Proteomic analysis of slow- and fast-twitch skeletal muscles

Skeletal muscles are composed of slow- and fast-twitch muscle fibers, which have high potential in aerobic and anaerobic ATP production, respectively. To investigate the molecular basis of the difference in their functions, we examined protein profiles of skeletal muscles using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis with pH 4-7 and 6-11 isoelectric focusing gels. A comparison between rat soleus and extensol digitorum longus (EDL) muscles that are predominantly slow- and fast-twitch fibers, respectively, showed that the EDL muscle had higher levels of glycogen phosphorylase, most glycolytic enzymes, glycerol 3-phosphate dehydrogenase, and creatine kinase; while the soleus muscle had higher levels of myoglobin, TCA cycle enzymes, electron transfer flavoprotein, and carbonic anhydrase III. The two muscles also expressed different isoforms of contractile proteins including myosin heavy and light chains. These protein patterns were further compared with those of red and white gastrochnemius as well as red and white quadriceps muscles. It was found that metabolic enzymes showed a concerted regulation dependent on muscle fiber types. On the other hand, expression of contractile proteins seemed to be independent of the metabolic characteristics of muscle fibers. These results suggest that metabolic enzymes and contractile proteins show different expression patterns in skeletal muscles.     

Mapping of alkaline proteins in bovine skeletal muscle

In agricultural sciences, proteomics has become the new hope for analyzing the meat quality traits that are closely related to the skeletal muscle traits. 2-DE muscle maps of many species have been recently reported and used to find molecular markers of meat quality traits. However, one limitation of 2-DE based analyses is due to the limited alkaline protein separation. Considering this problem, there is a need to use recent advances that have markedly improved the 2-DE based analysis of alkaline proteins. Hence, the present study provides additional information concerning the alkaline proteome of bovine skeletal muscle by using an appropriate protocol to characterize proteins over the entire range of pH 7-11. A total of 32 distinct gene products corresponding to 60 protein spots were identified by PMF and grouped in seven categories according to their main function. This 2-D map will contribute to muscle proteome studies since a significant portion of proteins is in the alkaline pH range.     

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 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.     

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.     

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.     

Exploring the priming mechanism of liver regeneration: proteins and protein complexes

The liver has the ability to restore its functional capacity following injury or resection and the priming of liver regeneration is a complex process that has not been completely elucidated. In the current research, to further reveal the priming mechanism of liver regeneration, hepatocyte total protein and hepatocyte cytosol of the rats at 4 h after 2/3 partial hepatectomy (PHx) were studied, respectively, by 2‐DE and 2‐D blue native gel electrophoresis. Seventeen unique differential proteins were identified in hepatocyte total protein samples. Nine differential protein complexes containing 41 protein components were identified in hepatocyte cytosol samples. For the first time, at the priming stage of liver regeneration, the variations of serine protease inhibitor 2c, sulfite oxidase and valosin‐containing protein (VCP) were presented and validated by Western blotting, and the VCP complex was further validated by antibody super‐shift experiments. The current results suggested that at 4 h after PHx, VCP complex was down‐regulated in hepatocyte cytosol, apoptosis pathways were inhibited, nuclear factor‐κB and interleukin 6 pathways worked together and triggered the liver regeneration.     

Proteomic analysis of the role of S-nitrosoglutathione reductase in lipopolysaccharide-challenged mice

S-Nitrosoglutathione reductase (GSNOR) is a key regulator of protein S-nitrosylation, the covalent modification of cysteine residues by nitric oxide that can affect activities of many proteins. We recently discovered that excessive S-nitrosylation from GSNOR deficiency in mice under inflammation inactivates the key DNA repair protein O(6) -alkylguanine-DNA alkyltransferase and promotes both spontaneous and carcinogen-induced hepatocellular carcinoma. To explore further the mechanism of tumorigenesis due to GSNOR deficiency, we compared the protein expression profiles in the livers of wild-type and GSNOR-deficient (GSNOR(-/-) ) mice that were challenged with lipopolysaccharide to induce inflammation and expression of inducible nitric oxide synthase (iNOS). Two-dimensional difference gel electrophoresis analysis identified 38 protein spots of significantly increased intensity and 31 protein spots of significantly decreased intensity in the GSNOR(-/-) mice compared to those in the wild-type mice. We subsequently identified 19 upregulated and 19 downregulated proteins in GSNOR(-/-) mice using mass spectrometry. Immunoblot analysis confirmed in GSNOR(-/-) mice a large increase in the expression of the pro-inflammatory mediator S100A9, a protein previously implicated in human liver carcinogenesis. We also found a decrease in the expression of multiple members of the protein disulfide-isomerase (PDI) family and an alteration in the expression pattern of the endoplasmic reticulum (ER) chaperones in GSNOR(-/-) mice. Furthermore, altered expression of these proteins from GSNOR deficiency was prevented in mice lacking both GSNOR and iNOS. In addition, we detected S-nitrosylation of two members of the PDI protein family. These results suggest that S-nitrosylation resulting from GSNOR deficiency may promote carcinogenesis under inflammatory conditions in part through the disruption of inflammatory and ER stress responses.     

