Proteomic analysis of near-isogenic sunflower varieties differing in seed oil traits

Near-isogenic sunflower lines containing 25% (inbred RHA280) and 48% (RHA801) oil by seed dry mass were comparatively analyzed in biological triplicate at 18 days after flowering using two-dimensional (both pI 3-10 and 4-7) Difference Gel Electrophoresis. Additionally, two inbred lines varying in oleic acid content, HA89 (18% oleic) and HA341 (89% oleic), were also analyzed in the same manner. Statistical analyses of these sunflower lines was performed beginning with fitting a mixed effects linear model to the log-transformed optical volume of each spot to account for gel variation, followed by testing the significance between varieties for mean transformed optical spot volumes. The p-values from the spot analysis procedures were then used to find the cutoff point for differential expression using a 10% false-discovery rate (FDR). Comparison of the oil content and oleic acid composition lines revealed 77 and 42 protein spots below the 10% FDR cutoff, respectively, and were therefore declared differentially expressed. Liquid chromatography-tandem mass spectrometry analysis of each of these protein spots resulted in assignments for 44 and 17 spots, respectively. Fructokinase, plastid phosphoglycerate kinase, and enolase proteins were determined to be up-regulated in the high oil line, while phosphofructokinase, cytosolic phosphoglucomutase, and cytsolic phosphoglycerate kinase were up-regulated in the low oil variety. Additionally, four activities involved in amino acid synthesis were up-regulated in the low oil variety in addition to 12S storage proteins and a protein similar to legumin storage protein. Interestingly, two 2-DE spots identified as 14-3-3 proteins were found to be up-regulated in high oleic acid variety. Alteration of glycolytic and amino acid biosynthetic enzymes, as well as storage protein levels, suggests seed oil content is tightly linked to carbohydrate metabolism and protein synthesis in a complex manner.     

2-D protein maps of rat gastrocnemius and soleus muscles: a tool for muscle plasticity assessment

Functional characterization of muscle fibers relies on ATPase activity and on differential measurements of metabolic proteins, including mitochondrial and glycolytic enzymes, glucose, lactate and lactic acid transporters, calcium cycling proteins and components of the contractile machinery. The recent introduction of microarray technology has enabled detailed gene expression studies under different physiological and pathological conditions, thus generating novel hypotheses on muscle function. However, microarray approaches are limited by the incomplete genome coverage of currently available chips, and by poor correlation between mRNA concentration and protein expression level. We have used 2-DE and MS to build a reference map of proteins from rat mixed gastrocnemius and soleus muscle, and to assess qualitative and quantitative differences in protein distribution between these two functionally dissimilar muscles. More than 800 spots on each gel were detected by silver staining, of which 167 were excised, digested in-gel with trypsin and analyzed by ESI-MS/MS. One hundred and twenty eight distinct gene products were identified, including metabolic, transport and contractile proteins. Forty one spots displayed differences in relative expression level between mixed gastrocnemius and soleus samples. These data not only enable differentiation of functionally distinct slow-twitch and fast-twitch fiber types, but also provide tools for investigating muscle plasticity in response to physiological and environmental conditions such as aging or hypoxia.     

Differential protein expression in brain capillary endothelial cells induced by hypoxia and posthypoxic reoxygenation

Cerebral ischemia causes functional alteration of the blood-brain barrier, formed by brain capillary endothelial cells (BCEC). Changes in protein expression and activity of selected differentially expressed enzymes were investigated in BCEC subjected to hypoxia (24 h) alone or followed by a 24-h reoxygenation. BCEC proteins were isolated, separated by 2-DE, and identified by MALDI-MS. Computer-based 2-D gel analysis identified 21 up-regulated proteins and 4 down-regulated proteins after hypoxia alone and 9 proteins that were further up-regulated after posthypoxic reoxygenation. The expression of the majority of hypoxia-induced proteins was reduced toward control levels during reoxygenation. The most prominent changes were identified for glycolytic enzymes (e.g., phosphoglycerate kinase), proteins of the ER (e.g., calreticulin), and cytoskeletal (e.g., vimentin) proteins. The results indicate that BCEC respond to hypoxia/reoxygenation by adaptive up-regulation of proteins involved in the glycolysis, protein synthesis, and stress response.     

