Sandra Murphy

Pathoproteomic profiling of the skeletal muscle matrisome in dystrophinopathy associated myofibrosis.

The gradual accumulation of collagen and associated proteins of the extracellular matrix is a crucial myopathological parameter of many neuromuscular disorders. Progressive tissue damage and fibrosis play a key pathobiochemical role in the dysregulation of contractile functions and often correlates with poor motor outcome in muscular dystrophies. Following a brief introduction into the role of the extracellular matrix in skeletal muscles, we review here the proteomic profiling of myofibrosis and its intrinsic role in X‐linked muscular dystrophy. Although Duchenne muscular dystrophy is primarily a disease of the membrane cytoskeleton, one of its most striking histopathological features is a hyperactive connective tissue and tissue scarring. We outline the identification of novel factors involved in the modulation of the extracellular matrix in muscular dystrophy, such as matricellular proteins. The establishment of novel proteomic markers will be helpful in improving the diagnosis, prognosis, and therapy monitoring in relation to fibrotic substitution of contractile tissue. In the future, the prevention of fibrosis will be crucial for providing optimum conditions to apply novel pharmacological treatments, as well as establish cell‐based approaches or gene therapeutic interventions. The elimination of secondary abnormalities in the matrisome promises to reduce tissue scarring and the loss of skeletal muscle elasticity.

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

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 full-length dystrophin protein isoform of 427 kDa (Dp427), the absence of which represents the principal abnormality in X-linked muscular dystrophy, is difficult to identify and characterize by routine proteomic screening approaches of crude tissue extracts. This is probably related to its large molecular size, its close association with the sarcolemmal membrane, and its existence within a heterogeneous glycoprotein complex. Here, we used a careful extraction procedure to isolate the total protein repertoire from normal versus dystrophic mdx-4cv skeletal muscles, in conjunction with label-free mass spectrometry, and successfully identified Dp427 by proteomic means. In contrast to a considerable number of previous comparative studies of the total skeletal muscle proteome, using whole tissue proteomics we show here for the first time that the reduced expression of this membrane cytoskeletal protein is the most significant alteration in dystrophinopathy. This agrees with the pathobiochemical concept that the almost complete absence of dystrophin is the main defect in Duchenne muscular dystrophy and that the mdx-4cv mouse model of dystrophinopathy exhibits only very few revertant fibers. Significant increases in collagens and associated fibrotic marker proteins, such as fibronectin, biglycan, asporin, decorin, prolargin, mimecan, and lumican were identified in dystrophin-deficient muscles. The up-regulation of collagen in mdx-4cv muscles was confirmed by immunofluorescence microscopy and immunoblotting. Thus, this is the first mass spectrometric study of crude tissue extracts that puts the proteomic identification of dystrophin in its proper pathophysiological context.

Comparative Skeletal Muscle Proteomics Using Two-Dimensional Gel Electrophoresis

The pioneering work by Patrick H. O’Farrell established two-dimensional gel electrophoresis as one of the most important high-resolution protein separation techniques of modern biochemistry (Journal of Biological Chemistry 1975, 250, 4007–4021). The application of two-dimensional gel electrophoresis has played a key role in the systematic identification and detailed characterization of the protein constituents of skeletal muscles. Protein changes during myogenesis, muscle maturation, fibre type specification, physiological muscle adaptations and natural muscle aging were studied in depth by the original O’Farrell method or slightly modified gel electrophoretic techniques. Over the last 40 years, the combined usage of isoelectric focusing in the first dimension and sodium dodecyl sulfate polyacrylamide slab gel electrophoresis in the second dimension has been successfully employed in several hundred published studies on gel-based skeletal muscle biochemistry. This review focuses on normal and physiologically challenged skeletal muscle tissues and outlines key findings from mass spectrometry-based muscle proteomics, which was instrumental in the identification of several thousand individual protein isoforms following gel electrophoretic separation. These muscle-associated protein species belong to the diverse group of regulatory and contractile proteins of the acto-myosin apparatus that forms the sarcomere, cytoskeletal proteins, metabolic enzymes and transporters, signaling proteins, ion-handling proteins, molecular chaperones and extracellular matrix proteins.

