Proteomic analysis of protein nitration in aging skeletal muscle and identification of nitrotyrosine-containing sequences in vivo by nanoelectrospray ionization tandem mass spectrometry

The nitration of protein tyrosine residues represents an important post-translational modification during development, oxidative stress, and biological aging. To rationalize any physiological changes with such modifications, the actual protein targets of nitration must be identified by proteomic methods. While several studies have used proteomics to screen for 3-nitrotyrosine-containing proteins in vivo, most of these studies have failed to prove nitration unambiguously through the actual localization of 3-nitrotyrosine to specific sequences by mass spectrometry. In this paper we have applied sequential solution isoelectric focusing and SDS-PAGE for the proteomic characterization of specific 3-nitrotyrosine-containing sequences of nitrated target proteins in vivo using nanoelectrospray ionization-tandem mass spectrometry. Specifically, we analyzed proteins from the skeletal muscle of 34-month-old Fisher 344/Brown Norway F1 hybrid rats, a well accepted animal model for biological aging. We identified the 3-nitrotyrosine-containing sequences of 11 proteins, including cytosolic creatine kinase, tropomyosin 1, glyceraldehyde-3-phosphate dehydrogenase, myosin light chain, aldolase A, pyruvate kinase, glycogen phosphorylase, actinin, gamma-actin, ryanodine receptor 3, and neurogenic locus notch homolog. For creatine kinase and neurogenic locus notch homolog, two 3-nitrotyrosine-containing sequences were identified, i.e. at positions 14 and 20 for creatine kinase and at positions 1175 and 1205 for the neurogenic locus notch homolog. The selectivity of the in vivo nitration of creatine kinase at Tyr14 and Tyr20 does not correspond to the product selectivity in vitro, where exclusively Tyr82 was nitrated when creatine kinase was exposed to peroxynitrite. The latter experiments demonstrate that the in vitro exposure of an isolated protein to peroxynitrite may not always be a good model to mimic protein nitration in vivo.     

Proteomic analysis of protein nitration in rat cerebellum: effect of biological aging

3-Nitrotyrosine (3-NT) is a useful biomarker of increasing oxidative stress and protein nitration during biological aging. The proteomic analysis of cerebellar homogenate from Fisher 344/Brown Norway (BN/F1) rats shows an age-dependent increase in protein nitration, monitored by western-blot analysis after two-dimensional gel electrophoresis (2DE), mainly in the acidic region. Analysis of in-gel digests by nanoelectrospray (NSI)-MS/MS resulted in the identification of 16 putatively nitrated proteins. The selective isolation of nitrated proteins using immunoprecipitation, followed by SDS-PAGE and in-gel digest/NSI-MS/MS analysis led to the identification of 22 putatively nitrated proteins, of which 7 were identical to those detected after 2DE. When proteins were separated by solution isoelectrofocusing and analyzed by NSI MS/MS, we obtained MS/MS spectra of 3-NT containing peptides of four proteins - similar to ryanodine receptor 3, low density lipoprotein related receptor 2, similar to nebulin-related anchoring protein isoform C and 2,3 cyclic nucleotide 3-phosphodiesterase. Although the functional consequences of protein nitration for these targets are not yet known, our proteomic experiments serve as a first screen for the more targeted analysis of nitrated proteins from aging cerebellum for functional characterization.     

Proteomic investigation of changes in rat skeletal muscle after exercise-induced fatigue

The mechanisms of exercise-induced fatigue have not been investigated using proteomic techniques, an approach that could improve our understanding and generate novel information regarding the effects of exercise. In this study, the proteom alterations of rat skeletal muscle were investigated during exercise-induced fatigue. The proteins were extracted from the skeletal muscle of SD rat thigh, and then analyzed by two-dimensional electrophoresis and PDQuest software. Compared to control samples, 10 significantly altered proteins were found in exercise samples, two of them were upregulated and eight of them were downregulated. These proteins were identified by MALDI TOF-MS. The two upregulated proteins were identified as MLC1 and myosin L2 (DTNB) regulatory light-chain precursors. The eight decreased proteins are Glyceraldehyde-3-phosphate Dehydrogenas (GAPDH); Beta enolase; Creatine kinase M chain (M-CK); ATP-AMP Transphosphorylase (AK1); myosin heavy chain (MHC); actin; Troponin I, fast-skeletal muscle (Troponin I fast-twitch isoform), fsTnI; Troponin T, fast-skeletal muscle isoforms (TnTF). In these proteins, four of the eight decreased proteins are related directly or indirectly to exercise induced fatigue. The other proteins represent diverse sets of proteins including enzymyes related to energy metabolism, skeletal muscle fabric protein and protein with unknown functions. They did not exhibit evident relationship with exercise-induced fatigue. Whereas the two identified increased proteins exhibit evident relationship with fatigue. These findings will help in understanding the mechanisms involved in exercise-induced fatigue.     