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.     

Direct chemiluminescent imaging detection of human serum proteins in two-dimensional polyacrylamide gel electrophoresis

A novel chemiluminescence (CL)-based imaging method capable of directly detecting proteins in polyacrylamide gels after electrophoresis is proposed. Human serum proteins are presently detected by a direct CL imaging method after native 2-D PAGE. As a consequence, some proteins, including haptoglobin (Hp), Hp precursor, hemopexin (Hpx) precursor, Ig alpha-1 chain C region, and Complement C3 precursor can be detected and identified by MS and MS/MS techniques. These proteins are all acute phase proteins, which have been defined as biomarkers for certain diseases. Moreover, serum proteins from healthy people and cirrhotic patients were analyzed. A decrease in Hp spots for cirrhotic patients could be confirmed. The CL imaging conditions were optimized, including the concentrations of H(2)O(2) and luminol. The process of CL detection of proteins is simple, and there is no need for specialized equipment. In comparison with the traditional CBB-R250 staining method, the detection sensitivity was improved and the detection period decreased about 70 times. Hence, this technique possesses potentials as a rapid, convenient, and inexpensive analytical technique for protein detection and for the diagnosis of diseases.     

The whereabouts of transmembrane proteins from rat brain synaptosomes during two-dimensional gel electrophoresis

Little is known about what happens to transmembrane proteins (TMP) in 2-DE. In order to obtain more insight into the whereabouts of these proteins we prepared membrane-enriched synaptosomes from rat frontal cortex and washed them with 7 M urea or Na(2)CO(3). From each preparation, 200 microg protein was loaded on 2-DE gels covering the 4-7 and 6-11 pH ranges, respectively. MALDI-MS/MS analysis detected only 3 TMP among 421 identified spots. However, when the samples had been washed with Na(2)CO(3), only few well-focused spots remained detectable on the gel covering the pH 6-11 range. Instead, a heavily ruthenium-stained smear became visible at the upper edge of the gel at the location where the samples had been applied by cup loading. LC-MS/MS analysis revealed that this smear contained 38 unfocused TMP with up to 12 transmembrane helices. After transfer to the second dimension, no major areas of protein staining were left on the IPG strips. This indicates that after extraction and denaturation the TMP may form high-molecular aggregates, due to their "hydrophobic interactions". These aggregates enter the IPG strips, but do not focus regularly. They are then transferred onto the 2-DE-gels, where they remain caught at the upper edge.     

Differential expression of proteins in response to the interaction between the pathogen Fusarium graminearum and its host, Hordeum vulgare

Using proteomic techniques, a study aimed at isolating and identifying proteins associated with resistance to fusarium head blight (FHB) was conducted on six barley genotypes of varying resistance. At anthesis, barley spikelets were point inoculated with Fusarium graminearum macroconidial suspensions or mock inoculum. In total, 43 acidic protein spots out of 600 were detected 3 days postinoculation to be differentially expressed due to FHB and were identified. Identification of proteins responsive to FHB included those associated with oxidative burst and oxidative stress response, such as malate dehydrogenase and peroxidases, and pathogenesis-related (PR). An increase in abundance of PR-3 or PR-5 could be associated with the resistant genotypes CI4196, Svansota, and Harbin, as well as the intermediate resistant genotype CDC Bold. On the contrary, the susceptible genotype Stander showed a decrease in abundance of these acidic PR-proteins. In the susceptible and intermediate resistant genotypes Stander and CDC Bold, as well as CI4196, the increased abundance of proteins associated with an oxidative response might have prepared the terrain for saprophytic fungal invasion. On the contrary, in the resistant sources Harbin and Svansota we did not observed change in abundance of these proteins. Not a single significant change in acidic protein abundance could be detected in Chevron. Three distinct response patterns are reported from these six barley genotypes.     

