Proteome-wide Alterations Research Articles

FLASHQuant: A Fast Algorithm for Proteoform Quantification in Top-Down Proteomics.

Accurate quantification of individual proteoforms is a crucial step in identifying proteome-wide alterations in different biological conditions. Intact proteoforms have been analyzed predominantly by liquid chromatography-mass spectrometry (LC-MS)-based top-down proteomics (TDP) and quantified primarily by the label-free quantification (LFQ) method, as it requires no additional costly labeling. In TDP, due to frequent coelution and complex signal structures, overlapping signals deriving from multiple proteoforms complicate accurate quantification. Here, we introduce FLASHQuant for MS1-level LFQ analysis in TDP, which is capable of automatically resolving and quantifying coeluting proteoforms. In benchmark tests performed with both spike-in proteins and proteome-level mixture data sets, FLASHQuant was shown to perform highly accurate and reproducible quantification in short runtimes of just a few minutes per LC-MS run. In particular, it was demonstrated that resolving overlapping proteoforms boosts the quantification accuracy. FLASHQuant is publicly available as platform-independent open-source software at https://openms.org/flashquant/, accompanied by the simple alignment algorithm ConsensusFeatureGroupDetector for multiple LC-MS runs.

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.

Sample Preparation and Protein Determination for 2D-DIGE Proteomics.

Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a widely employed method for efficient protein separation and the determination of abundance changes in distinct proteoforms. This makes this gel-based method a key technique of comparative approaches in top-down proteomics. For the appropriate screening of proteome-wide alterations, initial preparative steps involve sample handling, homogenization, subcellular fractionation, and the determination of protein concentration, which makes the optimal application of these techniques a crucial part of a successful initiation of a new 2D-DIGE-based analysis. This chapter describes sample homogenization and a standardized protein assay for the preparation of homogenates with a known protein concentration for subsequent differential fluorescent tagging and two-dimensional gel electrophoretic separation.

An integrative-omics analysis of an industrial clavulanic acid-overproducing Streptomyces clavuligerus.

Clavulanic acid (CA) is a clinically important secondary metabolite used to treat infectious diseases. We aimed to decipher complex regulatory mechanisms acting in CA biosynthesis by analyzing transcriptome- and proteome-wide alterations in an industrial CA overproducer Streptomyces clavuligerus strain, namely DEPA and its wild-type counterpart NRRL3585. A total of 924 differentially expressed genes (DEGs) and 271 differentially produced proteins (DPPs) were obtained by RNA-seq and nanoLC-MS/MS analyses, respectively. In particular, CA biosynthetic genes, namely, car (cad), cas2, oat2, pah, bls, ceas2, orf12, and claR, a cluster situated regulatory (CSR) gene, were significantly upregulated as shown by RNA-seq. Enzymes of clavam biosynthesis were downregulated considerably in the DEPA strain, while the genes involved in the arginine biosynthesis, one of the precursors of CA pathway, were overexpressed. However, the biosynthesis of the other CA precursor, glyceraldehyde-3-phosphate (G3P), was not affected. CA overproduction in the DEPA strain was correlated with BldD, BldG, BldM, and BldN (AdsA) overrepresentation. In addition, TetR, WhiB, and Xre family transcriptional regulators were shown to be significantly overrepresented. Several uncharacterized/unknown proteins differentially expressed in the DEPA strain await further studies for functional characterization. Correlation analysis indicated an acceptable degree of consistency between the transcriptome and proteome data. The study represents the first integrative-omics analysis in a CA overproducer S. clavuligerus strain, providing insights into the critical control points and potential rational engineering targets for a purposeful increase of CA yields in strain improvement. KEY POINTS: ∙ Transcriptome and proteome-wide alterations in industrial CA overproducer strain DEPA ∙ An acceptable degree of consistency between the transcriptome and proteome data ∙ New targets to be exploited for rational engineering.

Proteome-Wide Alterations of Asymmetric Arginine Dimethylation Associated With Pancreatic Ductal Adenocarcinoma Pathogenesis.