Phosphoenolpyruvate-dependent protein kinase activity in rat skeletal muscle

Phosphoenolpyruvate-dependent protein kinase activity has been demonstrated in the soluble fraction of rat skeletal muscle. The reaction was not due to the formation of ATP in the incubation mixture. Cyclic AMP, calcium, ATP and a number of phosphate acceptor proteins did not stimulate the reaction. One 32P-labelled protein (Mr 25000) was observed on SDS gels. The phosphorylated protein contained acid stable phosphoserine as a major phosphorylated amino acid. The phosphorylation reaction in crude extracts was not directly proportional to the amount of protein, but typical of a two-component system; i.e., kinase and substrate. The chromatography of soluble proteins on Ultrogel AcA44 separated the phosphate acceptor protein(s) from the phosphoenolpyruvate-dependent protein kinase activity.     

Quantitative identification of protein nitration sites

Several labelling strategies have been developed targeting specific amino acid residues and/or PTMs. Methods specifically tailored for the qualitative and sometimes quantitative determination of PTMs have emerged. Many research groups have focused their attention towards o‐nitrotyrosine residues, developing various methodologies for their identification, while direct quantification has remained elusive. So far the iTRAQ chemistry has been limited to primary amines. Here, we report a new strategy based on the use of iTRAQ reagents coupled to MS analysis for the selective labelling of o‐nitrotyrosine residues. This method was proved to lead to the simultaneous localisation and quantification of nitration sites both in model proteins and in biological systems.     

Proteomic identification of tyrosine nitration targets in kidney of spontaneously hypertensive rats

Nitrosative and oxidative stress are implicated in the development of hypertension. Events in the renal medulla may play a key role in the development and progression of hypertension. This may arise through disruption of nitric oxide signalling in the medulla and be accompanied by enhanced nitrosative and oxidative stress as indicated by the presence of proteins containing 3-nitrotyrosine. Here we demonstrate enhanced protein nitration in the medulla of spontaneously hypertensive rats. We have identified several nitrated proteins with both varied subcellular location and functional roles. These proteins are involved in nitric oxide signalling, antioxidant defense and energy metabolism. Moreover, increased nitration was observed in conjunction with enhanced oxidative damage as evidenced by the presence of protein carbonyl oxidative stress biomarkers. Our results suggest that kidney medulla is subject to enhanced nitrosative and oxidative stress, and that resulting protein modifications may contribute to the progression of hypertension.     

Proteomic analysis of bovine skeletal muscle hypertrophy

Myostatin plays a major role in muscle growth and development and animals with disruption of this gene display marked increases in muscle mass. Little is known about muscle physiological adaptations in relation to this muscle hypertrophy. To provide a more comprehensive view, we analyzed bovine muscles from control, heterozygote and homozygote young Belgian blue bulls for myostatin deletion, which results in a normal level of inactive myostatin. Heterozygote and homozygote animals were characterized by a higher proportion of fast-twitch glycolytic fibers in Semitendinosus muscle. Differential proteomic analysis of this muscle was performed using two-dimensional gel electrophoresis followed by mass spectrometry. Thirteen proteins, corresponding to 28 protein spots, were significantly altered in response to the myostatin deletion. The observed changes in protein expression are consistent with an increased fast muscle phenotype, suggesting that myostatin negatively controls mainly fast-twitch glycolytic fiber number. Finally, we demonstrated that differential mRNA splicing of fast troponin T is altered by the loss of myostatin function. The structure of mutually exclusive exon 16 appears predominantly expressed in muscles from heterozygote and homozygote animals. This suggests a role for exon 16 of fast troponin T in the physiological adaptation of the fast muscle phenotype.     

Proteomic profiling of giant skeletal muscle proteins

Introduction: Distinct subtypes of contractile fibres are highly diverse in their proteomic profile and greatly adaptable to physiological or pathological challenges. A striking biochemical feature of heterogeneous skeletal muscle tissues is the presence of a considerable number of extremely large protein species, which often present a bioanalytical challenge for the systematic separation and identification of muscle proteomes during large-scale screening surveys.