4周中等强度有氧运动诱导大鼠心房肌蛋白质组差异表达的研究

目的：探讨中等强度有氧运动对大鼠心房肌蛋白质组及其基因差异表达的影响,为运动心脏重塑和慢性心血管疾病康复研究提供研究依据。方法：20只雄性SD大鼠按照体重随机配对分为对照组、实验组（n=10）,实验组大鼠每次按照速度24 m·min^-1、持续训练40 min （负荷强度相当于60%~70% VO_2max）,每周训练6 d,持续训练4周中等强度有氧运动。应用双向凝胶电泳技术（2-DE）分离心房肌蛋白质点,串联飞行时间质谱仪技术鉴定电泳结果中表达量上调≥5倍以上,下调至1/5以下的13个备选目标蛋白质点。并对其中6个目标蛋白质用逆转录-聚合酶链式反应（RT-PCR）技术检测其mRNA。结果：通过软件分析,实验组与对照组比较,其中表达量下调至20%以下的点8个,上调5倍及以上点有5个,质谱鉴定分析其中的13个蛋白质点,最终鉴定出8种蛋白质和一个分子量为54 KDa的未知蛋白,包括：丙酮酸脱氢酶E1α1、线粒体乌头酸水合酶、蛋白质二硫键异构酶A3、甲基丙二酸半醛脱氢酶、线粒体二氢硫辛酸脱氢酶、异戊酰辅酶A脱氢酶、谷胱甘肽合成酶、丝裂素活化蛋白激酶3等。RT-PCR检测结果表明,与对照组相比,4周中等强度有氧运动后,大鼠心房肌中甲基丙二酸半醛脱氢酶的mRNA表达量降低（P﹤0.05）,线粒体二氢硫辛酸脱氢酶、蛋白质二硫键异构酶A3、线粒体乌头酸水合酶、谷胱甘肽合成酶的mRNA表达量降低（P>0.05）;异戊烯辅酶A脱氢酶的mRNA表达量增高（P>0.05）,表明mRNA表达水平与质谱鉴定结果的变化不完全一致。结论：4周的中等强度有氧运动诱导大鼠心房肌蛋白质组发生显著变化,有13个明显变化的目标蛋白,多数为能量物质代谢酶,这些目标蛋白质的变化与其mRNA表达量的变化并不完全一致,表明中等强度运动可能影响这些目标蛋白质上游基因转录的调控,也可影响下游翻译﹑修饰等的调控,导致表达的差异变化。     

采用定量蛋白质组学技术筛选小鼠肝癌淋巴道转移相关蛋白

淋巴道转移是上皮来源恶性肿瘤转移的早期阶段,其发生机制不清,一直是肿瘤学研究面临的难题,为寻找淋巴道转移相关蛋白,以一对来源于同一亲本细胞,且淋巴道转移潜能显著不同的小鼠肝癌腹水型细胞株为研究对象,其中Hca-F为高淋巴道转移力细胞株,Hca-P为低淋巴道转移力细胞株,采用定量蛋白质组学技术——荧光差异双向凝胶电泳,建立了高低淋巴道转移力小鼠肝癌细胞荧光差异蛋白表达图谱,高通量筛选与肿瘤淋巴道转移相关的蛋白质.经DeCyde软件分析,共得到163个有统计学差异的蛋白质点,选择2倍以上的差异性蛋白质点23个,经质谱鉴定得到17个蛋白质,在Hca-F中高表达的蛋白质有7个:转羟乙醛酶、波形蛋白、肌酸激酶(脑)、膜联蛋白7、膜联蛋白5、烯酰辅酶A水合酶1(过氧化物酶体)、核内异质核糖核蛋白A2/B1异构体1.而在Hca-F中低表达的蛋白质有10个:真核翻译延长因子2、Ero1样蛋白、乙醛脱氢酶2(线粒体)、苹果酸盐脱氢酶2(NAD)、β-内酰胺酶2、谷胱甘肽S转移酶"1、泛素C末端水解酶同工酶L3、内质网蛋白29(前体)、溶血磷脂酶1、微管不稳定蛋白.这些差异性蛋白质的功能涉及到代谢、蛋白质分泌、蛋白质结合、核苷酸结合,钙离子结合、凋亡和调节生长等过程.对这些蛋白质功能的进一步验证,将有助于解析肿瘤淋巴道转移的分子机制.     