Arginine methylation catalyzed by protein arginine methyltransferases (PRMTs) performs essential roles in regulating cancer initiation and progression, but its implication in pancreatic ductal adenocarcinoma (PDAC) requires further elucidation. In this study, asymmetric dimethylarginine (ADMA)-containing peptides in PDAC cell line PANC-1 were identified by label-free quantitative proteomics combined with affinity purification, using human non-cancerous pancreatic ductal epithelium cell line HPDE6c7 as the control. In total, 289 ADMA sites in 201 proteins were identified in HPDE6c7 and PANC-1 cells, including 82 sites with lower dimethylation and 37 sites with higher dimethylation in PANC-1 cells compared with HPDE6c7 cells. These ADMA-containing peptides demonstrated significant enrichment of glycine and proline residues in both cell lines. Importantly, leucine residues were significantly enriched in ADMA-containing peptides identified only in HPDE6c7 cells or showing lower dimethylation in PANC-1 cells. ADMA-containing proteins were significantly enriched in multiple biological processes and signaling cascades associated with cancer development, such as spliceosome machinery, the Wnt/β-catenin, Hedgehog, tumor growth factor beta (TGF-β), and mitogen-activated protein kinase (MAPK) signaling pathways. Moreover, PDAC cell lines with enhanced cell viability showed lower PRMT4 protein abundance and global ADMA-containing protein levels compared with HPDE6c7. PRMT4 overexpression partially recovered ADMA-containing protein levels and repressed viability in PANC-1 cells. These results revealed significantly altered ADMA-containing protein profiles in human pancreatic carcinoma cells, which provided a basis for elucidating the pathogenic roles of PRMT-mediated protein methylation in pancreatic cancer.

OR33-04 The Effect of Adiposity on the Fasting Serum Proteome in Normal Weight and Obese Men

In many individuals with obesity, adipose tissue is not only increased in mass but also exhibits altered function. Disordered adipokine secretion contributes to a pro-inflammatory milieu that may lead to obesity-related co-morbidities. Hypothesis-generating, high-throughput techniques can generate novel insights. Our objective was to identify proteome-wide alterations in serum proteins related to adiposity. We evaluated the fasting serum proteome in 25 men [13 normal weight (mean±SD body mass index (BMI 21.2±1.4 kg/m2) and 12 with obesity (BMI 31.5±4.8 kg/m2)]. Blood was drawn at 0, 15, 30, and 55 minutes for proteomic analysis after an overnight fast (SOMAScan, SomaLogic, Inc.). The number of proteins and pathways that significantly differed between the two groups was determined across all time points during the fasting period. Normalized protein levels were compared between adiposity groups using rank product testing. Overrepresentation of protein constituents of established biological pathways was evaluated by hypergeometric test. P-values were adjusted using the Benjamini-Hockberg method, and those with an associated false discovery rate (FDR) of <0.05 were considered statistically significant. A total of 4,785 protein isoforms were robustly identified in serum of normal weight and men with obesity, of which 226 protein isoforms were significantly higher in obesity (vs. normal weight), and 178 were significantly lower in obesity (vs. normal weight). Higher levels of leptin and lower levels of IGFBP1 and IGFBP2 were observed in individuals with obesity during fasting (all FDR < 0.019). Functional annotation using Gene Ontology indicated that the following pathways differed most between obese vs. normal weight men: complement and coagulation cascade activation, amine metabolism, phosphatase activity, cellular response to nutrients (lipids, alcohol, and vitamin D), organic acid catabolism, and regulation of inflammatory responses. The protein isoforms identified in serum likely reflect systemic tissue-level changes in metabolism that may yield insights into the pathogenesis of obesity-related comorbidities and rational targets for intervention.

Elevated carbon dioxide levels lead to proteome-wide alterations for optimal growth of a fast-growing cyanobacterium, Synechococcus elongatus PCC 11801

The environmental considerations attributing to the escalation of carbon dioxide emissions have raised alarmingly. Consequently, the concept of sequestration and biological conversion of CO2 by photosynthetic microorganisms is gaining enormous recognition. In this study, in an attempt to discern the synergistic CO2 tolerance mechanisms, metabolic responses to increasing CO2 concentrations were determined for Synechococcus elongatus PCC 11801, a fast-growing, novel freshwater strain, using quantitative proteomics. The protein expression data revealed that the organism responded to elevated CO2 by not only regulating the cellular transporters involved in carbon-nitrogen uptake and assimilation but also by inducing photosynthesis, carbon fixation and glycolysis. Several components of photosynthetic machinery like photosystem reaction centers, phycobilisomes, cytochromes, etc. showed a marked up-regulation with a concomitant downshift in proteins involved in photoprotection and redox maintenance. Additionally, enzymes belonging to the TCA cycle and oxidative pentose phosphate pathway exhibited a decline in their expression, further highlighting that the demand for reduced cofactors was fulfilled primarily through photosynthesis. The present study brings the first-ever comprehensive assessment of intricate molecular changes in this novel strain while shifting from carbon-limited to carbon-sufficient conditions and may pave the path for future host and pathway engineering for production of sustainable fuels through efficient CO2 capture.

