Proteomic identification of differentially expressed proteins in the anoxic rice coleoptile

Rice is the staple food for more than fifty percent of the world's population, and is therefore an important crop. However, its production is hindered by several biotic and abiotic stresses. Although rice is the only crop that can germinate even in the complete absence of oxygen (i.e. anoxia), flooding (low oxygen) is one of the major causes of reduced rice production. Rice germination under anoxia is characterized by the elongation of the coleoptile, but leaf growth is hampered. In this work, a comparative proteomic approach was used to detect and identify differentially expressed proteins in the anoxic rice coleoptile compared to the aerobic coleoptile. Thirty-one spots were successfully identified by MALDI-TOF MS analysis. The majority of the identified proteins were related to stress responses and redox metabolism. The expression levels of twenty-three proteins and their respective mRNAs were analyzed in a time course experiment.     

Proteomic identification of differentially expressed proteins in Arabidopsis in response to methyl jasmonate

Jasmonates (JAs) are the well characterized fatty acid-derived cyclopentanone signals involved in the plant response to biotic and abiotic stresses. JAs have been shown to regulate many aspects of plant metabolism, including glucosinolate biosynthesis. Glucosinolates are natural plant products that function in defense against herbivores and pathogens. In this study, we applied a proteomic approach to gain insight into the physiological processes, including glucosinolate metabolism, in response to methyl jasmonate (MeJA). We identified 194 differentially expressed protein spots that contained proteins that participated in a wide range of physiological processes. Functional classification analysis showed that photosynthesis and carbohydrate anabolism were repressed after MeJA treatment, while carbohydrate catabolism was up-regulated. Additionally, proteins related to the JA biosynthesis pathway, stress and defense, and secondary metabolism were up-regulated. Among the differentially expressed proteins, many were involved in oxidative tolerance. The results indicate that MeJA elicited a defense response at the proteome level through a mechanism of redirecting growth-related metabolism to defense-related metabolism.     

A simple method for staining nuclei of mature and germinated maize pollen

A sugar acetocarmine staining technique has been developed for staining the sperm and vegetative nucleus of mature and germinated maize pollen grains. This procedure is simple, stable and highly repeatable. The physiological properties of the mature maize pollen grains are first adjusted by using an in vitro germinating culture solution. This solution is 15% sucrose and contains 360 ppm calcium chloride dihydrate, and 120 ppm boric acid. One part fresh pollen grains is uniformly mixed with nine parts of the solution and left at room temperature for at least 5 hr. One part of this solution is then mixed with two parts of regular acetocarmine stain and left overnight. The color of this mixture is pinkish red or raspberry. The sugar in the mixture helps to increase color contrast between the pollen cytoplasm (light pink) and the nuclei (reddish purple), decreases the frequency of burst pollen, increases pollen expansion, stabilizes pollen figures and automatically seals the coverglass.     

Cell wall acyl-lipids, proteins and polysaccharides in mature and germinated olive pollen

Proteins, acyl-lipids and polysaccharides from cell walls of mature and germinated olive pollen were studied. In general, hemicelluloses are the most abundant polysaccharides, arabinose in mature and glucose in germinated pollen being the main components of these macromolecules. Protein content and its amino acid composition are very similar in walls from mature and germinated pollen, these compounds showing a weak acid character. Free-fatty acids are the most abundant lipid molecules in mature and germinated pollen walls and a decrease in acyl-lipids, especially in polar lipids, as well as a higher unsaturation of their fatty acid components are observed after germination.     

Proteomic analysis of differentially expressed serum proteins in lung cancer

Lung cancer continues to represent a major public health concern with high morbidity and mortality worldwide. Early detection of lung cancer is problematic due to a lack of diagnostic markers with high sensitivity and specificity. To determine the differently expressed proteins in the serum of lung cancer and identify the function of such proteins, two-dimensional electrophoresis (2DE) and liquid chromatography mass spectrometry (LC-MS) were used to screen the serum of lung cancer model induced by 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK). A total of 25 protein spots were qualitatively different and 6 were quantitatively different in the serum from rats bearing induced lung cancer when compared with normal controls. Two of the proteins that showed major changes in concentration in sera were identified to be Immunoglobulin γ 2A chain C region (heavy chain) and Transferrin by LC-MS/MS.     