Areas covered: This review outlines the proteomic characterization of skeletal muscles with a special focus on giant proteins of the sarcomere, the cytoskeleton and the sarcoplasmic reticulum. This includes an overview of the involvement of large muscle proteins, such as titin, nebulin, obscurin, plectin, dystrophin and the ryanodine receptor calcium release channel, during normal muscle functioning, swift adaptations to changed physiological demands and changes in relation to pathobiochemical insults.

Expert commentary: The proteomic screening and characterization of total muscle extracts and various subcellular fractions has confirmed the critical role of large skeletal muscle proteins in the regulation of ion homeostasis, the maintenance of contraction-relaxation cycles and fibre elasticity, and the stabilisation of supramolecular complexes of the muscle periphery and cytoskeletal networks of contractile fibres. These findings will be helpful for the future functional systems analysis of giant muscle proteins.     

Hypoxia affects mitochondrial protein expression in rat skeletal muscle

Hypoxia affects mammalian mitochondrial function, as well as mitochondria-based energy metabolism. The detail mechanism has not been fully understood. In this study, we detected protein expression levels in mitochondrial fractions of Wistar rats exposed to hypobaric hypoxia by use of proteomic methods. Adult male Wistar rats were randomized into an hypoxic (4,500?m, 30 days) group and a normoxic control group (sea level). Gastrocnemius muscles mitochondria were extracted and purified. Mitochondrial oxygen consumption was measured with a Clark oxygen electrode; mitochondrial transmembrane potential was detected with Rhodamine 123 as a fluoresce probe. Using 2-DE and MALDI-TOF MS analysis, we identified eight mitochondrial protein spots that were differentially expressed in the hypoxic group compared with the normoxic control. These proteins included Chain A of F1-ATPase, voltage dependent anion channel 1 (VDAC), hydroxyacyl Coenzyme A dehydrogenase α-subunit, mitochondrial F1 complex γ-subunit, androgen-regulated protein and tripartite motif protein 50. Two of the spots, VDAC and ATP synthase α-subunit, were confirmed by Western blotting analysis. Oxygen consumption during State 3 respiration, as well as the respiratory control ratio (RCR) was significantly higher in the control than that in the hypoxic group; mitochondrial transmembrane potential was significantly higher in hypoxic group than that in the control. With successful use of multiple proteomic analysis techniques, we demonstrates that 30 days hypoxia exposure has effects on the expression of mitochondrial proteins involved in ATP production and lipid metabolism, decrease the stability of mitochondrial membrane, and affect the mitochondrial electron transport chain.     

Contraction-associated translocation of protein kinase C in rat skeletal muscle

Electrical stimulation of the sciatic nerve of the anaesthetized rat in vivo led to a time-dependent translocation of protein kinase C from the muscle cytosol to the particulate fraction. Maximum activity of protein kinase C in the particulate fraction occurred after 2 min of intermittent short tetanic contractions of the gastrocnemius-plantaris-soleus muscle group and coincided with the loss of activity from the cytosol. Translocation of protein kinase C may imply a role for this kinase in contraction-initiated changes in muscle metabolism.     

Cytochrome c protein synthesis rate in rat skeletal muscle.

Endurance training is associated with increases in mitochondrial density, of which cytochrome c protein is an index. Increases in the synthesis rates of cytochrome c protein in skeletal muscle during endurance training have been inferred (Biochem. Biophys. Res. Commun. 66: 173, 1975; J. Biol. Chem. 252: 416, 1977). One purpose of the present study was to test these indirect approximations with direct measurements of the synthesis rates of cytochrome c protein in skeletal muscles postexercise. No change in the fractional synthesis rate of cytochrome c was detected in the red quadriceps muscle of rats either 2-7 h after a 104-min run on a motor-driven treadmill or 17-22 h after the final bout of 4 days of running 100 min/day. If the 16% increase in cytochrome c protein concentration in the red quadriceps muscle on the 5th day of training is used to calculate the nanomoles of cytochrome c synthesized per gram of wet muscle weight, the normalized rate of cytochrome c protein synthesis is increased 29% on the 5th day of training. The observation of no significant alteration in cytochrome c mRNA in the red quadriceps muscle of rats during the 1st wk of training implies that the initial increase in the synthesis rate of cytochrome c protein normalized per unit of muscle mass during treadmill training is likely to occur at a translational or posttranslational step. These results suggest that the control of increased cytochrome c expression in skeletal muscle during exercise training involves a complex mechanism.     