Proteomic evaluation of cadmium toxicity on the midge Chironomus riparius Meigen larvae

Heavy-metal pollution of aquatic ecosystems is a widespread phenomenon after industrial consumption. Whether aquatic organisms are adapted to the heavy-metal pollutants or not, such environmental stress causes changes in physiological responses. In this study, the aquatic midge, Chironomus riparius Meigen, was used to find changes of expression of proteins in relation to cadmium exposure. Dose-response relationships between cadmium concentrations and mortality of 3rd instar midge larvae were observed and the protein levels were compared using PD-Quest after 2-DE. Comparing the intensity of protein spots, 21 proteins decreased and 18 proteins increased in response to cadmium treatment. With increased proteins, three enzymes such as S-adenosylmethionine decarboxylase, O-methyltransferase, and aspartokinase were involved in the glutathione biosynthesis and a key enzyme regulating fatty acid biosynthesis, oleyl-acyl carrier protein thioesterase was also identified. According to the functional classification of decreased levels of proteins, they were involved in energy production, protein fate, nucleotide biosynthesis, cell division, transport and binding, signal transduction, and fatty acid and phospholipid metabolism in the cell. In addition, phenol hydroxylase, thioesterase, zinc metalloprotease, and aspartate kinase were newly expressed after cadmium exposure at the concentration of the LC(10 )value. Therefore, these proteins seem to be potential biomarkers for cadmium exposure in the aquatic ecosystems.     

Metabolic responses and arginine kinase expression under hypoxic stress of the kuruma prawn Marsupenaeus japonicus

In response to hypoxia at PO(2) 1.3-1.7 mg/L for 6 h, the kuruma prawn Marsupenaeus (Penaeus) japonicus showed a dramatic decrease in phosphoarginine storage in muscle, with normal levels restored during 4-h post-hypoxic recovery. Large stores of muscle glycogen only decreased between 4 and 6 h during hypoxia, but greatly diminished during recovery. Muscle ATP levels and energy charge decreased only slightly under hypoxia. Lactate levels increased slightly during hypoxia and promptly returned to control levels during recovery. These data indicate that phosphoarginine works in muscle as an ATP buffer during hypoxia and glycogen is utilized as an energy source during recovery. Under hypoxia, up- and down-regulated proteins were identified after 2D electrophoresis and partial sequences were obtained after protease digestion. Fructose bisphosphate aldolase was down-regulated during hypoxia, suggesting the suppression of glycolysis under hypoxia. Several partial sequences from three protein spots up-regulated under hypoxia were all assigned to arginine kinase, suggesting the existence of several isoforms of arginine kinase in the muscle of M. japonicus. This arginine kinase up-regulation under hypoxia may indicate a provision for oxygen re-supply after anaerobiosis. This is consistent with the prompt replenishment of phosphoarginine stores during recovery from hypoxia.     

A two-dimensional electrophoresis and mass spectrometry protein analysis of the antibiotic producer Nonomuraea sp. ATCC 39727 in different growth conditions

Nonomuraea sp. ATCC 39727 is an aerobic actinomycete, industrially important as a producer of the glycopeptide A40926, which is used as a precursor of the semi-synthetic antibiotic dalbavancin. Previous studies showed that the production of A40926 is depressed by calcium, but promoted by l-glutamine or l-asparagine. In this study, the protein expression changes of Nonomuraea sp. ATCC 39727 in these two different growth and antibiotic-production conditions have been investigated by two-dimensional electrophoresis and mass spectrometry (MS) analysis. Few protein spots show statistically significant expression changes, and, among this group of proteins, malate dehydrogenase (MDH) shows a significant decrease in the overproduction condition. The decrease of MDH is of particular interest because it is the first described significant change in the expression levels of enzymes of the central metabolism related with A40926 overproduction.     

Proteomic Analysis of Methylarginine-Containing Proteins in HeLa Cells by Two-Dimensional Gel Electrophoresis and Immunoblotting with a Methylarginine-Specific Antibody

Protein arginine methylation is found in many nucleic acid binding proteins affecting numerous cellular functions. In this study we identified methylarginine-containing proteins in HeLa cell extracts by two-dimensional electrophoresis and immunoblotting with a methylarginine-specific antibody. Protein spots with matched protein stain and blotting signals were analyzed by mass spectrometry. The identities of 12 protein spots as 11 different proteins were suggested. Known methylarginine-containing proteins such as hnRNP A2/B1, hnRNP A1, hnRNP G and FUS were identified, indicating the feasibility of our approach. However, four highly abundant metabolic enzymes that might co-electrophorese with methylarginine-containing proteins were also identified. Other nucleic acid binding proteins hnRNP M, hnRNP I and NonO protein were identified. Recombinant hnRNP M and a peptide with the RGG sequence in hnRNP M could be further methylated in vitro. The immunoblotting results of immunoprecipitated hnRNP I and NonO protein are consistent with arginine methylation in both proteins. In this study we identified methylarginine-containing proteins in HeLa cells through proteomic approaches and the method is fast and robust for further applications.     