Proteomic analysis of differentially expressed proteins in fungal elicitor-treated Arabidopsis cell cultures

Slow progress has been made in discovering plant genes governing the interaction of plant pathogens and their hosts using classical genetic approaches. Extensive studies employing DNA microarray techniques to identify global changes in gene expression during pathogen-host interaction have greatly enhanced discovery of genetic components regulating the plant defence response to pathogen attack. In this study, a complementary approach was used to identify changes in protein abundance during interaction of Arabidopsis cell cultures with a pathogen-derived elicitor. The soluble protein fractions were analysed by two-dimensional difference gel electrophoresis and proteins differentially expressed in response to treatment with fungal elicitor were identified via matrix-assisted laser desorption ionization-time of flight mass spectrometry. Elicitor responsive proteins included molecular chaperones, oxidative stress defence proteins, mitochondrial proteins, and enzymes of a diverse number of metabolic pathways. The findings, in combination with currently available microarray data, will form the basis of a filter to identify pivotal genes whose role in pathogen defence systems will require confirmation using gene knockout mutants.     

Proteomic analysis of differentially expressed skin proteins in iRhom2Uncv mice

A mouse homozygous for the spontaneous mutation uncovered (Uncv) has a hairless phenotype. A 309-bp non-frameshift deletion mutation in the N-terminal cytoplasmic domain of iRhom2 was identified in Uncv mice (iRhom2^Uncv) using target region sequencing. The detailed molecular basis for how the iRhom2 mutation causes the hairless phenotype observed in the homozygous iRhom2^Uncv mouse remains unknown. To identify differentially expressed proteins in the skin of wild-type and homozygous iRhom2^Uncv littermates at postnatal day 5, proteomic approaches, including two-dimensional gel electrophoresis and mass spectrometry were used. Twelve proteins were differentially expressed in the skin in a comparison between wild-type and homozygous iRhom2^Uncv mice. A selection of the proteomic results were tested and verified using qRT-PCR, western blot and immunohistochemistry. These data indicate that differentially expressed proteins, especially KRT73, MEMO1 and Coro-1, might participate in the mechanism by which iRhom2 regulates the development of murine skin. [BMB Reports 2015; 48(1): 19-24]     

Proteomic screening and identification of differentially distributed membrane proteins in Escherichia coli

Bacteria show asymmetric subcellular distribution of many proteins involved in diverse cellular processes such as chemotaxis, motility, actin polymerization, chromosome partitioning and cell division. In many cases, the specific subcellular localization of these proteins is critical for proper regulation and function. Although cellular organization of the bacterial cell clearly plays an important role in cell physiology, systematic studies to uncover asymmetrically distributed proteins have not been reported previously. In this study, we undertook a proteomics approach to uncover polar membrane proteins in Escherichia coli. We identified membrane proteins enriched in E. coli minicells using a combination of two-dimensional electrophoresis and mass spectrometry. Among a total of 173 membrane protein spots that were consistently detected, 36 spots were enriched in minicell membranes, whereas 15 spots were more abundant in rod cell membranes. The minicell-enriched proteins included the inner membrane proteins MCPs, AtpA, AtpB, YiaF and AcrA, the membrane-associated FtsZ protein and the outer membrane proteins YbhC, OmpW, Tsx, Pal, FadL, OmpT and BtuB. We immunolocalized two of the minicell-enriched proteins, OmpW and YiaF, and showed that OmpW is a bona fide polar protein whereas YiaF displays a patchy membrane distribution with a polar and septal bias.     

