Simultaneous Pathoproteomic Evaluation of the Dystrophin-Glycoprotein Complex and Secondary Changes in the mdx-4cv Mouse Model of Duchenne Muscular Dystrophy

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.

Concurrent Label-Free Mass Spectrometric Analysis of Dystrophin Isoform Dp427 and the Myofibrosis Marker Collagen in Crude Extracts from mdx-4cv Skeletal Muscles

“…The up-regulated collagen type VI is a major filament-forming collagen of the interstitial matrix that closely interacts with other collagens, fibronectin, biglycan, decorin, and integrins [104]. Hence, the dystrophinopathy-related accumulation of the fibrillar extracellular matrix is clearly associated with the concomitant increase in decorin, asporin, and prolargin, as shown in this report, as well as dermatopontin and the matricellular protein periostin, as previously shown by comparative subproteomic and proteomic studies [35,36,37,49]. Subunits of fibrinogen (Q8VCM7, Q8KOE8) were also identified as being increased in mdx-4cv hind limb muscles, as previously shown to occur in the aged and severely fibrotic mdx diaphragm [35].…”

“…All four proteins exist in high-molecular-mass complexes as integral protein assemblies and exhibit extensive hydrophobic peptide domains. The successful identification of these key physiological regulators demonstrates the improved protein coverage of label-free mass spectrometry as compared to purely gel-based comparative studies [49]. The increased concentration of the ion pumps and ion channels suggests major restructuring within the regulatory pathways of mdx-4cv hind limb muscles.…”

“…Skeletal muscle samples (100 mg wet weight) from combined muscles of the entire hind limbs (including bulk muscle from the lower and upper leg, but excluding the grasping foot-paw) of six-month-old dystrophic mdx-4cv mice ( n = 4) and age-matched wild-type ( wt ) mice ( n = 4) were finely chopped and homogenized in 10 volumes of homogenization buffer (20 mM sodium pyrophosphate, 20 mM sodium phosphate, 1 mM MgCl 2 , 0.303 M sucrose, 0.5 mM EDTA, pH 7.0), using a hand-held IKA T10 Basic Homogenizer (IKA Labortechnik, Staufen, Germany) [49]. This buffer was supplemented with a protease inhibitor cocktail from Roche Diagnostics (Mannheim, Germany) to minimize degradation of skeletal muscle proteins [61].…”

“…In order to verify alterations in protein expression in crude extracts from dystrophic skeletal muscles, as identified by label-free mass spectrometry, immunoblotting of select muscle proteins was carried out per standard procedure [49]. Electrophoretic separation of proteins was achieved using standard 10% polyacrylamide gels, followed by wet transfer at 100 V for 70 min at 4 °C to Whatman Protan nitrocellulose sheets in a Transblot Cell from Bio-Rad Laboratories (Hemel-Hempstead).…”

“…Previous proteomic studies with a focus on dystrophin have used pre-fractionation approaches, including immuno precipitation, elaborate density gradient centrifugation, and liquid chromatography procedures [44,45,46,47], or specialized mass spectrometric methodology with a stable isotope labelled dystrophin as a spike-in standard for the quantitation of select peptides representing dystrophin within a heterogeneous protein mixture [48]. The recent application of organelle proteomics, in conjunction with label-free mass spectrometry, has succeeded in the identification of dystrophin isoform Dp427, dystroglycan, δ-sarcoglycan, γ-sarcoglycan, and α1-syntrophin by decisively reducing sample complexity using differential centrifugation to enrich the microsomal fraction [49]. However, since subcellular fractionation steps may introduce artifacts in comparative proteomic studies, mainly due to the differences in membrane organization and myofibrosis, the identification of muscle-associated biomarker candidates in crude muscle extracts is more promising for the establishment of a superior marker signature.…”