Comparison of extraction procedures for proteome analysis of Streptococcus pneumoniae and a basic reference map

Streptococcus pneumoniae is an important human pathogen causing life-threatening invasive diseases such as pneumonia, meningitis and bacteraemia. Despite major advances in our understanding of pneumococcal mechanisms of pathogenicity obtained through genomic studies very little has been achieved on the characterisation of the proteome of this pathogen. The highly complex structure of its cell envelope particularly amongst the various capsular forms enables the cell to resist lysis by conventional mechanical methods. It is therefore highly desirable to develop a cellular lysis and protein solubilisation procedure that minimises protein losses and allows for maximum possible coverage of the proteome of S. pneumoniae. Here we have utilised various combinations of mechanical or enzymatic cell lysis with two protein solubilisation mixtures urea/CHAPS-based mixture or SDS/DTT-based mixture in order to achieve best quality protein profiles using two proteomic technologies surface-enhanced laser desorption ionisation (SELDI) TOF MS and 2-DE. While urea/CHAPS-based mixture combined with freeze/thawing provided enough material for good-quality SELDI TOF MS fingerprints, a combination of mechanical, enzymatic and chemical lysis was needed to be used to successfully extract the desired protein content for 2-DE analysis. The methods chosen were also assessed for reproducibility and tested on various capsular types of S. pneumoniae. As a result, good-quality and reproducible profiles were created using various ProteinChip arrays and more than 800 protein spots were separated on a single 2-D gel of S. pneumoniae. Twenty-five of the most abundant protein spots were identified using LC/MS/MS to create a reference map of S. pneumoniae. The proteins identified included glycolytic enzymes such as glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, enolase etc. Several fermentation enzymes were also present including two of the components of the arginine deiminase system. Proteins involved in protein synthesis, such as translation factors and ribosomal proteins, as well as several chaperone proteins were also identified.     

Lectin-based proteomic profiling of aged skeletal muscle: decreased pyruvate kinase isozyme M1 exhibits drastically increased levels of N-glycosylation

Since various neuromuscular diseases are associated with abnormal glycosylation, it was of interest to determine whether this key post-translational modification is also altered in aged skeletal muscle. Lectins represent highly versatile carbohydrate-binding proteins that are routinely employed for the characterization of glycoproteins. Here, we used the lectin wheat germ agglutinin (WGA) for the proteomic profiling of senescent fibers. WGA labeling of the soluble proteome from 3-month- versus 30-month-old rat gastrocnemius muscle, following two-dimensional gel electrophoretic separation, resulted in the identification of 13 distinct protein species. Analysis of WGA binding levels, in conjunction with mass spectrometric fingerprinting, revealed that one isoform of a major metabolic muscle protein exhibited a drastic alteration in the content of sialic acid and N-acetylglucosaminyl sugar residues. Pyruvate kinase isoform M1 with protein accession number gi|16757994|, exhibiting a pI of 6.6 and an apparent molecular mass of 57.8kDa, showed a six fold increase in N-glycosylation and a three fold decrease in protein expression. In contrast to comparable levels of N-glycosylated proteins in young adult versus senescent muscle, as judged by fluorescein-conjugated WGA labeling of transverse muscle cryosections, staining with antibodies to the M1 isoform of pyruvate kinase showed reduced expression of this cytosolic element. Furthermore, activity assays demonstrated a reduced activity of this glycolytic enzyme in senescent muscle. This agrees with the idea that abnormal post-translational modifications in key metabolic enzymes may be involved in the conversion of aged muscle to slower twitch patterns and a drastic shift to more aerobic-oxidative metabolism.     

Proteomic analysis of Pteris vittata fronds: two arbuscular mycorrhizal fungi differentially modulate protein expression under arsenic contamination

Arbuscular mycorrhizae (AM) are the most widespread mutualistic symbioses between the roots of most land plants and a phylum of soil fungi. AM are known to influence plant performance by improving mineral nutrition, protecting against pathogens and enhancing resistance or tolerance to biotic and abiotic stresses. The aim of this study was to investigate the frond proteome of the arsenic hyperaccumulator fern Pteris vittata in plants that had been inoculated with one of the two AM fungi (Glomus mosseae or Gigaspora margarita) with and without arsenic treatment. A protective role for AM fungi colonisation in the absence of arsenic was indicated by the down-regulation of oxidative damage-related proteins. Arsenic treatment of mycorrhizal ferns induced the differential expression of 130 leaf proteins with specific responses in G. mosseae- and Gi. margarita-colonised plants. Up-regulation of multiple forms of glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and enolase, primarily in G. mosseae-inoculated plants, suggests a central role for glycolytic enzymes in arsenic metabolism. Moreover, a putative arsenic transporter, PgPOR29, has been identified as an up-regulated protein by arsenic treatment.     

