Ashling Holland

Label-free mass spectrometric analysis of the mdx-4cv diaphragm identifies the matricellular protein periostin as a potential factor involved in dystrophinopathy-related fibrosis

Proteomic profiling plays a decisive role in the identification of novel biomarkers of muscular dystrophy and the elucidation of new pathobiochemical mechanisms that underlie progressive muscle wasting. Building on the findings of recent comparative analyses of tissue samples and body fluids from dystrophic animals and patients afflicted with Duchenne muscular dystrophy, we have used here label‐free MS to study the severely dystrophic diaphragm from the not extensively characterized mdx‐4cv mouse. This animal model of progressive muscle wasting exhibits less dystrophin‐positive revertant fibers than the conventional mdx mouse, making it ideal for the future monitoring of experimental therapies. The pathoproteomic signature of the mdx‐4cv diaphragm included a significant increase in the fibrosis marker collagen and related extracellular matrix proteins (asporin, decorin, dermatopontin, prolargin) and cytoskeletal proteins (desmin, filamin, obscurin, plectin, spectrin, tubulin, vimentin, vinculin), as well as decreases in proteins of ion homeostasis (parvalbumin) and the contractile apparatus (myosin‐binding protein). Importantly, one of the most substantially increased proteins was identified as periostin, a matricellular component and apparent marker of fibrosis and tissue damage. Immunoblotting confirmed a considerable increase of periostin in the dystrophin‐deficient diaphragm from both mdx and mdx‐4cv mice, suggesting an involvement of this matricellular protein in dystrophinopathy‐related fibrosis.

Proteomic profiling of cardiomyopathic tissue from the aged mdx model of Duchenne muscular dystrophy reveals a drastic decrease in laminin, nidogen and annexin

The majority of patients afflicted with Duchenne muscular dystrophy develop cardiomyopathic complications, warranting large‐scale proteomic studies of global cardiac changes for the identification of new protein markers of dystrophinopathy. The aged heart from the X‐linked dystrophic mdx mouse has been shown to exhibit distinct pathological aspects of cardiomyopathy. In order to establish age‐related alterations in the proteome of dystrophin‐deficient hearts, cardiomyopathic tissue from young versus aged mdx mice was examined by label‐free LC‐MS/MS. Significant age‐dependent alterations were established for 67 proteins, of which 28 proteins were shown to exhibit a lower abundance and 39 proteins were found to be increased in their expression levels. Drastic changes were demonstrated for 17 proteins, including increases in Ig chains and transferrin, and drastic decreases in laminin, nidogen and annexin. An immunblotting survey of young and old wild‐type versus mdx hearts confirmed these proteomic findings and illustrated the effects of natural aging versus dystrophin deficiency. These proteome‐wide alterations suggest a disintegration of the basal lamina structure and cytoskeletal network in dystrophin‐deficient cardiac fibres, increased levels of antibodies in a potential autoimmune reaction of the degenerating heart, compensatory binding of excess iron and a general perturbation of metabolic pathways in dystrophy‐associated cardiomyopathy.

Proteomics of the dystrophin-glycoprotein complex and dystrophinopathy.

The largest human gene is represented by the X-chromosomal dystrophin gene of 2.4 million bases, which encodes for the membrane cytoskeletal protein dystrophin. The dystrophin isoform Dp427 has a subsarcolemmal location and forms a supramolecular membrane assembly with a variety of glycoproteins. In healthy muscle fibres, dystrophin acts as an actin-binding protein that links the cytoskeleton via the α/β-dystroglycan complex to the extracellular matrix protein laminin. This trans-sarcolemmal complex is believed to stabilize the muscle surface and thus prevents membrane rupturing during excitation-contraction-relaxation cycles. In the highly progressive muscle wasting disease Duchenne muscular dystrophy, the primary deficiency in dystrophin causes a drastic reduction in dystrophin-associated glycoproteins, which renders muscle fibres more susceptible to necrosis. Following the biochemical and cell biological characterization of the dystrophin-glycoprotein complex, several mass spectrometry-based proteomic studies have investigated global changes in dystrophin-deficient muscle tissues. This review briefly outlines the basic domain structure of Dp427 and the composition of the dystrophin-associated glycoprotein complex from skeletal muscle. A detailed discussion of recent proteomic analyses of the purified dystrophin-glycoprotein complex is included, as well as a summary of mass spectrometric surveys of dystrophic specimens. The study of these new areas of muscle proteomics tends to improve our understanding of the normal function of dystrophin in contractile fibres and better define the molecular mechanism of X-linked muscular dystrophy.

