Comparative proteome analysis of Aspergillus oryzae 3.042 and A. oryzae 100-8 strains: Towards the production of different soy sauce flavors

Aspergillus oryzae plays a central role in soybean fermentation, particularly in its contribution to the flavor of soy sauce. We present a comparative assessment of the intracellular differences between wild-type strain 3.042 and mutant strain A100-8, at the proteome level. 522 different protein spots were identified by MALDI-TOF MS, with 134 spots being confirmed by MALDI-TOF MS/MS. Of these, 451 were differentially expressed proteins (DEPs). There was at least a two-fold increase for 288 spots, and at least a two-fold decrease for 163 spots, in strain A100-8 when compared to 3.042. Further analysis showed that 63 of the more abundant proteins were involved in glycolysis and the citrate cycle; 43 more abundant proteins and 10 less abundant proteins were related to amino acid biosynthesis and metabolism; two of the more abundant proteins were involved in vitamin biosynthesis; and five of the more abundant proteins and four of the less abundant proteins were related to secondary metabolites. Moreover, quantitative real time PCR showed that the mRNA expression levels of six typical genes we selected were consistent with changes in protein expression. We postulate that there may be a relationship between DEPs and the flavor formation mechanism in A. oryzae.     

The instantly released Drosophila immune proteome is infection-specific

In this study, we analyzed the hemolymph proteome of Drosophila third instar larvae, which were induced with a suspension of Gram-positive bacteria or yeast. Profiling of the hemolymph proteins of infected versus non-infected larvae was performed by two-dimensional difference gel electrophoresis. Infection with Micrococcus luteus or Saccharomyces cerevisiae induced, respectively, 20 and 19 differential protein spots. The majority of the spots are specifically regulated by one pathogen, whereas only a few spots correspond to proteins altered in all cases of challenging (including after challenge with lipopolysaccharides). All of the upregulated proteins can be assigned to specific aspects of the immune system, as they did not increase in the hemolymph of sterile pricked larvae. Next to known immune proteins, unannotated proteins were identified such as CG4306 protein, which has homologues with unknown function in all metazoan genome databases available today.     

Attenuation of fungal infection in thermoregulating Locusta migratoria is accompanied by changes in hemolymph proteins

Hemolymph proteins in the locust, Locusta migratoria migratorioides infected with the fungus Metarhizium anisopliae var acridum were analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Under conditions that allowed locusts to thermoregulate, 2 proteins, ITB1 (ca. 18kDa) and ITB2 (ca. 13kDa) were induced 48h post inoculation. In contrast, under non-thermoregulating conditions, only 1 band, INTB1 (ca. 18kDa) was induced with similar molecular mass to ITB1. ITB1 and ITB2 were N-terminally sequenced but showed little homology to known proteins. The induction of hemolymphal proteins in infected, thermoregulating locusts and implication in insect immune defence are discussed.     

肝癌亚细胞结构的蛋白质组分比较分析

运用亚细胞蛋白质组学的研究策略,分离纯化亚细胞结构,可以提高低丰度蛋白质在双向电泳中检出的数量。通过对比分析肝癌细胞与正常肝细胞线粒体、细胞核蛋白质组的差异表达情况,为肝癌发病机理的研究提供更多、更有价值的信息。以体外培养的人体肝癌细胞QGY-7703与正常肝细胞LO2为研究模型,通过超离心的方法分离细胞的线粒体和细胞核。双向电泳分离线粒体和细胞核的蛋白质,图像分析筛选差异表达蛋白斑点,MALDI-TOF-MS鉴定蛋白质。从线粒体、细胞核的蛋白质电泳图谱中筛选出54个候选差异表达的蛋白质斑点,质谱鉴定出22种差异表达蛋白质,其中17种在肝癌细胞中表达上调,5种在肝癌细胞中表达下调。筛选出的差异表达蛋白质涉及到细胞的能量代谢、蛋白质合成、细胞骨架与核骨架的改变、mRNA的加工成熟及凋亡调控等许多方面,表明癌变细胞的组织结构和代谢状态都发生了很大的变化。     

Separation and identification of soybean leaf proteins by two-dimensional gel electrophoresis and mass spectrometry

To establish a proteomic reference map for soybean leaves, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 260 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. Fifty-three of these protein spots were identified by searching NCBInr and SwissProt databases using the Mascot search engine. Sixty-seven spots that were not identified by MALDI-TOF-MS analysis were analyzed with liquid chromatography tandem mass spectrometry (LC-MS/MS), and 66 of these spots were identified by searching against the NCBInr, SwissProt and expressed sequence tag (EST) databases. We have identified a total of 71 unique proteins. The majority of the identified leaf proteins are involved in energy metabolism. The results indicate that 2D-PAGE, combined with MALDI-TOF-MS and LC-MS/MS, is a sensitive and powerful technique for separation and identification of soybean leaf proteins. A summary of the identified proteins and their putative functions is discussed.     

