not precisely known [1], the findings from systematic cataloguing studies suggest that several thousand protein species constitute the protein repertoire of the skeletal muscle proteome [2,3,4,5,6]. Proteomic surveys indicate that several hundred proteins are differentially expressed in contractile tissue with a differing fibre specification [12,13,14,15] This includes, especially, the contractile and regulatory elements of the actomyosin apparatus, such as the many isoforms of myosin heavy chains, myosin light chains, actins, troponins and tropomyosins, and many metabolic enzymes, signalling proteins, and ion-handling proteins [16,17,18]
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