Proteomic reference map for sarcopenia research: mass spectrometric identification of key muscle proteins of organelles, cellular signaling, bioenergetic metabolism and molecular chaperoning.

During the natural aging process, frailty is often associated with abnormal muscular performance. Although inter-individual differences exit, in most elderly the tissue mass and physiological functionality of voluntary muscles drastically decreases. In order to study age-related contractile decline, animal model research is of central importance in the field of biogerontology. Here we have analyzed wild type mouse muscle to establish a proteomic map of crude tissue extracts. Proteomics is an advanced and large-scale biochemical method that attempts to identify all accessible proteins in a given biological sample. It is a technology-driven approach that uses mass spectrometry for the characterization of individual protein species. Total protein extracts were used in this study in order to minimize the potential introduction of artefacts due to excess subcellular fractionation procedures. In this report, the proteomic survey of aged muscles has focused on organellar marker proteins, as well as proteins that are involved in cellular signaling, the regulation of ion homeostasis, bioenergetic metabolism and molecular chaperoning. Hence, this study has establish a proteomic reference map of a highly suitable model system for future aging research.

Proteomic reference map for sarcopenia research: mass spectrometric identification of key muscle proteins located in the sarcomere, cytoskeleton and the extracellular matrix.

Sarcopenia of old age is characterized by the progressive loss of skeletal muscle mass and concomitant decrease in contractile strength. Age-related skeletal muscle dysfunctions play a key pathophysiological role in the frailty syndrome and can result in a drastically diminished quality of life in the elderly. Here we have used mass spectrometric analysis of the mouse hindlimb musculature to establish the muscle protein constellation at advanced age of a widely used sarcopenic animal model. Proteomic results were further analyzed by systems bioinformatics of voluntary muscles. In this report, the proteomic survey of aged muscles has focused on the expression patterns of proteins involved in the contraction-relaxation cycle, membrane cytoskeletal maintenance and the formation of the extracellular matrix. This includes proteomic markers of the fast versus slow phenotypes of myosin-containing thick filaments and actin-containing thin filaments, as well as proteins that are associated with the non-sarcomeric cytoskeleton and various matrisomal layers. The bioanalytical usefulness of the newly established reference map was demonstrated by the comparative screening of normal versus dystrophic muscles of old age, and findings were verified by immunoblot analysis.

Mass spectrometry-based proteomic characterization of the middle-aged mouse brain for animal model research of neuromuscular diseases.

Neuromuscular diseases with primary muscle wasting symptoms may also display multi-systemic changes in the body and exhibit secondary pathophysiological alterations in various non-muscle tissues. In some cases, this includes proteome-wide alterations and/or adaptations in the central nervous system. Thus, in order to provide an improved bioanalytical basis for the comprehensive evaluation of animal models that are routinely used in muscle research, this report describes the mass spectrometry-based proteomic characterization of the mouse brain. Crude tissue extracts were examined by bottom-up proteomics and detected 4558 distinct protein species. The detailed analysis of the brain proteome revealed the presence of abundant cellular proteoforms in the neuronal cytoskeleton, as well as various brain region enriched proteins, including markers of the cerebral cortex, cerebellum, hippocampus and the olfactory bulb. Neuroproteomic markers of specific cell types in the brain were identified in association with various types of neurons and glia cells. Markers of subcellular structures were established for the plasmalemma, nucleus, endoplasmic reticulum, mitochondria and other crucial organelles, as well as synaptic components that are involved in presynaptic vesicle docking, neurotransmitter release and synapse remodelling.

Extracellular Matrix Proteomics: The mdx-4cv Mouse Diaphragm as a Surrogate for Studying Myofibrosis in Dystrophinopathy.