MON-LB021 Effects of Intranasal Oxytocin on the Fasting Serum Proteome in Healthy Lean and Obese Men

Background: The neuropeptide oxytocin (OT) may have metabolic effects, including regulation of energy intake, fat oxidation, and insulin sensitivity. However, the mechanisms underlying the metabolic effects of OT are not well understood. Objective: To examine proteome-wide alterations in response to intranasal OT in serum of healthy lean and obese men. Methods: 25 healthy men, [13 lean (mean±SD body mass index (BMI) 21.2±1.4 kg/m2) and 12 obese (BMI 31.5±4.8 kg/m2)] mean age 27.1±7.8 years, completed a randomized, placebo-controlled crossover study of a single-dose of 24 IU intranasal OT (1). Fasting blood was drawn immediately prior (T0) and at 15, 30, and 55 minutes after OT/placebo administration for proteomic analysis (SOMAScan, SomaLogic, Inc.). The number of proteins and pathways that significantly changed from T0 to the mean post-dose, fasting measurement (T15-T55) in OT vs. placebo was determined. Normalized protein levels were compared between treatment and adiposity groups using rank product testing. Overrepresentation of known pathways containing proteins with significantly different concentrations was evaluated by hypergeometric test. P-values were adjusted by the Benjamini-Hockberg method to determine false discovery rates (FDR). Significance at FDR of 5%. Results: A total of 4,785 proteins were identified in serum of lean and obese men. In response to OT, a total of 710 pathways were altered. In both lean and obese men, OT-responsive biological pathways included regulation of inflammatory pathways via IL4, IL5, IL6, and IL7 (e.g., STAT1 and NFkB), insulin signaling (e.g., G-protein coupled receptor 2 and AKT), leptin signaling (e.g., STAT1, STAT3, and JAK2); and regulation of lipid metabolic processes (e.g., phospholipase and protein kinase C). In obese men, OT-responsive pathways included regulation of white adipocyte differentiation (e.g., cyclin dependent kinase) and IL3 signaling (e.g., tyrosine and serine/threonine kinase). In lean men, OT-responsive pathways included regulation of the acute inflammatory response (e.g., C-reactive protein and IL6 signal transducer) and lipid localization (e.g., apolipoproteins B, F, and L1). Conclusions: Our results suggest that OT modulates metabolic and inflammatory processes in men, and the specific effects may vary by adiposity status. Further research is required to confirm these exploratory findings. A clearer understanding of the effects of OT on the serum proteome will improve the translational capacity of OT-based therapeutics for use in obesity and other metabolic diseases.

Neuronal Apolipoprotein E4 Expression Results in Proteome-Wide Alterations and Compromises Bioenergetic Capacity by Disrupting Mitochondrial Function.

Apolipoprotein (apo) E4, the major genetic risk factor for Alzheimer’s disease (AD), alters mitochondrial function and metabolism early in AD pathogenesis. When injured or stressed, neurons increase apoE synthesis. Because of its structural difference from apoE3, apoE4 undergoes neuron-specific proteolysis, generating fragments that enter the cytosol, interact with mitochondria, and cause neurotoxicity. However, apoE4’s effect on mitochondrial respiration and metabolism is not understood in detail. Here we used biochemical assays and proteomic profiling to more completely characterize the effects of apoE4 on mitochondrial function and cellular metabolism in Neuro-2a neuronal cells stably expressing apoE4 or apoE3. Under basal conditions, apoE4 impaired respiration and increased glycolysis, but when challenged or stressed, apoE4-expressing neurons had 50% less reserve capacity to generate ATP to meet energy requirements than apoE3-expressing neurons. ApoE4 expression also decreased the NAD+/NADH ratio and increased the levels of reactive oxygen species and mitochondrial calcium. Global proteomic profiling revealed widespread changes in mitochondrial processes in apoE4 cells, including reduced levels of numerous respiratory complex subunits and major disruptions to all detected subunits in complex V (ATP synthase). Also altered in apoE4 cells were levels of proteins related to mitochondrial endoplasmic reticulum–associated membranes, mitochondrial fusion/fission, mitochondrial protein translocation, proteases, and mitochondrial ribosomal proteins. ApoE4-induced bioenergetic deficits led to extensive metabolic rewiring, but despite numerous cellular adaptations, apoE4-expressing neurons remained vulnerable to metabolic stress. Our results provide insights into potential molecular targets of therapies to correct apoE4-associated mitochondrial dysfunction and altered cellular metabolism.