Drosophila melanogaster larval hemolymph protein mapping

With the completion of the genome sequence of Drosophila melanogaster the importance of constructing a proteome map is to be considered. Therefore, with the application of recent advances in proteomic analysis approaches, a protein map of D. melanogaster larvae hemolymph proteins was obtained using 2-DE in the range of pH 3-10. After Coomassie colloidal detection of 289 spots, a total of 105 were excised from the gel and digested with trypsin. Identification was done based on a combination of MALDI-TOF/TOF MS and MS/MS spectra. The 99 proteins identified using this approach include a large number of metabolic enzymes, translational apparatus components, and structural proteins. Among these we emphasize the identification of proteins with molecular chaperone properties (heat shock proteins and PPIases) and protein spots involved in defense responses such as antioxidant and immunological defense mechanisms (thioredoxin, prophenoloxidase, and serine proteases), as well as in signal transduction pathways.     

Specific activity of a Bacillus thuringiensis strain against Locusta migratoria manilensis

Bacillus thuringiensis (Bt) has played an important role in biocontrol of pests. However, insecticidal activity of B. thuringiensis against locusts has been rarely reported. Bt strain BTH-13 exhibiting specific activity to locusts was isolated from a soil sample in China and characterized. Its bipyramidal parasporal crystal is mainly composed of a protein of 129 kDa, and produces a mature toxin of 64 kDa after activation. The pattern of total DNA from BTH-13 showed a large and three small plasmid bands. Known δ-endotoxin genes, cry1Aa, cry1Ab, cry1Ac, cry1C, cry3, cry4 and cry7Aa were not found from strain BTH-13 by PCR amplification. The sequence analysis of a DNA fragment produced by PCR amplification with degenerate cry-selective primers revealed that the fragment encoded a δ-endotoxin segment, which exhibited some similarity to several Cry proteins (41% of the highest similarity to Cry7Ba1). Toxicity tests were performed against Locusta migratoria manilensis, and the results demonstrated that trypsin-treated sporulated cultures and crystal proteins had high toxicity to larval and adult locusts. Cry toxin of BTH-13 was detected on the midguts of treated locusts using immunofluorescent technology, which confirmed the site of action of the crystal proteins in their toxicity for locusts.     

Proteomics in Drosophila melanogaster: first 2D database of larval hemolymph proteins

A proteomic approach was used for the identification of larval hemolymph proteins of Drosophila melanogaster. We report the initial establishment of a two-dimensional gel electrophoresis reference map for hemolymph proteins of third instar larvae of D. melanogaster. We used immobilized pH gradients of pH 4-7 (linear) and a 12-14% linear gradient polyacrylamide gel. The protein spots were silver-stained and analyzed by nanoLC-Q-Tof MS/MS (on-line nanoscale liquid chromatography quadrupole time of flight tandem mass spectrometry) or by Matrix assisted laser desorption time of flight MS (MALDI-TOF MS). Querying the SWISSPROT database with the mass spectrometric data yielded the identity of the proteins in the spots. The presented proteome map lists those protein spots identified to date. This map will be updated continuously and will serve as a reference database for investigators, studying changes at the protein level in different physiological conditions.     

A proteomic approach to identifying proteins differentially expressed in conidia and mycelium of the entomopathogenic fungus Metarhizium acridum

Metarhizium spp. is an important worldwide group of entomopathogenic fungi used as an interesting alternative to chemical insecticides in programs of agricultural pest and disease vector control. Metarhizium conidia are important in fungal propagation and also are responsible for host infection. Despite their importance, several aspects of conidial biology, including their proteome, are still unknown. We have established conidial and mycelial proteome reference maps for Metarhizium acridum using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS). In all, 1130±102 and 1200±97 protein spots were detected in ungerminated conidia and fast-growing mycelia, respectively. Comparison of the two protein-expression profiles reveled that only 35% of the protein spots were common to both developmental stages. Out of 94 2-DE protein spots (65 from conidia, 25 from mycelia and two common to both) analyzed using mass spectrometry, seven proteins from conidia, 15 from mycelia and one common to both stages were identified. The identified protein spots exclusive to conidia contained sequences similar to known fungal stress-protector proteins (such as heat shock proteins (HSP) and 6-phosphogluconate dehydrogenase) plus the fungal allergen Alt a 7, actin and the enzyme cobalamin-independent methionine synthase. The identified protein spots exclusive to mycelia included proteins involved in several cell housekeeping biological processes. Three proteins (HSP 90, 6-phosphogluconate dehydrogenase and allergen Alt a 7) were present in spots in conidial and mycelial gels, but they differed in their locations on the two gels.     