The progressive degeneration of the skeletal musculature in Duchenne muscular dystrophy is accompanied by reactive myofibrosis, fat substitution, and chronic inflammation. Fibrotic changes and reduced tissue elasticity correlate with the loss in motor function in this X-chromosomal disorder. Thus, although dystrophinopathies are due to primary abnormalities in the DMD gene causing the almost-complete absence of the cytoskeletal Dp427-M isoform of dystrophin in voluntary muscles, the excessive accumulation of extracellular matrix proteins presents a key histopathological hallmark of muscular dystrophy. Animal model research has been instrumental in the characterization of dystrophic muscles and has contributed to a better understanding of the complex pathogenesis of dystrophinopathies, the discovery of new disease biomarkers, and the testing of novel therapeutic strategies. In this article, we review how mass-spectrometry-based proteomics can be used to study changes in key components of the endomysium, perimysium, and epimysium, such as collagens, proteoglycans, matricellular proteins, and adhesion receptors. The mdx-4cv mouse diaphragm displays severe myofibrosis, making it an ideal model system for large-scale surveys of systematic alterations in the matrisome of dystrophic fibers. Novel biomarkers of myofibrosis can now be tested for their appropriateness in the preclinical and clinical setting as diagnostic, pharmacodynamic, prognostic, and/or therapeutic monitoring indicators.

Comparing the efficacy in reducing brain injury of different neuroprotective agents following neonatal hypoxia-ischemia in newborn rats: a multi-drug randomized controlled screening trial.

Intrapartum hypoxia-ischemia leading to neonatal encephalopathy (NE) results in significant neonatal mortality and morbidity worldwide, with > 85% of cases occurring in low- and middle-income countries (LMIC). Therapeutic hypothermia (HT) is currently the only available safe and effective treatment of HIE in high-income countries (HIC); however, it has shown limited safety or efficacy in LMIC. Therefore, other therapies are urgently required. We aimed to compare the treatment effects of putative neuroprotective drug candidates following neonatal hypoxic-ischemic (HI) brain injury in an established P7 rat Vannucci model. We conducted the first multi-drug randomized controlled preclinical screening trial, investigating 25 potential therapeutic agents using a standardized experimental setting in which P7 rat pups were exposed to unilateral HI brain injury. The brains were analysed for unilateral hemispheric brain area loss after 7 days survival. Twenty animal experiments were performed. Eight of the 25 therapeutic agents significantly reduced brain area loss with the strongest treatment effect for Caffeine, Sonic Hedgehog Agonist (SAG) and Allopurinol, followed by Melatonin, Clemastine, ß-Hydroxybutyrate, Omegaven, and Iodide. The probability of efficacy was superior to that of HT for Caffeine, SAG, Allopurinol, Melatonin, Clemastine, ß-hydroxybutyrate, and Omegaven. We provide the results of the first systematic preclinical screening of potential neuroprotective treatments and present alternative single therapies that may be promising treatment options for HT in LMIC.

Neutrophil Extracellular Traps Release following Hypoxic-Ischemic Brain Injury in Newborn Rats Treated with Therapeutic Hypothermia.

The peripheral immune system plays a critical role in neuroinflammation of the central nervous system after an insult. Hypoxic-ischemic encephalopathy (HIE) induces a strong neuroinflammatory response in neonates, which is often associated with exacerbated outcomes. In adult models of ischemic stroke, neutrophils infiltrate injured brain tissue immediately after an ischemic insult and aggravate inflammation via various mechanisms, including neutrophil extracellular trap (NETs) formation. In this study, we used a neonatal model of experimental hypoxic-ischemic (HI) brain injury and demonstrated that circulating neutrophils were rapidly activated in neonatal blood. We observed an increased infiltration of neutrophils in the brain after exposure to HI. After treatment with either normothermia (NT) or therapeutic hypothermia (TH), we observed a significantly enhanced expression level of the NETosis marker Citrullinated H3 (Cit-H3), which was significantly more pronounced in animals treated with TH than in those treated with NT. NETs and NLR family pyrin domain containing 3 (NLRP-3) inflammasome assembly are closely linked in adult models of ischemic brain injury. In this study, we observed an increase in the activation of the NLRP-3 inflammasome at the time points analyzed, particularly immediately after TH, when we observed a significant increase in NETs structures in the brain. Together, these results suggest the important pathological functions of early arriving neutrophils and NETosis following neonatal HI, particularly after TH treatment, which is a promising starting point for the development of potential new therapeutic targets for neonatal HIE.