Olfactory proteotyping: towards the enlightenment of the neurodegeneration.

Olfactory proteotyping: towards the enlightenment of the neurodegeneration.

Proteomic profiling of liver tissue from the mdx-4cv mouse model of Duchenne muscular dystrophy

BackgroundDuchenne muscular dystrophy is a highly complex multi-system disease caused by primary abnormalities in the membrane cytoskeletal protein dystrophin. Besides progressive skeletal muscle degeneration, this neuromuscular disorder is also associated with pathophysiological perturbations in many other organs including the liver. To determine potential proteome-wide alterations in liver tissue, we have used a comparative and mass spectrometry-based approach to study the dystrophic mdx-4cv mouse model of dystrophinopathy.MethodsThe comparative proteomic profiling of mdx-4cv versus wild type liver extracts was carried out with an Orbitrap Fusion Tribrid mass spectrometer. The distribution of identified liver proteins within protein families and potential protein interaction patterns were analysed by systems bioinformatics. Key findings on fatty acid binding proteins were confirmed by immunoblot analysis and immunofluorescence microscopy.ResultsThe proteomic analysis revealed changes in a variety of protein families, affecting especially fatty acid, carbohydrate and amino acid metabolism, biotransformation, the cellular stress response and ion handling in the mdx-4cv liver. Drastically increased protein species were identified as fatty acid binding protein FABP5, ferritin and calumenin. Decreased liver proteins included phosphoglycerate kinase, apolipoprotein and perilipin. The drastic change in FABP5 was independently verified by immunoblotting and immunofluorescence microscopy.ConclusionsThe proteomic results presented here indicate that the intricate and multifaceted pathogenesis of the mdx-4cv model of dystrophinopathy is associated with secondary alterations in the liver affecting especially fatty acid transportation. Since FABP5 levels were also shown to be elevated in serum from dystrophic mice, this protein might be a useful indicator for monitoring liver changes in X-linked muscular dystrophy.

Obesity Proteomics: An Update on the Strategies and Tools Employed in the Study of Human Obesity.

Proteomics has become one of the most important disciplines for characterizing cellular protein composition, building functional linkages between protein molecules, and providing insight into the mechanisms of biological processes in a high-throughput manner. Mass spectrometry-based proteomic advances have made it possible to study human diseases, including obesity, through the identification and biochemical characterization of alterations in proteins that are associated with it and its comorbidities. A sizeable number of proteomic studies have used the combination of large-scale separation techniques, such as high-resolution two-dimensional gel electrophoresis or liquid chromatography in combination with mass spectrometry, for high-throughput protein identification. These studies have applied proteomics to comprehensive biochemical profiling and comparison studies while using different tissues and biological fluids from patients to demonstrate the physiological or pathological adaptations within their proteomes. Further investigations into these proteome-wide alterations will enable us to not only understand the disease pathophysiology, but also to determine signature proteins that can serve as biomarkers for obesity and related diseases. This review examines the different proteomic techniques used to study human obesity and discusses its successful applications along with its technical limitations.