Proteomic analyses of Oryza sativa mature pollen reveal novel proteins associated with pollen germination and tube growth

As a highly reduced organism, pollen performs specialized functions to generate and carry sperm into the ovule by its polarily growing pollen tube. Yet the molecular genetic basis of these functions is poorly understood. Here, we identified 322 unique proteins, most of which were not reported previously to be in pollen, from mature pollen of Oryza sativa L. ssp japonica using a proteomic approach, 23% of them having more than one isoform. Functional classification reveals that an overrepresentation of the proteins was related to signal transduction (10%), wall remodeling and metabolism (11%), and protein synthesis, assembly and degradation (14%), as well as carbohydrate and energy metabolism (25%). Further, 11% of the identified proteins are functionally unknown and do not contain any conserved domain associated with known activities. These analyses also identified 5 novel proteins by de novo sequencing and revealed several important proteins, mainly involved in signal transduction (such as protein kinases, receptor kinase-interacting proteins, guanosine 5'-diphosphate dissociation inhibitors, C2 domain-containing proteins, cyclophilins), protein synthesis, assembly and degradation (such as prohibitin, mitochondrial processing peptidase, putative UFD1, AAA+ ATPase), and wall remodeling and metabolism (such as reversibly glycosylated polypeptides, cellulose synthase-like OsCsLF7). The study is the first close investigation, to our knowledge, of protein complement in mature pollen, and presents useful molecular information at the protein level to further understand the mechanisms underlying pollen germination and tube growth.     

Proteomic analysis of differentially expressed proteins in Lactobacillus brevis NCL912 under acid stress

Protein expression of Lactobacillus brevis NCL912 under acid stress was analysed by two-dimensional gel electrophoresis and MS. Twenty-five proteins were differentially expressed under acid stress. Among them, eight protein spots were identified by matrix-assisted laser desorption/ionization time-of-flight MS, of which seven were upregulated and one was downregulated. The function of the downregulated protein was unknown and the putative functions of the upregulated proteins were categorized as stress response, DNA repair, protein synthesis and glycolysis. Quantitative real-time PCR was used to further validate these differentially expressed proteins at the mRNA level and a positive correlation between the content of the proteins and their mRNA levels was found. The results suggest that these proteins are involved in the acid stress response mechanisms of this bacterium.     

Proteomic profiling of differentially expressed proteins from Bax inhibitor-1 knockout and wild type mice

Bax inhibitor-1 (BI-1) is an anti-apoptotic protein located in the endoplasmic reticulum (ER). The role of BI-1 has been studied in different physiopathological models including ischemia, diabetes, liver regeneration and cancer. However, fundamental knowledge about the effects of BI-1 deletion on the proteome is lacking. To further explore this protein, we compared the levels of different proteins in bi-1 (-/-) and bi-1 (+/+) mouse tissues by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). In several bi-1 (-/-) mice, glucose-regulated protein 75 (GRP75/mortalin/ PBP74/mthsp70), peroxiredoxin6 (Prx6) and fumarylacetoacetate hydrolase (FAH) showed a pI shift that could be attributed to post-translational modifications. Selenium-binding protein 2 (SBP2) and ferritin light chain 1 levels were significantly increased. Phosphatidylethanolamine-binding protein-1 (PEBP-1) was dramatically decreased in bi-1 (-/-) mice, which was confirmed by Western blotting. The phosphorylation of GRP75, Prx6 and FAH were compared between bi-1 (+/+) and bi-1 (-/-) mice using liver tissue lysates. Of these three proteins, only one exhibited modified phosphorylation; Tyr phosphorylation of Prx6 was increased in bi-1 (-/-) mice. Our protein profiling results provide fundamental knowledge about the physiopathological function of BI-1.     