Bioinformatic Analysis of the Subproteomic Profile of Cardiomyopathic Tissue.

Following large-scale protein separation by two-dimensional gel electrophoresis or liquid chromatography, mass spectrometry-based proteomics can be used for the swift identification and characterization of cardiac proteins and their various proteoforms. Comparative cardiac proteomics has been widely applied for the systematic analysis of heart disease and the establishment of novel diagnostic protein biomarkers. The X-linked neuromuscular disorder Duchenne muscular dystrophy is a multisystemic disease that is characterized by late-onset cardiomyopathy. This chapter outlines the bioinformatic analysis of the subproteomic profile of cardiac tissue from wild-type versus the dystrophic mdx-4cv mouse model of dystrophinopathy.

Verification of Protein Changes Determined by 2D-DIGE Based Proteomics Using Immunofluorescence Microscopy.

Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a key biochemical method for the comparative analysis of complex protein mixtures. The technique focuses on the identification and characterization of individual protein species following gel electrophoretic separation making it an important analytical tool of top-down proteomics. In order to verify changes in the expression levels of a particular protein, as determined by 2D-DIGE analysis, and evaluate the subcellular localization of the proteoform of interest, immunofluorescence microscopy is very well suited. This chapter describes in detail the preparation of tissue specimens and the process of cryo-sectioning, as well as incubation with primary antibodies and fluorescently labeled secondary antibodies, followed by image analysis. As illustrative examples, the co-detection of immuno-labeled dystrophin and the Y-chromosome in skeletal muscle are shown, and the localization of calbindin in the cerebellum is presented.

Histological and Histochemical Microscopy Used to Verify 2D-DIGE Pathoproteomics.

Comparative gel electrophoretic analyses of normal versus pathological specimens can swiftly identify proteome-wide changes in the concentration of specific protein isoforms. The application of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) can be employed for the characterization of complex protein populations in health and disease. In order to verify pathoproteomic findings and correlate them to histopathological alterations, standardized histological and histochemical methodology can be applied for the cell biological analysis of normal versus pathological tissue samples. This chapter outlines the usage of histochemical ATPase staining of fast and slow fiber types in normal versus dystrophic skeletal muscles, as well as the application of hematoxylin and eosin staining of nuclei and the cellular body in kidney cells, and Sudan black staining of lipids in cryo-sections.

Proteomic Identification of Markers of Membrane Repair, Regeneration and Fibrosis in the Aged and Dystrophic Diaphragm.

Deficiency in the membrane cytoskeletal protein dystrophin is the underlying cause of the progressive muscle wasting disease named Duchenne muscular dystrophy. In order to detect novel disease marker candidates and confirm the complexity of the pathobiochemical signature of dystrophinopathy, mass spectrometric screening approaches represent ideal tools for comprehensive biomarker discovery studies. In this report, we describe the comparative proteomic analysis of young versus aged diaphragm muscles from wild type versus the dystrophic mdx-4cv mouse model of X-linked muscular dystrophy. The survey confirmed the drastic reduction of the dystrophin-glycoprotein complex in the mdx-4cv diaphragm muscle and concomitant age-dependent changes in key markers of muscular dystrophy, including proteins involved in cytoskeletal organization, metabolite transportation, the cellular stress response and excitation-contraction coupling. Importantly, proteomic markers of the regulation of membrane repair, tissue regeneration and reactive myofibrosis were detected by mass spectrometry and changes in key proteins were confirmed by immunoblotting. Potential disease marker candidates include various isoforms of annexin, the matricellular protein periostin and a large number of collagens. Alterations in these proteoforms can be useful to evaluate adaptive, compensatory and pathobiochemical changes in the intracellular cytoskeleton, myofiber membrane integrity and the extracellular matrix in dystrophin-deficient skeletal muscle tissues.

