Early changes in the liver-soluble proteome from mice fed a nonalcoholic steatohepatitis inducing diet

Despite the increasing incidence of nonalcoholic steatohepatitis (NASH) with the rise in lifestyle-related diseases such as the metabolic syndrome, little is known about the changes in the liver proteome that precede the onset of inflammation and fibrosis. Here, we investigated early changes in the liver-soluble proteome of female C57BL/6N mice fed an NASH-inducing diet by 2D-DIGE and nano-HPLC-MS/MS. In parallel, histology and measurements of hepatic content of triglycerides, cholesterol and intermediates of the methionine cycle were performed. Hepatic steatosis manifested itself after 2 days of feeding, albeit significant changes in the liver-soluble proteome were not evident before day 10 in the absence of inflammatory or fibrotic signs. Proteomic alterations affected mainly energy and amino acid metabolism, detoxification processes, urea cycle, and the one-carbon/S-adenosylmethionine pathways. Additionally, intermediates of relevant affected pathways were quantified from liver tissue, confirming the findings from the proteomic analysis.     

Proteomic analysis of salt stress-responsive proteins in rice root

Salt stress is one of the major abiotic stresses in agriculture worldwide. We report here a systematic proteomic approach to investigate the salt stress-responsive proteins in rice (Oryza sativa L. cv. Nipponbare). Three-week-old seedlings were treated with 150 mM NaCl for 24, 48 and 72 h. Total proteins of roots were extracted and separated by two-dimensional gel electrophoresis. More than 1100 protein spots were reproducibly detected, including 34 that were up-regulated and 20 down-regulated. Mass spectrometry analysis and database searching helped us to identify 12 spots representing 10 different proteins. Three spots were identified as the same protein, enolase. While four of them were previously confirmed as salt stress-responsive proteins, six are novel ones, i.e. UDP-glucose pyrophosphorylase, cytochrome c oxidase subunit 6b-1, glutamine synthetase root isozyme, putative nascent polypeptide associated complex alpha chain, putative splicing factor-like protein and putative actin-binding protein. These proteins are involved in regulation of carbohydrate, nitrogen and energy metabolism, reactive oxygen species scavenging, mRNA and protein processing, and cytoskeleton stability. This study gives new insights into salt stress response in rice roots and demonstrates the power of the proteomic approach in plant biology studies.     

Proteomic analysis and protein carbonylation profile in trained and untrained rat muscles

Understanding the relationship between physical exercise, reactive oxygen species and skeletal muscle modification is important in order to better identify the benefits or the damages that appropriate or inappropriate exercise can induce. Unbalanced ROS levels can lead to oxidation of cellular macromolecules and a major class of protein oxidative modification is carbonylation. The aim of this investigation was to study muscle protein expression and carbonylation patterns in trained and untrained animal models. We analyzed two muscles characterized by different metabolisms: tibialis anterior and soleus. Whilst tibialis anterior is mostly composed of fast-twitch fibers, the soleus muscle is mostly composed of slow-twitch fibers. By a proteomic approach we identified 15 protein spots whose expression is influenced by training. Among them in tibialis anterior we observed a down-regulation of several glycolitic enzymes. Concerning carbonylation, we observed the existence of a high basal level of protein carbonylation. Although this level shows some variation among individual animals, several proteins (mostly involved in energy metabolism, muscle contraction, and stress response) appear carbonylated in all animals and in both types of skeletal muscle. Moreover we identified 13 spots whose carbonylation increases after training.     

Proteomic analysis of the effect of heat stress on hexaploid wheat grain: Characterization of heat-responsive proteins from total endosperm

High temperatures during grain filling have been reported to be one of the factors that can affect the dough properties and quality characteristics of wheat. Responses to high temperature have been related to changes in protein composition at both quantitative and qualitative levels. The present study was conducted to determine the influence of high temperature during grain filling on the protein composition of bread wheat evaluated by proteomic tools. Plants were grown in the field and transferred to cabinets soon after flowering. They were subjected to two thermal regimes 18 degrees C/10 degrees C (day/night) and 34 degrees C/10 degrees C. Total proteins were extracted from control grains and treated plants at three different post-anthesis stages. The proteins were separated by two-dimensional gel electrophoresis and analysed by Melanie 3 software. Of the total number of mature wheat grain proteins, 37 were identified as significantly changed by heat treatment. Analysis by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry coupled with database searching allowed the characterization of 25 heat-induced proteins and only one heat-decreased protein spot. To learn more about the function of the identified proteins, we examined their expression during treatment.     