Proteomic analysis of differentially expressed proteins between Xiangyou 15 variety and the mutant M15

A high oleic acid rapeseed material MI5 （derived from Xiangyou 15 variety） has been received more attention for its significant effect for human health. And it has almost the same physiological characteristic with Xiangyou 15 variety. To find out the difference between high oleic acid rapeseed material and Xiangyou 15 seedling, a comparative proteomic approach based on 2-DE and mass spectrometry was adopted. A total of 277 protein spots showed a significant change in intensity by more than 2.0-fold from M15 compared with Xiangyou 15 variety. Among them, 48 spots that changed at least 3.0-fold were excised from gels and successfully identified by MALDI-TOF/TOF MS. The identified proteins involved in metabolism of carbohydrate and energy （75%）, stress and defense （8.3%）, photosynthesis （6.3%）, protein metabolism （2.1%） and other functions （8.3%）. Then real-time quantitative PCR （qPCR） analysis was used to verify the expression levels of differentially expressed proteins, but the results did well agree with the proteomic results. In this work, most of the proteins involved in metabolism of carbohydrate and energy have higher expression in M15, which may reveal M15 has higher metabolism ability. These results provided much information to understand the differences between high oleic acid rapeseed material and Xiangyou 15 variety, which will be useful to screen high oleic rapeseed materials in seedling period.     

Proteomic analysis of differentially expressed proteins in Penaeus vannamei hemocytes upon Taura syndrome virus infection

To understand molecular responses of crustacean hemocytes to virus infection, we applied 2-DE proteomics approach to investigate altered proteins in hemocytes of Penaeus vannamei during Taura syndrome virus (TSV) infection. At 24 h postinfection, quantitative intensity analysis and nano-LC-ESI-MS/MS revealed 11 forms of 8 proteins that were significantly up-regulated, whereas 9 forms of 5 proteins were significantly down-regulated in the infected shrimps. These altered proteins play important roles in host defense (hemocyanin, catalase, carboxylesterase, transglutaminase, and glutathione transferase), signal transduction (14-3-3 zeta), carbohydrate metabolism (acetylglucosamine pyrophosphorylase), cellular structure and integrity (beta-tubulin, beta-actin, tropomyosin, and myosin), and ER-stress response (protein disulfide isomerase). Semiquantitative RT-PCR and Western blot analysis confirmed the upregulation of 14-3-3 at both mRNA and protein levels. Interestingly, several altered protein spots were identified as fragments of hemocyanin. Mass spectrometric analysis showed that the hemocyanin spots at acidic and basic regions represented the C- and N-terminal hemocyanin fragments, respectively. As three-quarters of C-terminal fragments were up-regulated, whereas two-thirds of N-terminal hemocyanin fragments were down-regulated, we therefore hypothesize that C- and N-terminal hemocyanin fragments may have differential roles in hemocytes. Further investigation of these data may lead to better understanding of the molecular responses of crustacean hemocytes to TSV infection.     

Proteomic analysis of differentially expressed proteins induced by rice blast fungus and elicitor in suspension-cultured rice cells

We used two-dimensional electrophoresis (2-DE) and other proteomic approaches to identify proteins expressed in suspension-cultured rice cells in response to the rice blast fungus, Magnaporthe grisea. Proteins were extracted from suspension-cultured cells at 24 and 48 h after rice blast fungus inoculation or treatment with elicitor or other signal molecules such as jasmonic acid (JA), salicylic acid, and H(2)O(2). The proteins were then polyethylene glycol fractionated before separation by 2-DE. Fourteen protein spots were induced or increased by the treatments, which we analyzed by N-terminal or internal amino acid sequencing. Twelve proteins from six different genes were identified. Rice pathogen-related protein class 10 (OsPR-10), isoflavone reductase like protein, beta-glucosidase, and putative receptor-like protein kinase were among those induced by rice blast fungus; these have not previously been reported in suspension-cultured rice cells. Six isoforms of probenazole-inducible protein (PBZ1) and two isoforms of salt-induced protein (SalT) that responded to blast fungus, elicitor, and JA were also resolved on a 2-DE gel and identified by proteome analysis. The expression level of these induced proteins both in suspension-cultured cells and in leaves of whole plants was analyzed by Western blot. PBZ1, OsPR-10, and SalT proteins from incompatible reactions were induced earlier and to a greater extent than those in compatible reactions. Proteome analysis can thus distinguish differences in the timing and amount of protein expression induced by pathogens and other signal molecules in incompatible and compatible interactions.