Proteomic Analysis of Interactions Between the Generalist Herbivore <Emphasis Type="Italic">Spodoptera exigua</Emphasis> (Lepidoptera: Noctuidae) and <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In this study, comparative proteomics was used to investigate the interaction of Spodoptera exigua and Arabidopsis thaliana. By using 2-D electrophoresis of differentially expressed proteins, combined with high-throughput matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF/TOF MS, the changes in the abundance of proteins induced by insect feeding were studied in A. thaliana. More than 1,100 protein spots were reproducibly detected on each gel. The intensities of 30 protein spots in particular changed significantly, showing differences in volume of at least twofold. Among these, 17 protein spots were upregulated, and 13 were downregulated following an 8-h insect feeding period. Nineteen insect-feeding-responsive proteins were identified, all of which were involved in metabolic regulation, binding functions or cofactor requirement of protein, cell rescue, and defense and virulence, as assessed by Munich Information Center for Protein Sequences function category. About 50% of these were involved in metabolism, including transketolase, S-adenosylmethionine synthase 3, 2,3-biphosphoglycerate-independent phosphoglycerate mutase, beta-ureidopropionase, GDP-d-mannose 3′,5′-epimerase, and fatty acid synthase. The identification of insect-feeding-responsive proteins on Arabidopsis provides not only new insights into insect stress but also a good start for further investigation of their functions. Understanding how the plant responses to insects in the proteomic level will provide tools for a better management of insect pest in the field.     

Proteomic analysis of two Trypanosoma cruzi zymodeme 3 strains

Two Trypanosoma cruzi Z3 strains, designated as 3663 and 4167, were previously isolated from insect vectors captured in the Brazilian Amazon region. These strains exhibited different infection patterns in Vero, C6/36, RAW 264.7 and HEp-2 cell lineages, in which 3663 trypomastigote form was much less infective than 4167 ones. A proteomic approach was applied to investigate the differences in the global patterns of protein expression in these two Z3 strains. Two-dimensional (2D) protein maps were generated and certain spots were identified by mass spectrometry (MS). Our analyses revealed a significant difference in the expression profile of different proteins between strains 3663 and 4167. Among them, cruzipain, an important regulator of infectivity. This data was corroborated by flow cytometry analysis using anti-cruzipain antibody. This difference could contribute to the infectivity profiles observed for each strain by in vitro assay using different cell lines.     

Cryptosporidium parvum: Radiation-induced alteration of the oocyst proteome

Cryptosporidium parvum is a waterborne protozoan parasite that is found intracellularly in host animals, including humans, and causes severe diarrhea, which can lead to the death of an immunocompromised individual. Previously, we found that this organism is highly radioresistant as it can productively infect mice after exposure to a 10-kGy dose of γ-radiation.To understand how C. parvum avoids radiation damage, we characterized its protein expression patterns 6, 24, and 48 h after a 10-kGy dose of γ-radiation using two-dimensional PAGE. The gels showed 10 silver-stained spots that increased or decreased in size following γ-irradiation. Five proteins contained in these spots were identified using MALDI-TOF MS peptide fingerprinting, and two of these showed an increase in expression after γ-irradiation. These proteins were identified by LC–MS/MS as proteasome subunit alpha type 4 (NTN hydrolase fold) and thioredoxin peroxidase-like protein. The roles of these two upregulated proteins as related to the radioresistance of C. parvum remain to be evaluated.     