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 analysis of the extremely thermoacidophilic archaeon Picrophilus torridus at pH and temperature values close to its growth limit

The thermoacidophilic archaeon Picrophilus torridus belongs to the Thermoplasmatales order and is the most acidophilic organism known to date, growing under extremely acidic conditions around pH 0 (pH(opt) 1) and simultaneously at high temperatures up to 65°C. Some genome features that may be responsible for survival under these harsh conditions have been concluded from the analysis of its 1.55 megabase genome sequence. A proteomic map was generated for P. torridus cells grown to the mid-exponential phase. The soluble fraction of the cells was separated by isoelectric focusing in the pH ranges 4-7 and 3-10, followed by a two dimension (2D) on SDS-PAGE gels. A total of 717 Coomassie collodial-stained protein spots from both pH ranges (pH 4-7 and 3-10) were excised and subjected to LC-MS/MS, leading to the identification of 665 soluble protein spots. Most of the enzymes of the central carbon metabolism were identified on the 2D gels, corroborating biochemically the metabolic pathways predicted from the P. torridus genome sequence. The 2D master gels elaborated in this study represent useful tools for physiological studies of this thermoacidophilic organism. Based on quantitative 2D gel electrophoresis, a proteome study was performed to find pH- or temperature-dependent differences in the proteome composition under changing growth conditions. The proteome expression patterns at two different temperatures (50 and 70°C) and two different pH conditions (pH 0.5 and 1.8) were compared. Several proteins were up-regulated under most stress stimuli tested, pointing to general roles in coping with stress.     

Proteomic analysis reveals the spatial heterogeneity of immobilized Taxus cuspidata cells in support matrices

A proteomic approach was used to study the responses of Taxus cuspidata cells to local microenvironments in different zones of immobilized support matrices. Analysis of protein spots by 2-DE revealed significant differences in the abundance of 31 spots, 28 spots, and 23 spots in outer, middle, and central zone cells between the immobilized and suspended cells. Six of these proteins, identified by MALDI-TOF-MS, were involved in the regulation of carbohydrate, nitrogen, and sulfur metabolisms. Immobilization triggered an increase in taxol production of the immobilized cells in the middle and central zones compared to that of the suspended cells. A negative relation between taxol production and the mitotic index was observed in the cells in the immobilization support matrix. Cells in the outer zone had high mitotic index and low taxol production, while cells in the middle and central zones showed low mitotic index and high taxol production. The abundance of S-adenosylmethionine synthetase, which was identified as one of the differentially expressed proteins, was positively correlated to the cell division activity in the immobilized cell cultures.     

Differential expression of sarcoplasmic proteins in four heterogeneous ovine skeletal muscles

Fiber-type distribution is known to vary widely within and between muscles according to differences in muscle functions. 2-DE and MALDI-MS were used to investigate the molecular basis of muscle fiber type-related variability. We compared four lamb skeletal muscles with heterogeneous fiber-type composition that are relatively rich in fast-twitch fiber types, i.e., the semimembranosus, vastus medialis, longissimus dorsi, and tensor fasciae latae (TL). Our results clearly showed that none of the glycolytic metabolism enzymes detected, including TL which was most strongly glycolytic, made intermuscular differentiation possible. Muscle differentiation was based on the differential expression of proteins involved in oxidative metabolism, including not only citric acid cycle enzymes but also other classes of proteins with functions related to oxidative metabolism, oxidative stress, and probably to higher protein turnover. Detected proteins were involved in transport (carbonate dehydratase, myoglobin, fatty acid-binding protein), repair of misfolding damage (heat shock protein (HSP) 60 kDa, HSP-27 kDa, alpha-crystallin beta subunit, DJ1, stress-induced phosphoprotein), detoxification or degradation of impaired proteins (GST-Pi, aldehyde dehydrogenase, peroxiredoxin, ubiquitin), and protein synthesis (tRNA-synthetase). The fractionating method led to the detection of proteins involved in different functions related to oxidative metabolism that have not previously been shown concomitancy.     