Proteomic Changes in Chick Brain Proteome Post Treatment with Lathyrus Sativus Neurotoxin, \u03b2-N-Oxalyl-L-\u03b1,\u03b2-Diaminopropionic Acid (L-ODAP): A Better Insight to Transient Neurolathyrism

Neurolathyrism is a neurodegenerative disorder characterized by spastic paraplegia resulting from the excessive consumption of Lathyrus sativus (Grass pea). β-N-Oxalyl-L-α,β-diaminopropionic acid (L-ODAP) is the primary neurotoxic component in this pea. The present study attempted to evaluate the proteome-wide alterations in chick brain 2 hr and 4 hr post L-ODAP treatment. Proteomic analysis of chick brain homogenates revealed several proteins involved in cytoskeletal structure, signaling, cellular metabolism, free radical scavenging, oxidative stress and neurodegenerative disorders were initially up-regulated at 2 hr and later recovered to normal levels by 4 hr. Since L-ODAP mediated neurotoxicity is mainly by excitotoxicity and oxidative stress related dysfunctions, this study further evaluated the role of L-ODAP in apoptosis in vitro using human neuroblastoma cell line, IMR-32. The in vitro studies carried out at 200 μM L-ODAP for 4 hr indicate minimal intracellular ROS generation and alteration of mitochondrial membrane potential though not leading to apoptotic cell death. L-ODAP at low concentrations can be explored as a stimulator of various reactive oxygen species (ROS) mediated cell signaling pathways not detrimental to cells. Insights from our study may provide a platform to explore the beneficial side of LODAP at lower concentrations. This study is of significance especially in view of the Government of India lifting the ban on cultivation of low toxin Lathyrus varieties and consumption of this lentil.

Anthracene drives sub-cellular proteome-wide alterations in the degradative system of Penicillium oxalicum

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed in polluted environments and are included in the priority list of toxic compounds. Previous studies have shown that the fungus Penicillium oxalicum, isolated from a hydrocarbon-polluted pond, has a great capability to transform different PAHs in short periods under submerged fermentation conditions. Although cytochrome p450s (CYPs) seems to be the main responsible enzyme in this process, changes in proteome profile remains poorly understood. The aim of this work was to characterise molecular disturbances in the cytosolic and microsomal sub-proteomes of P. oxalicum by applying two-dimensional (2D) gel electrophoresis and label-free quantitative proteomics during anthracene biodegradation. Our results showed that by using 2D-gels, 10 and 8 differential proteins were over-expressed in the cytosolic and microsomal fractions, respectively. Most of them were related to stress response. Shotgun proteomics allowed the identification of 158 and 174 unique protein species that differentially accumulated during anthracene biotransformation, such as CYPs, epoxide hydrolases and transferases enzymes, belonging to Phase I and Phase II of the metabolism of xenobiotics, contributing to the anthracene biodegradation pathway. These results confirm the biological significance of ascomycetes fungi the rol of CYPs on biodegradation and the need of a deeper knowledge on fungal proteomics for the application of the appropriate microorganisms in biodegradation processes.

Proteome-wide alterations in an industrial clavulanic acid producing strain of Streptomyces clavuligerus

The usefulness of genetic/metabolic engineering for further improvement of industrial strains is subject of discussion because of the general lack of knowledge on genetic alterations introduced by iterative cycles of random mutagenesis in such strains. An industrial clavulanic acid (CA)-overproducer Streptomyces clavuligerus DEPA was assessed to understand proteome-wide changes that have occurred in a local industrial CA overproducer developed through succesive mutagenesis programs. The proteins that could be identified corresponded to 33 distinct ORFs for underrepresented ones and 60 ORFs for overrepresented ones. Three CA biosynthetic enzymes were overrepresented in S. clavuligerus DEPA; carboxyethylarginine synthase (Ceas2), clavaldehyde dehydrogenase (Car) and carboxyethyl-arginine beta-lactam-synthase (Bls2) whereas the enzymes of two other secondary metabolites were underrepresented along with two important global regulators [two-component system (TCS) response regulator (SCLAV_2102) and TetR-family transcriptional regulator (SCLAV_3146)] that might be related with CA production and/or differentiation. γ-butyrolactone biosynthetic protein AvaA2 was 2.6 fold underrepresented in S. clavuligerus DEPA. The levels of two glycolytic enzymes, 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase and phosophoglycerate kinase were found decreased while those of dihydrolipoyl dehydrogenase (E3) and isocitrate dehydrogenase, with two isoforms were found as significantly increased. A decrease of amino acid metabolism, methionine biosynthesis in particular, as well as S-adenosylmethionine synthetase appeared as one of the prominent mechanisms of success of S. clavuligerus DEPA strain as a prolific producer of CA. The levels of two enzymes of shikimate pathway that leads to the production of aromatic amino acids and aromatic secondary metabolites were also underrepresented. Some of the overrepresented stress proteins in S. clavuligerus DEPA included polynucleotide phosphorylase/polyadenylase (PNPase), ATP-dependent DNA helicase, two isoforms of an anti-sigma factor and thioredoxin reductase. Downregulation of important proteins of cell wall synthesis and division was recorded and a protein with β-lactamase domain (SCLAV_p1007) appeared in 12 isoforms, 5 of which were drastically overrepresented in DEPA strain. These results described herein provide useful information for rational engineering to improve CA production in Streptomyces clavuligerus.