Comparative Label-Free Mass Spectrometric Analysis of Mildly versus Severely Affected mdx Mouse Skeletal Muscles Identifies Annexin, Lamin, and Vimentin as Universal Dystrophic Markers

The primary deficiency in the membrane cytoskeletal protein dystrophin results in complex changes in dystrophic muscles. In order to compare the degree of secondary alterations in differently affected subtypes of skeletal muscles, we have conducted a global analysis of proteome-wide changes in various dystrophin-deficient muscles. In contrast to the highly degenerative mdx diaphragm muscle, which showed considerable alterations in 35 distinct proteins, the spectrum of mildly to moderately dystrophic skeletal muscles, including interosseus, flexor digitorum brevis, soleus, and extensor digitorum longus muscle, exhibited a smaller number of changed proteins. Compensatory mechanisms and/or cellular variances may be responsible for differing secondary changes in individual mdx muscles. Label-free mass spectrometry established altered expression levels for diaphragm proteins associated with contraction, energy metabolism, the cytoskeleton, the extracellular matrix and the cellular stress response. Comparative immunoblotting verified the differences in the degree of secondary changes in dystrophin-deficient muscles and showed that the up-regulation of molecular chaperones, the compensatory increase in proteins of the intermediate filaments, the fibrosis-related increase in collagen levels and the pathophysiological decrease in calcium binding proteins is more pronounced in mdx diaphragm as compared to the less severely affected mdx leg muscles. Annexin, lamin, and vimentin were identified as universal dystrophic markers.

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.

Mass Spectrometry-Based Identification of Muscle-Associated and Muscle-Derived Proteomic Biomarkers of Dystrophinopathies

The optimization of large-scale screening procedures of pathological specimens by genomic, proteomic and metabolic methods has drastically increased the bioanalytical capability for swiftly identifying novel biomarkers of inherited disorders, such as neuromuscular diseases. X-linked muscular dystrophy represents the most frequently inherited muscle disease and is characterized by primary abnormalities in the membrane cytoskeletal protein dystrophin. Mass spectrometry-based proteomics has been widely employed for the systematic analysis of dystrophin-deficient muscle tissues, using patient samples and animal models of dystrophinopathy. Both, gel-based methods and label-free mass spectrometric techniques have been applied in comparative analyses and established a large number of altered proteins that are associated with muscle contraction, energy metabolism, ion homeostasis, cellular signaling, the cytoskeleton, the extracellular matrix and the cellular stress response. Although these new indicators of muscular dystrophy have increased our general understanding of the molecular pathogenesis of dystrophinopathy, their application as new diagnostic or prognostic biomarkers would require the undesirable usage of invasive methodology. Hence, to reduce the need for diagnostic muscle biopsy procedures, more recent efforts have focused on the proteomic screening of suitable body fluids, such as plasma, serum or urine, for the identification of changed concentration levels of muscle-derived peptides, protein fragments or intact proteins. The occurrence of muscular dystrophy-related protein species in biofluids will be extremely helpful for the future development of cost-effective and non-invasive diagnostic procedures. Novel biomarker signatures of dystrophinopathies will be indispensible for the swift evaluation of innovative therapeutic approaches, such as exon skipping, codon-read-through or stem cell therapy.