Proteomic analysis of rat pheochromocytoma PC12 cells

PC12 cell line is well documented and widely applied as many kinds of models in neurobiological and neurochemical studies. Yet a thorough proteomic analysis has not been performed so far. Here we report the construction of a large-scale 2-D protein database for PC12 cells. The proteins extracted from PC12 cells were separated by 2-DE and identified by MALDI-TOF/TOF MS. A total of 1080 protein spots, excised from three different 2-D gels, were identified with high confidence. These proteins represent 474 different gene products, mainly binding proteins and enzymes. Three hundred and seven identified protein spots were located in the low-molecular weight region below 20 kDa. This database today represents one of the largest 2-D databases for higher eukaryotic cell proteomes and for low-molecular weight proteins. In addition, fragment ion spectra obtained by TOF/TOF confirmed that calcylin in PC12 cells was N-acetylated. The database of PC12 proteome is expected to be a powerful tool for neuroscientists.     

Proteomic analysis of anti-Francisella tularensis LVS antibody response in murine model of tularemia

Francisella tularensis live vaccine strain infection of mice has been established as an experimental model of tularemia that is suitable for studies of immune mechanisms against the intracellular pathogen. In this study, the model was used to explore immunogenic repertoire of F. tularensis with the aim of identifying new molecules able to activate the host immune system, potential bacterial markers with vaccine, and diagnostic applications. Immunoproteomic approach based on the combination of two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry was applied. Globally, 36 different proteins were identified, which strongly reacted with sera from experimentally infected mice, including several putative virulence markers of intracellular pathogens as nucleoside diphosphate kinase, isocitrate dehydrogenase, RNA-binding protein Hfq, and molecular chaperone ClpB. Of them, 27 proteins are described for the first time as immunorelevant Francisella proteins. When comparing murine immunoproteome of F. tularensis with our previous data from human patients, 25 of the total of 50 identified murine sera immunoreactive spots were recognized by human sera collected from patients suffering from tularemia, as well. Immune sera from two Lps gene congenic strains of mice, C3H/HeN (Lpsn) and C3H/HeJ (Lpsd), represented murine immunoproteome in this study. The spectrum of immunoreactive spots detected by two-dimensional immunoblotting varied throughout the course of infection depending on murine strain. Nevertheless, the antibody patterns of the two strains showed significant homogeneity in being directed against almost identical subset of antigens.     

Proteomic analysis of Burkholderia cepacia MBA4 in the degradation of monochloroacetate

Burkholderia cepacia MBA4 is a bacterium that degrades 2-haloacids by removing the halogen and subsequent metabolism of the product for energy. In this study, 2-DE, MS/MS, and N-terminal amino acid sequencing were used to investigate the protein expression profiles of MBA4 grown in a 2-haloacid (monochloroacetate, MCA) and in the corresponding metabolic product (glycolate). Glycolate was used as a control to eliminate the proteins induced by it. Five proteins were found to be up-regulated and five proteins were down-regulated in response to MCA. The differentially expressed proteins were examined, seven of them were identified by MS/MS and two of them were sequenced by Edman degradation. Our results definitely provide an insight for understanding the physiology of B. cepacia MBA4 in response to organohalide contaminated site.     

Analysis of proteome and frost tolerance in chromosome 5A and 5B reciprocal substitution lines between two winter wheats during long-term cold acclimation

Dynamics of cold tolerance and crown proteome composition has been analysed in a set of two winter wheat cultivars Mironovskaya 808 and Bezostaya 1 and four reciprocal substitution lines with interchanged chromosomes 5A and 5B during a long-term cold-acclimation (CA) treatment. Proteome analysis has revealed 298 differently abundant spots during experiment. Most of them (260) were changed due to CA process and only 52 spots displayed differences between genotypes. Two hundred and seven protein spots were successfully identified by tandem mass spectrometry. Comparison of samples before and after vernalization fulfillment by a combination of ANOVA and Student' T-test displayed ten differentially abundant protein spots (e.g. chopper chaperones). However, differences in the accumulation of these spots did not reflect differences in vernalization requirement of genotypes. Therefore, our results indicate that vernalization process has not influenced total proteome of CA wheat crowns. A few protein spots (14 spots; e.g. malate dehydrogenase) revealed differential accumulation levels between the individual genotypes or their groups possessing chromosome 5A or 5B from Mironovskaya 808 versus Bezostaya 1. The study has shown the effect of chromosome 5A on physiological traits and also proteome in winter wheat. Putative candidate protein markers for cold tolerance in wheat are discussed.     