Proteome-wide alterations on adipose tissue from obese patients as age-, diabetes- and gender-specific hallmarks.

Obesity is a main global health issue and an outstanding cause of morbidity and mortality predisposing to type 2 diabetes (T2DM) and cardiovascular diseases. Huge research efforts focused on gene expression, cellular signalling and metabolism in obesity have improved our understanding of these disorders; nevertheless, to bridge the gap between the regulation of gene expression and changes in signalling/metabolism, protein levels must be assessed. We have extensively analysed visceral adipose tissue from age-, T2DM- and gender-matched obese patients using high-throughput proteomics and systems biology methods to identify new biomarkers for the onset of T2DM in obesity, as well as to gain insight into the influence of aging and gender in these disorders. About 250 proteins showed significant abundance differences in the age, T2DM and gender comparisons. In diabetic patients, remarkable gender-specific hallmarks were discovered regarding redox status, immune response and adipose tissue accumulation. Both aging and T2DM processes were associated with mitochondrial remodelling, albeit through well-differentiated proteome changes. Systems biology analysis highlighted mitochondrial proteins that could play a key role in the age-dependent pathophysiology of T2DM. Our findings could serve as a framework for future research in Translational Medicine directed at improving the quality of life of obese patients.

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.

Proteomic profiling of the dystrophin-deficient mdx phenocopy of dystrophinopathy-associated cardiomyopathy.

Cardiorespiratory complications are frequent symptoms of Duchenne muscular dystrophy, a neuromuscular disorder caused by primary abnormalities in the dystrophin gene. Loss of cardiac dystrophin initially leads to changes in dystrophin-associated glycoproteins and subsequently triggers secondarily sarcolemmal disintegration, fibre necrosis, fibrosis, fatty tissue replacement, and interstitial inflammation. This results in progressive cardiac disease, which is the cause of death in a considerable number of patients afflicted with X-linked muscular dystrophy. In order to better define the molecular pathogenesis of this type of cardiomyopathy, several studies have applied mass spectrometry-based proteomics to determine proteome-wide alterations in dystrophinopathy-associated cardiomyopathy. Proteomic studies included both gel-based and label-free mass spectrometric surveys of dystrophin-deficient heart muscle from the established mdx animal model of dystrophinopathy. Comparative cardiac proteomics revealed novel changes in proteins associated with mitochondrial energy metabolism, glycolysis, signaling, iron binding, antibody response, fibre contraction, basal lamina stabilisation, and cytoskeletal organisation. This review summarizes the importance of studying cardiomyopathy within the field of muscular dystrophy research, outlines key features of the mdx heart and its suitability as a model system for studying cardiac pathogenesis, and discusses the impact of recent proteomic findings for exploring molecular and cellular aspects of cardiac abnormalities in inherited muscular dystrophies.

P.13.11 Proteomic analysis of cardiomyopathic tissue from the aged mdx model of Duchenne muscular dystrophy

Senescent heart tissue from the mdx mouse model of Duchenne muscular dystrophy exhibits distinct pathological aspects of X-linked cardiomyopathy. In order to establish proteome-wide alterations in dystrophin-deficient hearts during aging, cardiomyopathic tissue from young versus aged mdx mice was examined by label-free LC–MS/MS analysis. Significant age-dependent alterations were established for 67 proteins, of which 28 proteins were found to be decreased and 39 proteins were shown to be increased in their expression levels. Drastic changes were demonstrated for proteins involved in ion transportation, the immune response, the cellular stress response, muscle contraction, metabolic regulation and the extracellular matrix. An immunoblotting 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. The proteomic technologies and analytical tools used in this study to identify significant proteins of interest have the potential to be brought forward and provide new therapeutic and diagnostic biomarkers for dystrophinopathy-related cardiomyopathy.

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.