Proteomic profiling of the dystrophin complex and membrane fraction from dystrophic mdx muscle reveals decreases in the cytolinker desmoglein and increases in the extracellular matrix stabilizers biglycan and fibronectin

The almost complete loss of the membrane cytoskeletal protein dystrophin and concomitant drastic reduction in dystrophin-associated glycoproteins are the underlying mechanisms of the highly progressive neuromuscular disorder Duchenne muscular dystrophy. In order to identify new potential binding partners of dystrophin or proteins in close proximity to the sarcolemmal dystrophin complex, proteomic profiling of the isolated dystrophin–glycoprotein complex was carried out. Subcellular membrane fractionation and detergent solubilisation, in combination with ion exchange, lectin chromatography and density gradient ultracentrifugation, was performed to isolate a dystrophin complex-enriched fraction. Following gradient gel electrophoresis and on-membrane digestion, the protein constituents of the dystrophin fraction were determined by peptide mass spectrometry. This proteomic strategy resulted in the novel identification of desmoglein and desmoplakin, which act as cytolinker proteins and possibly exist in close proximity to the dystrophin complex in the sarcolemma membrane. Interestingly, comparative immunoblotting showed a significant reduction in desmoglein in dystrophin-deficient mdx skeletal muscles, reminiscent of the pathobiochemical fate of the dystrophin-associated core proteins in muscular dystrophy. Comparative membrane proteomics was used to correlate this novel finding to large-scale changes in the dystrophic phenotype. A drastic increase in the extracellular stabilizers biglycan and fibronectin was shown by both mass spectrometric analysis and immunoblotting. The reduced expression of desmoglein in dystrophin-deficient skeletal muscles, and simultaneous increase in components of the extracellular matrix, suggest that muscular dystrophy is associated with plasmalemmal disintegration, loss of cellular linkage and reactive myofibrosis.

The Extracellular Matrix Complexome from Skeletal Muscle

The various layers of the extracellular matrix, forming the endomysium, perimysium and epimysium of skeletal muscles, provide essential structural and mechanical support to contractile fibres. Crucial aspects of muscle elasticity and fibre contractility are depend‐ ent on proper cell–matrix interactions. A complex network of collagen fibres, nonfibrillar collagens, proteoglycans, matricellular proteins, matrix metalloproteinases, adhesion receptors and signalling molecules maintain the physical structure for force transmission within motor units, embed critical cellular structures such as capillaries and motor neurons, and enable essential sarcolemma-matrix adhesion processes and signalling cascades. The systems biological concept of protein complexomes, which assumes the existence of interconnectivities between large protein assemblies, can be readily applied to the proteins within the extracellular space of muscles. Recent proteomic studies confirm that the extracellular matrix complexome has considerable influence on the integrity and cellular functions of skeletal muscle fibres. Adaptations or changes in the organization of the extracellular matrix play a crucial role during fibre regeneration following injury, extensive neuromuscular activity or pathophysiological insults. This chapter outlines the molecular components of the matrisome from skeletal muscles and discusses the extracellular matrix in relation to myogenesis, maturation of motor units, adaptation to changed functional demands and myofibrosis in muscular disorders.

DIGE Analysis of ProteoMiner TM Fractionated Serum/Plasma Samples

The discovery of clinically relevant biomarkers using gel-based proteomics has proven extremely challenging, principally because of the large dynamic range of protein abundances in biofluids such as blood and the fact that only a small number of proteins constitute the vast majority of total blood protein mass. Various separation, depletion, enrichment, and quantitative developments coupled with improvements in gel-based protein quantification technologies, specifically difference gel electrophoresis (DIGE), have contributed to significant improvements in the detection and identification of lower abundance proteins. One of these enrichment technologies, Proteominer, will be the focus of this chapter. The Proteominer technology a utilizes hexapeptide bead library with huge diversity to bind and enrich low-abundance proteins but at the same time suppressing the concentration of high-abundance proteins in subsequent analysis.

Subcellular Fractionation for DIGE-Based Proteomics

Mass spectrometry-based protein methodologies have revolutionized the field of analytical biochemistry and enable the identification of hundreds to thousands of proteins in biological fluids, cell lines, and tissue. This methodology requires the initial separation of a protein constellation and this has been successfully achieved using gel-based techniques, particularly that of two-dimensional difference gel electrophoresis (2D-DIGE). However, given the complexity of the proteome, fractionation techniques may be required to optimize the detection of low-abundance proteins, which are often under-represented, but which may represent important players in health and disease. Such subcellular fractionation protocols typically utilize density-gradient centrifugation and have enabled the enrichment of crude microsomes, the cytosol, the plasmalemma, the nuclei, and the mitochondria. In this chapter, we describe the experimental steps involved in the enrichment of crude microsomes from skeletal muscle using differential centrifugation and subsequent verification of enrichment by gel electrophoresis and immunoblotting, prior to comparative DIGE analysis.