Comprehensive analysis of the extracellular proteins from Xanthomonas campestris pv. campestris B100

The extracellular proteome of Xanthomonas campestris pv. campestris (Xcc) cultivated in minimal medium was isolated from the cell-free culture supernatant and separated by two-dimensional gel electrophoresis. This technique resolved 97 clearly visible protein spots, which were excised, digested with trypsin and identified on the basis of their peptide mass fingerprints generated by matrix assisted laser desorption/ionisation-time of flight-mass spectrometry. Using this approach 87 different proteins could be distinguished. The Signal P software predicted putative signal peptides for 53% of the extracellular proteins. These proteins are probably transported over the inner membrane and are localized in the periplasm, the outer membrane or secreted into the extracellular space. Among the secreted proteins are 11 degradative enzymes, which are involved in pathogenesis of Xcc. The proteins without obvious secretion signals are known to serve functions in the cytosol. How the cytosolic proteins are delivered to the extracellular space remains unclear.     

Partial characterization of the proteome of the mouse striatum

Many diseases of the mammalian CNS, including Parkinson's (PD) and Lesch Nyhan disease (LND), are associated with programmatic neurodegeneration or dysfunction of dopaminergic neurons in the mesencephalon, the nigrostriatal pathway, and its projections in the striatum [1-4]. Proteomic studies on brain tissue of both animal models and human PD patients have provided evidence for dysfunction and damage of many pathways, including oxidative stress-related damage, ubiquitin-proteasome dysfunction, mitochondrial energy metabolism deficiencies, and synaptic function [5-11]. To date no such proteomic studies have been reported in the related and rare basal ganglia disorder LND, a developmental rather than a neurodegenerative neurological disorder caused by deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) that regulates a major step in the purine salvage pathway [12]. Many studies have demonstrated that the both human LND patients and a mouse knockout model of HPRT deficiency have significantly reduced levels and uptake of dopamine in the striatum [4, 13-16] that is likely to be the principal cause of the CNS disorder. The precise molecular and cellular mechanisms that underlie this neurotransmitter defect are unknown.     

Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis

The solubilizing power of various nonionic and zwitterionic detergents as membrane protein solubilizers for two-dimensional electrophoresis was investigated. Human red blood cell ghosts and Arabidopsis thaliana leaf membrane proteins were used as model systems. Efficient detergents could be found in each class, i.e. with oligooxyethylene, sugar or sulfobetaine polar heads. Among the commercially available nonionic detergents, dodecyl maltoside and decaethylene glycol mono hexadecyl ether proved most efficient. They complement the more classical sulfobetaine detergents to widen the scope of useful detergents for the solubilization of membrane proteins in proteomics.     

Immunoproteomics of outer membrane proteins and extracellular proteins of Shigella flexneri 2a 2457T

Shigella flexneri 2a is an important pathogen causing bacillary dysentery in humans. In order to investigate any potential vaccine candidate proteins present in outer membrane proteins (OMPs) and extracellular proteins of S. flexneri 2a 2457T, we use the proteome mapping and database analyzing techniques. A subproteome map and database of OMPs were established first. One hundred and nine of the total 126 marked spots were cut out and processed to MALDI-TOF-MS and PMF. Eighty-seven spots were identified and they represented 55 OMP entries. Furthermore, immunoproteomics analysis of OMPs and extracellular proteins were performed. Total of 34 immunoreactive spots were identified, in which 22 and 12 were from OMPs and extracellular proteins, respectively. Eight novel antigens were found and some of these antigens may be potential vaccine candidate proteins. These results are useful for future studying of pathogenicity, vaccine, and novel antibacterial drugs. Maps and tables of all identified proteins are available on the Internet at www.proteomics.com.cn.     

Proteomic identification of putative plasmodesmatal proteins from Chara corallina

Plasmodesmata are channels that bridge the cell walls of plant cells, allowing regulated transport of molecules between neighbouring cells. We have used a proteomic strategy to identify putative plasmodesmata-associated proteins in the giant-celled green alga Chara corallina. Proteins were extracted from the plasmodesmata-rich nodal complexes and the middle of the long internodal cells, which do not contain plasmodesmata. Comparison of protein spot patterns generated by two-dimensional gel electrophoresis of both the soluble and cell wall fractions from the two cell types was done. Fifty-eight spots that were common to the nodal and internodal soluble fractions were analysed by matrix assisted laser desorption/ionisation-time of flight mass spectrometry, and peptide mass fingerprint data were used to search the database. Matches were made to four of these spots, in each case to housekeeping proteins. Further, a number of nodal specific spots were identified, 11 from the soluble fraction and nine from the wall fraction. These spots were excised from the gels and analysed by liquid chromatography tandem mass spectrometry to obtain peptide sequence. Database searches suggest that these spots include homologues to previously identified plasmodesmata-associated proteins cp-wap13 and heat shock cognate 70, as well as RNA-binding proteins, eukaryotic initiation factor 4A and a beta-1,3-glucanase. Several spots remained unidentified providing exciting new candidate plasmodesmata-associated proteins.