Long term anoxia in rainbow trout investigated by 2-DE and MS/MS

Twenty-four hours of N(2) induced anoxia induced global perturbations on protein expression in rainbow trout hypodermal fibroblasts cell line. Anoxia was obtained by depleting the medium of O(2) by flushing with N(2), and protein changes were studied by 2-DE coupled with MS providing quantitative measurements of a large number of proteins in one single study. The anoxic insult changed the level of 33 protein spots: 22 of these were up-regulated compared to the control situation and 11 were down-regulated. Using MS/MS sequencing 19 of the 33 protein spots that changed were identified, corresponding to a success rate of more than 50%. The identified proteins included two proteins involved in energy metabolism namely phosphoglycerate mutase and isocitrate dehydrogenase. In addition we observed the up-regulation of a cluster of proteins that contribute to cytoskeleton function. These are calpain, EB1, and Rho GDP dissociation inhibitor (GDI). The up-regulation of Rho GDI was shown to develop in a time dependent manner with no significant increase for up to 8 h of anoxia. In conclusion, this study provides a thorough investigation of the effect of anoxia in a cell line from rainbow trout.     

Regulated expression of sterol carrier protein 2 in the ovary: a key role for cyclic AMP.

Sterol carrier protein 2 (SCP2) is believed to play an important role in the intracellular movement of cholesterol in steroidogenic cells. We examined the distribution of SCP2 gene expression in the rat ovary and the role of gonadotropins and cyclic AMP in the regulation of SCP2 mRNA levels. In situ hybridization revealed that the most steroidogenically active ovarian compartments (e.g., corpora lutea and theca cells) contain significant amounts of SCP2 mRNA whereas granulosa cells have modest levels. Gonadotropins, which promote follicular growth and luteinization, increased the ovarian content of SCP2 mRNA as assessed by Northern blotting along with increases in cytochrome P450scc mRNA. Using steroidogenic transformed rat granulosa cells (Grs-21), a cyclic AMP analogue (8-Br-cAMP) was found to increase SCP2 mRNA and protein levels within 24 h of treatment. P450scc mRNA was also induced whereas actin mRNA levels were not affected. The 8-Br-cAMP stimulation of SCP2 mRNA accumulation was completely inhibited by actinomycin D and cycloheximide. The cyclic AMP analogue also increased SCP2 mRNA levels in a non-steroid hormone producing transformed rat granulosa cell line Gs-8. We conclude that SCP2 gene expression in the ovary is correlated with the state of differentiation of granulosa cells. Gonadotropic hormones which stimulate luteinization of the cells increase SCP2 gene expression. These actions of gonadotropins appear to be mediated at least in part by cyclic AMP through a mechanism requiring ongoing RNA and protein synthesis. However, SCP2 gene expression is not obligatorily coupled to steroidogenic activity, as cyclic AMP analogues can increase SCP2 mRNA in a line of transformed ovarian granulosa cells incapable of synthesizing hormones.     

Changes in liver protein abundance in inbred alcohol-preferring rats due to chronic alcohol exposure, as measured through a proteomics approach

This study compares the total liver proteome of inbred alcohol‐preferring line (iP) rats exposed to alcohol with iP rats without alcohol experience. Rat liver proteins were extracted using a three‐step procedure. Each of the three solutions solubilizes a different set of proteins. The extracted proteins were separated by 2‐DE. Scanned gels of two sample groups, alcohol‐exposed iP and alcohol‐naïve iP, were compared, revealing many protein spots with significantly higher or lower densities. These spots were cut from the gel, destained, and subjected to trypsin digestion and subsequent identification by LC‐MS/MS. Twenty‐four individual rats, 12 alcohol‐naïve, and 12 alcohol‐exposed, were used in this study. Two groups, each containing six naïve and six exposed animals, were created for statistical comparison. For the first group, 64 spots were observed to have statistically significant intensity differences upon alcohol exposure across all three extracts while 118 such spots were found in the second group. There were 113 unique proteins in both groups together. The majority of these proteins were enzymes. Significant changes are observed for three major metabolic pathways: glycolysis, gluconeogenesis, and fatty acid β‐oxidation. In addition, enzymes involved in protein synthesis and antioxidant activity show significant changes in abundance in response to alcohol exposure.