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Extraocular muscles (EOMs) represent a specialized type of contractile tissue with unique cellular, physiological, and biochemical properties. In Duchenne muscular dystrophy, EOMs stay functionally unaffected in the course of disease progression. Therefore, it was of interest to determine their proteomic profile in dystrophinopathy. The proteomic survey of wild type mice and the dystrophic mdx-4cv model revealed a broad spectrum of sarcomere-associated proteoforms, including components of the thick filament, thin filament, M-band and Z-disk, as well as a variety of muscle-specific markers. Interestingly, the mass spectrometric analysis revealed unusual expression levels of contractile proteins, especially isoforms of myosin heavy chain. As compared to diaphragm muscle, both proteomics and immunoblotting established isoform MyHC14 as a new potential marker in wild type EOMs, in addition to the previously identified isoforms MyHC13 and MyHC15. Comparative proteomics was employed to establish alterations in the protein expression profile between normal EOMs and dystrophin-lacking EOMs. The analysis of mdx-4cv EOMs identified elevated levels of glycolytic enzymes and molecular chaperones, as well as decreases in mitochondrial enzymes. These findings suggest a process of adaptation in dystrophin-deficient EOMs via a bioenergetic shift to more glycolytic metabolism, as well as an efficient cellular stress response in EOMs in dystrophinopathy.

The neuromuscular disorder Duchenne muscular dystrophy is a multi-systemic disease that is caused by a primary abnormality in the X-chromosomal Dmd gene. Although progressive skeletal muscle wasting and cardio-respiratory complications are the most serious symptoms that are directly linked to the almost complete loss of the membrane cytoskeletal protein dystrophin, dystrophic patients also suffer from gastrointestinal dysfunction. In order to determine whether proteome-wide changes potentially occur in the gastrointestinal system due to dystrophin deficiency, total tissue extracts from the interface between the stomach wall and the pancreas of the mdx-4cv model of dystrophinopathy were analysed by mass spectrometry. Following the proteomic establishment of both smooth muscle markers of the gastrointestinal system and key enzymes of the pancreas, core members of the dystrophin-glycoprotein complex, including dystrophin, dystroglycans, sarcoglycans, dystrobrevins and syntrophins were identified in this tissue preparation. Comparative proteomics revealed a drastic reduction in dystrophin, sarcoglycan, dystroglycan, laminin, titin and filamin suggesting loss of cytoskeletal integrity in mdx-4cv smooth muscles. A concomitant increase in various mitochondrial enzymesisindicative of metabolic disturbances. These findings agree with abnormal gastrointestinal function in dystrophinopathy

The proteomic data presented in this article provide supporting information to the related research article "Proteomic and cell biological profiling of the renal phenotype of the mdx-4cv mouse model of Duchenne muscular dystrophy" (Dowling et al., 2019) [1]. This article supplies additional datasets on protein species with increased versus decreased concentration in the kidney from the dystrophic mdx-4cv mouse, as well as tables with mass spectrometrically identified kidney marker proteins that exhibit characteristic tissue distributions, subcellular localizations and physiological functions. Information is provided on the underlying multi-consensus protein listings from the proteomic screening of both wild type and mdx-4cv mouse kidneys. The data article provides comprehensive information on the systematic and mass spectrometric identification of the mouse kidney proteome.

This protocol describes the comparative proteomic profiling of the spleen of wild type versus mdx-4cv mouse, a model of dystrophinopathy. We detail sample preparation for bottom-up proteomic mass spectrometry experiments, including homogenization of tissue, protein concentration measurements, protein digestion, and removal of interfering chemicals. We then describe the steps for mass spectrometric analysis and bioinformatic evaluation. For complete details on the use and execution of this protocol, please refer to Dowling et al. (2020).

The mass spectrometric analysis of skeletal muscle proteins has used both peptide-centric and protein-focused approaches. The term ‘top-down proteomics’ is often used in relation to studying purified proteoforms and their post-translational modifications. Two-dimensional gel electrophoresis, in combination with peptide generation for the identification and characterization of intact proteoforms being present in two-dimensional spots, plays a critical role in specific applications of top-down proteomics. A decisive bioanalytical advantage of gel-based and top-down approaches is the initial bioanalytical focus on intact proteins, which usually enables the swift identification and detailed characterisation of specific proteoforms. In this review, we describe the usage of two-dimensional gel electrophoretic top-down proteomics and related approaches for the systematic analysis of key components of the contractile apparatus, with a special focus on myosin heavy and light chains and their associated regulatory proteins. The detailed biochemical analysis of proteins belonging to the thick and thin skeletal muscle filaments has decisively improved our biochemical understanding of structure-function relationships within the contractile apparatus. Gel-based and top-down proteomics has clearly established a variety of slow and fast isoforms of myosin, troponin and tropomyosin as excellent markers of fibre type specification and dynamic muscle transition processes.

Introduction: Progressive skeletal muscle wasting is the manifesting symptom of Duchenne muscular dystrophy, an X-linked inherited disorder triggered by primary abnormalities in the DMD gene. The almost complete loss of dystrophin isoform Dp427 causes a multi-system pathology that features in addition to skeletal muscle weakness also late-onset cardio-respiratory deficiencies, impaired metabolism and abnormalities in the central nervous system. Areas covered: This review focuses on the mass spectrometry-based proteomic characterization of X-linked muscular dystrophy with special emphasis on the identification of novel biomarker candidates in skeletal muscle tissues, as well as non-muscle tissues and various biofluids. Individual sections focus on molecular and cellular aspects of the pathogenic changes in dystrophinopathy, proteomic workflows used in biomarker research, the proteomics of the dystrophin-glycoprotein complex and the potential usefulness of newly identified protein markers involved in fibre degeneration, fibrosis and inflammation. Expert opinion: The systematic application of large-scale proteomic surveys has identified a distinct cohort of both tissue- and biofluid-associated protein species with considerable potential for improving diagnostic, prognostic and therapy-monitoring procedures. Novel proteomic markers include components involved in fibre contraction, cellular signalling, ion homeostasis, cellular stress response, energy metabolism and the immune response, as well as maintenance of the cytoskeletal and extracellular matrix.

The proteomic data presented in this article provide supporting information to the related research article "Proteomic analysis of the sarcolemma-enriched fraction from dystrophic mdx-4cv skeletal muscle" (Murphy et al., 2018) [1]. In the associated research article, the sarcolemma from normal versus dystrophic skeletal muscle was analyzed by mass spectrometry-based proteomics. Sarcolemma vesicles were enriched by a lectin agglutination method and then analyzed by liquid chromatography tandem mass spectrometry. Here we provide additional datasets on proteins with decreased versus increased abundance in dystrophin-deficient muscle plasma membranes.