Salinity-induced changes in protein expression in the halophytic plant Nitraria sphaerocarpa

Salinity is a major abiotic stress that inhibits plant growth and development. Plants have evolved complex adaptive mechanisms that respond to salinity stress. However, an understanding of how plants respond to salinity stress is far from being complete. In particular, how plants survive salinity stress via alterations to their intercellular metabolic networks and defense systems is largely unknown. To delineate the responses of Nitraria sphaerocarpa cell suspensions to salinity, changes in their protein expression patterns were characterized by a comparative proteomic approach. Cells that had been treated with 150 mM NaCl for 1, 3, 5, 7, or 9 days developed several stress-related phenotypes, including those affecting morphology and biochemical activities. Of ~1100 proteins detected in 2-DE gel patterns, 130 proteins showed differences in abundance with more than 1.5-fold when cells were stressed by salinity. All but one of these proteins was identified by MS and database searching. The 129 spots contained 111 different proteins, including those involved in signal transduction, cell rescue/defense, cytoskeleton and cell cycle, protein folding and assembly, which were the most significantly affected. Taken together, our results provide a foundation to understand the mechanism of salinity response.     

Proteomics of Arabidopsis redox proteins in response to methyl jasmonate

Protein redox regulation is increasingly recognized as an important switch of protein activity in yeast, bacteria, mammals and plants. In this study, we identified proteins with potential thiol switches involved in jasmonate signaling, which is essential for plant defense. Methyl jasmonate (MeJA) treatment led to enhanced production of hydrogen peroxide in Arabidopsis leaves and roots, indicating in vivo oxidative stress. With monobromobimane (mBBr) labeling to capture oxidized sulfhydryl groups and 2D gel separation, a total of 35 protein spots that displayed significant redox and/or total protein expression changes were isolated. Using LC–MS/MS, the proteins in 33 spots were identified in both control and MeJA-treated samples. By comparative analysis of mBBr and SyproRuby gel images, we were able to determine many proteins that were redox responsive and proteins that displayed abundance changes in response to MeJA. Interestingly, stress and defense proteins constitute a large group that responded to MeJA. In addition, many cysteine residues involved in the disulfide dynamics were mapped based on tandem MS data. Identification of redox proteins and their cysteine residues involved in the redox regulation allows for a deeper understanding of the jasmonate signaling networks.     

An efficient extraction method to enhance analysis of low abundant proteins from soybean seed

Large amounts of the major storage proteins, β-conglycinin and glycinin, in soybean (Glycine max) seeds hinder the isolation and characterization of less abundant seed proteins. We investigated whether isopropanol extraction could facilitate resolution of the low abundant proteins, different from the main storage protein fractions, in one-dimensional polyacrylamide gel electrophoresis (1D-PAGE) and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). 1D-PAGE of proteins extracted by different concentrations (10%, 20%, 30%, 40%, 50%, 60%, 70% and 80%) of isopropanol showed that greater than 30% isopropanol was suitable for preferential enrichment of low abundant proteins. Analysis of 2D-PAGE showed that proteins which were less abundant or absent by the conventional extraction procedure were clearly seen in the 40% isopropanol extracts. Increasing isopropanol concentration above 40% resulted in a decrease in the number of less abundant protein spots. We have identified a total of 107 protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrophotometry (MALDI-TOF-MS) and liquid chromatography-mass spectrometry (LC-MS/MS). Our results suggest that extraction of soybean seed powder with 40% isopropanol enriches lower abundance proteins and is a suitable method for 2D-PAGE separation and identification. This methodology could potentially allow the extraction and characterization of low abundant proteins of other legume seeds containing highly abundant storage proteins.     

Mermis nigrescens: physiological relationship with its host, the adult desert locust Schistocerca gregaria

Adult desert locusts were experimentally infected per os with 30, 50, or 60 Mermis nigrescens eggs, and changes in the host physiology were recorded. Larval nematodes were recovered from the hemocoel and counted at appropriate times after infection. The food consumption and blood volume of the host were unaffected by the parasitism, but the nematode significantly impaired the ability of male locusts to excrete the injected dye, amaranth, from their hemolymph. The total carbohydrate in the hemolymph of infected male and female locusts was severely depleted during the active growth period of the nematode and the possible utilization of these carbohydrates by the nematode are discussed. The total amino acid and protein levels in the blood of the host were unaffected by the nematode development, although concomittant changes in the levels of both these blood metabolites occurred in all locusts throughout the experimental period. However, although changes of this nature reflected the normal pattern of protein synthesis during oocyte development and oviposition in control locusts, the nematode suppressed oocyte development and caused oocyte resorption in the female host. The nematode did not significantly affect the level of total protein and amino acids in the flight muscles of male and female locusts, but a significant decrease in the level of fat body proteins and amino acids was recorded in infected hosts 16 and 21 days after infection. The possible effect of the nematode on protein metabolism by the host fat body is discussed in relation to the nutritional requirements of the nematode and involvement of the host endocrine system.