Impact of Hypoxia-Ischemia on Neurogenesis and Structural and Functional Outcomes in a Mild-Moderate Neonatal Hypoxia-Ischemia Brain Injury Model.

Hypoxic-ischemic encephalopathy (HIE) is a common type of brain injury caused by a lack of oxygen and blood flow to the brain during the perinatal period. The incidence of HIE is approximately 2−3 cases per 1000 live births in high-income settings; while in low- and middle-income countries, the incidence is 3−10-fold higher. Therapeutic hypothermia (TH) is the current standard treatment for neonates affected by moderate−severe HIE. However, more than 50% of all infants with suspected HIE have mild encephalopathy, and these infants are not treated with TH because of their lower risk of adverse outcomes. Despite this, several analyses of pooled data provide increasing evidence that infants who initially have mild encephalopathy may present signs of more significant brain injury later in life. The purpose of this study was to expand our knowledge about the effect of mild−moderate hypoxia-ischemia (HI) at the cellular, structural, and functional levels. An established rat model of mild−moderate HI was used, where postnatal day (P) 7 rats were exposed to unilateral permanent occlusion of the left carotid artery and 90 min of 8% hypoxia, followed by TH or normothermia (NT) treatment. The extent of injury was assessed using histology (P14 and P42) and MRI (P11 and P32), as well as with short-term and long-term behavioral tests. Neurogenesis was assessed by BrdU staining. We showed that mild−moderate HI leads to a progressive loss of brain tissue, pathological changes in MRI scans, as well as an impairment of long-term motor function. At P14, the median area loss assessed by histology for HI animals was 20% (p < 0.05), corresponding to mild−moderate brain injury, increasing to 55% (p < 0.05) at P42. The data assessed by MRI corroborated our results. HI led to a decrease in neurogenesis, especially in the hippocampus and the lateral ventricle at early time points, with a delayed partial recovery. TH was not neuroprotective at early time points following mild−moderate HI, but prevented the increase in brain damage over time. Additionally, rats treated with TH showed better long-term motor function. Altogether, our results bring more light to the understanding of pathophysiology following mild-moderate HI. We showed that, in the context of mild-moderate HI, TH failed to be significantly neuroprotective. However, animals treated with TH showed a significant improvement in motor, but not cognitive long-term function. These results are in line with what is observed in some cases where neonates with mild HIE are at risk of neurodevelopmental deficits in infancy or childhood. Whether TH should be used as a preventive treatment to reduce adverse outcomes in mild-HIE remains of active interest, and more research has to be carried out in order to address this question.

Temporal Characterization of Microglia-Associated Pro- and Anti-Inflammatory Genes in a Neonatal Inflammation-Sensitized Hypoxic-Ischemic Brain Injury Model.

Hypoxic-ischemic encephalopathy (HIE) mainly affects preterm and term newborns, leading to a high risk of brain damage. Coexisting infection/inflammation and birth asphyxia are key factors associated with intracerebral increase of proinflammatory cytokines linked to HIE. Microglia are key mediators of inflammation during perinatal brain injury, characterized by their phenotypic plasticity, which may facilitate their participation in both the progression and resolution of injury-induced inflammation. The purpose of this study was to investigate the temporal expression of genes associated with pro- and anti-inflammatory cytokines as well as the nucleotide-binding domain, leucine-rich repeat protein (NLRP-3) inflammasome from microglia cells. For this purpose, we used our established neonatal rat model of inflammation-sensitized hypoxic-ischemic (HI) brain injury in seven-day-old rats. We assessed gene expression profiles of 11 cytokines and for NLRP-3 using real-time PCR from sorted CD11b/c microglia of brain samples at different time points (3.5 h after LPS injection and 0, 5, 24, 48, and 72 hours post HI) following different treatments: vehicle, E. coli lipopolysaccharide (LPS), vehicle/HI, and LPS/HI. Our results showed that microglia are early key mediators of the inflammatory response and exacerbate the inflammatory response following HI, polarizing into a predominant proinflammatory M1 phenotype in the early hours post HI. The brains only exposed to HI showed a delay in the expression of proinflammatory cytokines. We also demonstrated that NLRP-3 plays a role in the inflammatory resolution with a high expression after HI insult. The combination of both, a preinfection/inflammation condition and hypoxia-ischemia, resulted in a higher proinflammatory cytokine storm, highlighting the significant contribution of acute inflammation sensitizing prior to a hypoxic insult on the severity of perinatal brain damage.