Identification of toxicological biomarkers of di(2‐ethylhexyl) phthalate in proteins secreted by HepG2 cells using proteomic analysis

The effects of di(2‐ethylhexyl) phthalate (DEHP) on proteins secreted by HepG2 cells were studied using a proteomic approach. HepG2 cells were exposed to various concentrations of DEHP (0, 2.5, 5, 10, 25, 50, 100, and 250 μM) for 24 or 48 h. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) and comet assays were then conducted to determine the cytotoxicity and genotoxicity of DEHP, respectively. The MTT assay showed that 10 μM DEHP was the maximum concentration that did not cause cell death. In addition, the DNA damage in HepG2 cells exposed to DEHP was found to increase in a dose‐ and time‐dependent fashion. Proteomic analysis using two different pI ranges (4–7 and 6–9) and large size 2‐DE revealed the presence of 2776 protein spots. A total of 35 (19 up‐ and 16 down‐regulated) proteins were identified as biomarkers of DEHP by ESI‐MS/MS. Several differentiated protein groups were also found. Proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and motility were found to be up‐ or down‐regulated. Among these, the identities of cystatin C, Rho GDP inhibitor, retinol binding protein 4, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, cofilin‐1, and haptoglobin‐related protein were confirmed by Western blot assay. Therefore, these proteins could be used as potential biomarkers of DEHP and human disease associated with DEHP.     

Proteomic analysis of a filamentous fungal endophyte using EST datasets

Proteomic analysis of many species of fungi, particularly filamentous fungi, is difficult due to the lack of publicly available genome sequence data and the problems associated with cross‐species comparisons. Furthermore, the detection of fungal proteins in biological systems where there are a greater number of proteins present from other eukaryote species provides additional challenges. We present an EST‐based approach for identifying proteins from a fungal endophyte of temperate grasses and demonstrate that this method is well suited for fungi with minimal sequence data.     

Identification of membrane-associated proteins from Campylobacter jejuni strains using complementary proteomics technologies

Campylobacter jejuni is the leading cause of food- and water-borne illness world-wide. The membrane-associated proteome of a recent C. jejuni gastrointestinal isolate (JHH1) was generated by sodium carbonate precipitation and ultracentrifugation followed by 2-DE and MALDI-TOF MS as well as 2-DLC (strong cation exchange followed by RP chromatography) of trypsin digests coupled to MS/MS (2-DLC/MS/MS). 2-DE/MS identified 77 proteins, 44 of which were predicted membrane proteins, while 2-DLC/MS/MS identified 432 proteins, of which 206 were predicted to be membrane associated. A total of 453 unique proteins (27.4% of the C. jejuni theoretical proteome), including 187 bona fide membrane proteins were identified in this study. Membrane proteins were also compared between C. jejuni JHH1 and ATCC 700297 to identify factors potentially associated with increased gastrointestinal virulence. We identified 28 proteins that were significantly (>two-fold) more abundant in, or unique to, JHH1, including eight proteins involved in chemotaxis signal transduction and flagellar motility, the amino acid-binding surface antigens CjaA and CjaC, and four outer membrane proteins (OMPs) of unknown function (Cj0129c, Cj1031, Cj1279c, and Cj1721c). Immunoblotting using convalescent patient sera generated post-gastrointestinal infection revealed 13 (JHH1) and 12 (ATCC 700297) immunoreactive proteins. These included flagellin (FlaA) and CadF as well as Omp18, Omp50, Cj1721c, PEB1A, PEB2, and PEB4A. This study provides a comprehensive analysis of membrane-associated proteins from C. jejuni.