Comparative profiling of the sperm proteome.

The highly complex and species‐selective mechanism of fertilization is a central theme of developmental biology. Gametogenesis, sperm activation, and egg–sperm recognition are fundamental biological processes, warranting detailed studies into the molecular composition of gametes. Biological MS has been instrumental for the comprehensive itemizing of gamete proteomes. The protein constellation of sperm cells and its subcellular structures has been established for a variety of animal species. Spermatogenesis and the crucial activation of sperm cells as a prerequisite of successful fertilization and physiological adaptations to external stressors was investigated using proteomics, as well as the underlying mechanisms of male infertility with respect to proteome‐wide alterations. This review outlines recent achievements of sperm proteomics and exemplifies the usefulness of gel‐based surveys by outlining the comparative analysis of abnormal spermatozoa in globozoospermia. Besides label‐free MS techniques and cell‐based labeling methodology, high‐resolution fluorescence 2DE has been shown to be highly suitable as a proteomic biomarker discovery tool in sperm protein research. The appropriateness of novel protein markers for improving our understanding of normal spermatogenesis and sperm activation versus the molecular pathogenesis of male infertility will be discussed. New biomarker candidates might be useful to improve diagnostic, prognostic, and therapeutic aspects of infertility.

Pathoproteomics of testicular tissue deficient in the GARP component VPS54: The wobbler mouse model of globozoospermia

In human globozoospermia, round‐headed spermatozoa lack an acrosome and therefore cannot properly interact with oocytes. In the wobbler (WR) mouse, an L967Q missense mutation in the vesicular protein‐sorting factor VPS54 causes motor neuron degeneration and globozoospermia. Although electron microscopy of WR testis shows all major components of spermatogenesis, they appear in a deranged morphology that prevents the formation of the acrosome. In order to determine proteome‐wide changes, affected testes were analysed by 2D‐DIGE and MS. The concentration of 8 proteins was increased and that of 35 proteins decreased as compared to wild type. Mass spectrometric analysis identified proteins with an altered concentration to be associated with metabolite transport, fatty acid metabolism, cellular interactions, microtubule assembly and stress response (chaperones Hsp70–2 and Hsp90α). Minor changes were observed for proteins involved in cell redox homeostasis, cytoskeleton formation, PTMs, detoxification and metabolism. The most dramatically decreased protein in WR testis was identified as fatty acid binding protein FABP3, as confirmed by immunoblot analysis. We conclude that a missense mutation in VPS54, an essential component of the Golgi‐associated retrograde protein complex, not only prevents the formation of an acrosome but also initiates a cascade of metabolic abnormalities and a stress reaction.

Comparative Testis Tissue Proteomics Using 2-Dye Versus 3-Dye DIGE Analysis

Comparative tissue proteomics aims to analyze alterations of the proteome in response to a stimulus. Two-dimensional difference gel electrophoresis (2D-DIGE) is a modified and advanced form of 2D gel electrophoresis. DIGE is a powerful biochemical method that compares two or three protein samples on the same analytical gel, and can be used to establish differentially expressed protein levels between healthy normal and diseased pathological tissue sample groups. Minimal DIGE labeling can be used via a 2-dye system with Cy3 and Cy5 or a 3-dye system with Cy2, Cy3, and Cy5 to fluorescently label samples with CyDye flours pre-electrophoresis. DIGE circumvents gel-to-gel variability by multiplexing samples to a single gel and through the use of a pooled internal standard for normalization. This form of quantitative high-resolution proteomics facilitates the comparative analysis and evaluation of tissue protein compositions. Comparing tissue groups under different conditions is crucially important for advancing the biomedical field by characterization of cellular processes, understanding pathophysiological development and tissue biomarker discovery. This chapter discusses 2D-DIGE as a comparative tissue proteomic technique and describes in detail the experimental steps required for comparative proteomic analysis employing both options of 2-dye and 3-dye DIGE minimal labeling.