Histopathology of Duchenne muscular dystrophy in correlation with changes in proteomic biomarkers.

Duchenne muscular dystrophy is an inherited disorder of early childhood that affects multiple systems in the body. Besides late-onset cardio-respiratory syndrome and various body-wide pathophysiological changes, X-linked muscular dystrophy is primarily classified as a disorder of the skeletal musculature. This is reflected by severe histopathological alterations in voluntary contractile tissues, including progressive fibre degeneration, fat substitution, reactive myofibrosis and chronic inflammation. The underlying cause for dystrophinopathy are genetic abnormalities in the DMD gene, which can result in the almost complete loss of the membrane cytoskeletal protein dystrophin, which triggers the collapse of the dystrophin-associated glycoprotein complex and disintegration of sarcolemmal integrity. This in turn results in an increased frequency of membrane micro-rupturing and abnormal calcium ion fluxes through the impaired plasmalemma, which renders muscle fibres more susceptible to enhanced proteolytic degradation and necrosis. This review focuses on the complexity of skeletal muscle changes in X-linked muscular dystrophy and outlines cell biological and histological alterations in correlation to proteome-wide variations as judged by mass spectrometric analyses. This includes a general outline of sample handling, subcellular fraction protocols and modern proteomic approaches using gel electrophoretic and liquid chromatographic methods for efficient protein separation prior to mass spectrometry. The proteomic profiling of the dystrophic and highly fibrotic diaphragm muscle is described as an example to swiftly identify novel proteomic markers of complex histopathological changes during skeletal muscle degeneration. The potential usefulness of new protein markers is examined in relation to key histopathological hallmarks for establishing improved diagnostic, prognostic and therapy-monitoring approaches in the field of dystrophinopathy.

Proteomic profiling of carbonic anhydrase CA3 in skeletal muscle.

INTRODUCTION: Carbonic anhydrase (CA) is a key enzyme that mediates the reversible hydration of carbon dioxide. Skeletal muscles contain high levels of the cytosolic isoform CA3. This enzyme has antioxidative function and plays a crucial role in the maintenance of intracellular pH homeostasis. AREAS COVERED: Since elevated levels of serum CA3, often in combination with other muscle-specific proteins, are routinely used as a marker of general muscle damage, it was of interest to examine recent analyses of this enzyme carried out by modern proteomics. This review summarizes the mass spectrometry-based identification and evaluation of CA3 in normal, adapting, dystrophic, and aging skeletal muscle tissues. EXPERT OPINION: The mass spectrometric characterization of CA3 confirmed this enzyme as a highly useful marker of both physiological and pathophysiological alterations in skeletal muscles. Cytosolic CA3 is clearly enriched in slow-twitching type I fibers, which makes it an ideal marker for studying fiber type shifting and muscle adaptations. Importantly, neuromuscular diseases feature distinct alterations in CA3 in skeletal muscle tissues versus biofluids, such as serum. Characteristic changes of CA3 in age-related muscle wasting and dystrophinopathy established this enzyme as a suitable biomarker candidate for differential diagnosis and monitoring of disease progression and therapeutic impact.

Mass Spectrometric Profiling of Extraocular Muscle and Proteomic Adaptations in the mdx-4cv Model of Duchenne Muscular Dystrophy.

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.

Proteomic profiling of the interface between the stomach wall and the pancreas 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 enzymes is indicative of metabolic disturbances. These findings agree with abnormal gastrointestinal function in dystrophinopathy.