Proteomic identification of secreted proteins from human skeletal muscle cells and expression in response to strength training

Regular physical activity protects against several types of diseases. This may involve altered secretion of signaling proteins from skeletal muscle. Our aim was to identify the most abundantly secreted proteins in cultures of human skeletal muscle cells and to monitor their expression in muscles of strength-training individuals. A total of 236 proteins were detected by proteome analysis in medium conditioned by cultured human myotubes, which was narrowed down to identification of 18 classically secreted proteins expressed in skeletal muscle, using the SignalP 3.0 and Human Genome Expression Profile databases together with a published mRNA-based reconstruction of the human skeletal muscle secretome. For 17 of the secreted proteins, expression was confirmed at the mRNA level in cultured human myotubes as well as in biopsies of human skeletal muscles. RT-PCR analyses showed that 15 of the secreted muscle proteins had significantly enhanced mRNA expression in m. vastus lateralis and/or m. trapezius after 11 wk of strength training among healthy volunteers. For example, secreted protein acidic and rich in cysteine, a secretory protein in the membrane fraction of skeletal muscle fibers, was increased 3- and 10-fold in m. vastus lateralis and m. trapezius, respectively. Identification of proteins secreted by skeletal muscle cells in vitro facilitated the discovery of novel responses in skeletal muscles of strength-training individuals.     

Proteomic identification of altered proteins in skeletal muscle during chronic potassium depletion: Implications for hypokalemic myopathy

Prolonged potassium depletion is a well-known cause of myopathy. The pathophysiology of hypokalemic myopathy, however, remains unclear. We performed a gel-based, differential proteomics study to define altered proteins in skeletal muscles during chronic potassium depletion. BALB/c mice were fed with normal chow (0.36% K+) or K+-depleted (KD) diet (<0.001% K+) for 8 weeks (n = 5 in each group). Left gastrocnemius muscles were surgically removed from each animal. Histopathological examination showed mild-degree infiltration of polymornuclear and mononuclear cells at the interstitium of the KD muscles. Extracted proteins were resolved with two-dimensional electrophoresis (2-DE), and visualized with Coomassie Brilliant Blue R-250 stain. Quantitative intensity analysis revealed 16 up-regulated protein spots in the KD muscles, as compared to the controls. These differentially expressed proteins were subsequently identified by peptide mass fingerprinting and by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS). Most of the altered proteins induced by chronic potassium depletion were muscle enzymes that play significant roles in several various metabolic pathways. Other up-regulated proteins included myosin-binding protein H, alpha-B Crystallin, and translationally controlled tumor protein (TCTP). These findings may lead to a new roadmap for research on hypokalemic myopathy, to better understanding of the pathophysiology of this medical disease, and to biomarker discovery.     

The kinetics of myofibrillar protein breakdown in perfused rat skeletal muscle

N^t-Methylhistidine, a non-reutilised amino acid present in some myofibrillar proteins, was radioactively labelled in vito with $\text{[}{}^{\mathrm{Me}}\text{-}{}^3\text{H}\text{]}\text{methionine}$. The specific radioactivities of protein-bound methylhistidine and free methylhistidine in perfusate after perfusion of rat hind limbs taken from prelabelled rats was determined. The decrease in urinary methylhistidine activity with time was determined for rats similarly labelled. Comparison of the specific activities of free and bound methylhistidine and the non-linear semilogarithmic plot of urinary methylhistidine activity suggest that the myofibrillar protein catabolism, as indicated by methylhistidine release, may not be a simple exponential process. The possibility of non-random decay is discussed and an alternative model proposed.     

Proteomic analysis of age-dependent changes in protein solubility identifies genes that modulate lifespan

While it is generally recognized that misfolding of specific proteins can cause late‐onset disease, the contribution of protein aggregation to the normal aging process is less well understood. To address this issue, a mass spectrometry‐based proteomic analysis was performed to identify proteins that adopt sodium dodecyl sulfate (SDS)‐insoluble conformations during aging in Caenorhabditis elegans. SDS‐insoluble proteins extracted from young and aged C. elegans were chemically labeled by isobaric tagging for relative and absolute quantification (iTRAQ) and identified by liquid chromatography and mass spectrometry. Two hundred and three proteins were identified as being significantly enriched in an SDS‐insoluble fraction in aged nematodes and were largely absent from a similar protein fraction in young nematodes. The SDS‐insoluble fraction in aged animals contains a diverse range of proteins including a large number of ribosomal proteins. Gene ontology analysis revealed highly significant enrichments for energy production and translation functions. Expression of genes encoding insoluble proteins observed in aged nematodes was knocked down using RNAi, and effects on lifespan were measured. 41% of genes tested were shown to extend lifespan after RNAi treatment, compared with 18% in a control group of genes. These data indicate that genes encoding proteins that become insoluble with age are enriched for modifiers of lifespan. This demonstrates that proteomic approaches can be used to identify genes that modify lifespan. Finally, these observations indicate that the accumulation of insoluble proteins with diverse functions may be a general feature of aging.     