The Dystrophin Node as Integrator of Cytoskeletal Organization, Lateral Force Transmission, Fiber Stability and Cellular Signaling in Skeletal Muscle.

The systematic bioanalytical characterization of the protein product of the DMD gene, which is defective in the pediatric disorder Duchenne muscular dystrophy, led to the discovery of the membrane cytoskeletal protein dystrophin. Its full-length muscle isoform Dp427-M is tightly linked to a sarcolemma-associated complex consisting of dystroglycans, sarcoglyans, sarcospan, dystrobrevins and syntrophins. Besides these core members of the dystrophin-glycoprotein complex, the wider dystrophin-associated network includes key proteins belonging to the intracellular cytoskeleton and microtubular assembly, the basal lamina and extracellular matrix, various plasma membrane proteins and cytosolic components. Here, we review the central role of the dystrophin complex as a master node in muscle fibers that integrates cytoskeletal organization and cellular signaling at the muscle periphery, as well as providing sarcolemmal stabilization and contractile force transmission to the extracellular region. The combination of optimized tissue extraction, subcellular fractionation, advanced protein co-purification strategies, immunoprecipitation, liquid chromatography and two-dimensional gel electrophoresis with modern mass spectrometry-based proteomics has confirmed the composition of the core dystrophin complex at the sarcolemma membrane. Importantly, these biochemical and mass spectrometric surveys have identified additional members of the wider dystrophin network including biglycan, cavin, synemin, desmoglein, tubulin, plakoglobin, cytokeratin and a variety of signaling proteins and ion channels.

Protocol for the Bottom-Up Proteomic Analysis of Mouse Spleen.

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).

Proteome-wide Changes in the mdx-4cv Spleen due to Pathophysiological Cross Talk with Dystrophin-Deficient Skeletal Muscle.

Duchenne muscular dystrophy is primarily characterized by progressive muscle wasting due to deficiency in the membrane cytoskeletal protein dystrophin but is also associated with body-wide cellular disturbances in a variety of non-muscle tissues. In this study, we have focused on the comparative proteomic analysis of the spleen and established considerable changes in this crucial secondary lymphoid organ from the genetic mdx-4cv mouse model of dystrophinopathy. An apparent short isoform of dystrophin and associated glycoproteins were identified in spleen by mass spectrometry but appear not be affected in muscular dystrophy. In contrast, the mdx-4cv spleen showed significant proteome-wide changes in other protein species that are involved in metabolism, signaling, and cellular architecture. Since the spleen plays a key role in the immune response, these proteomic alterations may reflect pathophysiological cross talk between the lymphoid system and dystrophic muscles, which are affected by both fiber degeneration and inflammation.

Identification of marker proteins of muscular dystrophy in the urine proteome from the mdx-4cv model of dystrophinopathy.

Since the protein constituents of urine present a dynamic proteome that can reflect a variety of disease-related alterations in the body, the mass spectrometric survey of proteome-wide changes in urine promises new insights into pathogenic mechanisms. Urine can be investigated in a completely non-invasive way and provides valuable biomedical information on body-wide changes. In this report, we have focused on the urine proteome in X-linked muscular dystrophy using the established mdx-4cv mouse model of dystrophinopathy. In order to avoid potential artefacts due to the manipulation of the biofluid proteome prior to mass spectrometry, crude urine specimens were analyzed without the prior usage of centrifugation steps or concentration procedures. Comparative proteomics revealed 21 increased and 8 decreased proteins out of 870 identified urinary proteoforms using 50 µl of biofluid per investigated sample, i.e. 14 wild type versus 14 mdx-4cv specimens. Promising marker proteins that were almost exclusively found in mdx-4cv urine included nidogen, parvalbumin and titin. Interestingly, the mass spectrometric identification of urine-associated titin revealed a wide spread of peptides over the sequence of this giant muscle protein. The newly established urinomic signature of dystrophinopathy might be helpful for the design of non-invasive assays to improve diagnosis, prognosis, therapy-monitoring and evaluation of potential harmful side effects of novel treatments in the field of muscular dystrophy research.