Proteomic responses of skeletal and cardiac muscle to exercise

Regular exercise is effective in the prevention of chronic diseases and confers a lower risk of death in individuals displaying risk factors such as hypertension and dyslipidemia. Thus, knowledge of the molecular responses to exercise provides a valuable contrast for interpreting investigations of disease and can highlight novel therapeutic targets. While exercise is an everyday experience and can be conceptualized in simple terms, it is also a complex physiological phenomenon and investigation of exercise responses requires sophisticated analytical techniques and careful standardization of the exercise stimulus. Proteomic investigation of exercise is in its infancy but the ability to link changes in function with comprehensive changes in protein expression and post-translational modification holds great promise for advancing physiology. This article highlights recent pioneering work investigating the effects of exercise in skeletal and cardiac muscle that has uncovered novel mechanisms underlying the benefits of physical activity.     

Proteomic DIGE analysis of the mitochondria‐enriched fraction from aged rat skeletal muscle

Skeletal muscle aging is associated with a loss in tissue mass and contractile strength, as well as fiber type shifting and bioenergetic adaptation processes. Since mitochondria represent the primary site for energy generation via oxidative phosphorylation, we investigated potential changes in the expression pattern of the mitochondrial proteome using the highly sensitive DIGE approach. The comparative analysis of the mitochondria‐enriched fraction from young adult versus aged muscle revealed an age‐related change in abundance for 39 protein species. MS technology identified the majority of altered proteins as constituents of muscle mitochondria. An age‐dependent increase was observed for NADH dehydrogenase, the mitochondrial inner membrane protein mitofilin, peroxiredoxin isoform PRX‐III, ATPase synthase, succinate dehydrogenase, mitochondrial fission protein Fis1, succinate‐coenzyme A ligase, acyl‐coenzyme A dehydrogenase, porin isoform VDAC2, ubiquinol‐cytochrome c reductase core I protein and prohibitin. Immunoblotting, enzyme testing and confocal microscopy were used to validate proteomic findings. The DIGE‐identified increase in key mitochondrial elements during aging agrees with the concept that sarcopenia is associated with a shift to a slower contractile phenotype and more pronounced aerobic‐oxidative metabolism. This suggests that mitochondrial markers are reliable candidates that should be included in the future establishment of a biomarker signature of skeletal muscle aging.     

AMP-activated protein kinase activity and glucose uptake in rat skeletal muscle

The AMP-activated protein kinase (AMPK) has been hypothesized to mediate contraction and 5-aminoimidazole-4-carboxamide 1-beta-D-ribonucleoside (AICAR)-induced increases in glucose uptake in skeletal muscle. The purpose of the current study was to determine whether treadmill exercise and isolated muscle contractions in rat skeletal muscle increase the activity of the AMPK alpha 1 and AMPK alpha 2 catalytic subunits in a dose-dependent manner and to evaluate the effects of the putative AMPK inhibitors adenine 9-beta-D-arabinofuranoside (ara-A), 8-bromo-AMP, and iodotubercidin on AMPK activity and 3-O-methyl-D-glucose (3-MG) uptake. There were dose-dependent increases in AMPK alpha 2 activity and 3-MG uptake in rat epitrochlearis muscles with treadmill running exercise but no effect of exercise on AMPK alpha1 activity. Tetanic contractions of isolated epitrochlearis muscles in vitro significantly increased the activity of both AMPK isoforms in a dose-dependent manner and at a similar rate compared with increases in 3-MG uptake. In isolated muscles, the putative AMPK inhibitors ara-A, 8-bromo-AMP, and iodotubercidin fully inhibited AICAR-stimulated AMPK alpha 2 activity and 3-MG uptake but had little effect on AMPK alpha 1 activity. In contrast, these compounds had absent or minimal effects on contraction-stimulated AMPK alpha 1 and -alpha 2 activity and 3-MG uptake. Although the AMPK alpha 1 and -alpha 2 isoforms are activated during tetanic muscle contractions in vitro, in fast-glycolytic fibers, the activation of AMPK alpha 2-containing complexes may be more important in regulating exercise-mediated skeletal muscle metabolism in vivo. Development of new compounds will be required to study contraction regulation of AMPK by pharmacological inhibition.