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 human skeletal muscle (<Emphasis Type="Italic">m. vastus lateralis</Emphasis>) proteins: Identification of 89 gene expression products

Proteins from bioptates and autoptates of human skeletal muscle m. vastus lateralis were separated by O’Farrell two-dimensional gel electrophoresis (2DE). MALDI-TOF MS and MS/MS enabled identification of 89 protein spots as expression products of 55 genes. A modification of the O’Farrell’s method including non-equilibrium electrophoresis in a pH gradient allowed detection — among major sarcomeric, mitochondrial, and cytosolic proteins — of several proteins, such as PDZ- and LIM domain-containing ones (pI > 8.70), fragments of known proteins, and a stable complex of heavy and light ferritin chains. The data underlie further studies of human skeletal muscle proteins in terms of molecular mechanisms of some physiological and pathological processes.     

Stress proteins in the cytoplasmic membrane fraction of <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Stress proteomes of the cytoplasmic membrane fraction of Bacillus subtilis trp _C2-exposed to acid pH and ethanol were characterized. Although these stress factors impair the cell function in a specific manner, they share the ability to denature proteins. Therefore, specific and general stress proteins in the membranes were investigated. Both ethanol (3 %) and pH 5.0 increase the doubling time from 17 to 25 min. Isolated cytoplasmic membranes were subjected to an optimized 2D PAGE analysis which permitted the separation and analysis of ≈450 distinct protein spots. Two alternative methods of protein detection were compared, i.e. silver staining and ³⁵S-l-methionine pulse labeling; the stress induced proteins were identified by MALDI-TOF MS. After ethanol stress, five proteins were increased, viz. YdaP, Ctc, YfhM, YjcH and YwaC. Acid stress proteins were AcoB, YkwC, SodA, YjcH and YwaC. Proteins YjcH and YwaC were increased after ethanol as well as acid pH treatment.     

A proteomic approach analysing the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> response to virulent and avirulent <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> strains

The present work is directed at studying changes at the proteome level in Arabidopsis thaliana leaves in response to Pseudomonas syringae virulent (Pst) and avirulent (Pst avrRpt2) strains. Arabidopsis leaves were sampled from challenged plants at 4, 8 and 24 h post inoculation. Proteins were TCA–acetone–phenol extracted and subjected to 2-DE (5–8 pH range) and MS/MS (MALDI–TOF–TOF) analysis. Out of 800 matched spots on each of the 36 gels analysed, 147 spots were either absent in at least one of the conditions studied (time or treatments; qualitative variable spots) or differentially accumulated between time and treatments (quantitative variable spots). Out of the 24 proteins successfully identified over TAIR10 database, 23 have not been reported previously in similar proteomics studies of the Arabidopsis thaliana–Pseudomonas syringae interaction. The exhaustive statistical analysis performed, including principal component and heat map, showed that 24 h post inoculation can clearly discriminate the challenged plants from the control. The protein change occurred early (4 h post inoculation) following the virulent pathogen infection, whereas the change occurred later (24 h post inoculation) following the avirulent pathogen inoculation. Concerning the variable proteins, three behavioural groups can be observed: group 1 (common protein changes in response to virulent and avirulent pathogen infection), group 2 (protein changes in response to virulent pathogen infection) and group 3 (protein changes in response to avirulent pathogen infection). Differential identified proteins following the pathogen infection belonged to different groups including those of oxidative stress defence, enzymes of metabolic pathways and molecular chaperones.     

Analysis of Protein Expression Profiles of <Emphasis Type="Italic">Halobacillus dabanensis</Emphasis> D-8<Superscript>T</Superscript> Under Optimal and High Salinity Conditions

Two-dimensional (2-D) gel electrophoresis was employed to display the expression profiles of proteins of Halobacillus dabanensis D-8^T under 1%, 10%, and 20% salinities. Approximately 700 protein spots could be detected in the 2-D gels by Imagemaster™ 2D Platinum software. The molecular masses of the majority of intracellular proteins were distributed in the range of 17.5 kDa–66 kDa and isoelectric points of 4.0–5.9. In total 133 protein spots were observed with a changed expression level under different salinity conditions. Sixty-two protein spots showed upregulation and 26 new protein spots were found under high salinity conditions, while 25 protein spots were downregulated and 20 spots disappeared. Twenty-seven proteins with a markedly changed expression in hypersaline environments were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF/MS) and MASCOT. A changed expression pattern was observed for proteins related to energy-producing pathways, stress regulators, and proteins involved in the survival of strain D-8^T under high salt challenges. Many proteins play necessary roles in the adaptation to high salt or as a general stress protein, and some proteins are salt-stressed specific proteins that improve the capability of salt-tolerance of strain D-8^T growth under extremely hypersaline condition.     

Proteomic analysis of endogenous nitrotryptophan-containing proteins in rat hippocampus and cerebellum

Nitration of tryptophan residues is a novel post-translational modification. In the present study, we examined whether NO₂Trp (nitrotryptophan)-containing proteins are produced in the hippocampus and cerebellum of the adult rat under physiological conditions in vivo. Using Western blot analysis with anti-6-NO₂Trp-specific antibody, we found many similar immunoreactive spots in the protein extracts from both regions. These spots were subsequently subjected to trypsin digestion and LC-ESI-MS/MS (LC-electrospray ionization-tandem MS) analysis. We identified several cytoskeletal proteins and glycolytic enzymes as NO₂Trp-containing proteins and determined the position of nitrated tryptophan residues with significant ion score levels (P<0.05) in several proteins in both regions. We also observed that the total amount of NO₂Trp-containing proteins in the cerebellum was significantly greater than that in the hippocampus (P<0.05). Moreover, IP (immunoprecipitation) assays using anti-aldolase C antibody showed that the relative intensity of immunostaining for NO₂Trp over aldolase C was much higher in cerebellum than in hippocampus. The amounts of nNOS (neuronal nitric oxide synthase) and eNOS (endothelial nitric oxide synthase) were much greater in cerebellum than in hippocampus. This is the first evidence of several specific sites of nitrated tryptophan in proteins under physiological conditions in vivo.     

Proteomic profiling of cerebrospinal fluid identifies prostaglandin D2 synthase as a putative biomarker for pediatric medulloblastoma: A pediatric brain tumor consortium study

The aims of this study were to demonstrate the feasibility of centrally collecting and processing high-quality cerebrospinal fluid (CSF) samples for proteomic studies within a multi-center consortium and to identify putative biomarkers for medulloblastoma in CSF. We used 2-DE to investigate the CSF proteome from 33 children with medulloblastoma and compared it against the CSF proteome from 25 age-matched controls. Protein spots were subsequently identified by a combination of in-gel tryptic digestion and MALDI-TOF TOF MS analysis. On average, 160 protein spots were detected by 2-DE and 76 protein spots corresponding to 25 unique proteins were identified using MALDI-TOF. Levels of prostaglandin D2 synthase (PGD2S) were found to be six-fold decreased in the tumor samples versus control samples (p<0.00001). These data were further validated using ELISA. Close examination of PGD2S spots revealed the presence of complex sialylated carbohydrates at residues Asn(78) and Asn(87) . Total PGD2S levels are reduced six-fold in the CSF of children with medulloblastoma most likely representing a host response to the presence of the tumor. In addition, our results demonstrate the feasibility of performing proteomic studies on CSF samples collected from patients at multiple institutions within the consortium setting.     

Proteome analysis of <Emphasis Type="Italic">Aspergillus ochraceus</Emphasis>

Genome sequencing for many important fungi has begun during recent years; however, there is still some deficiency in proteome profiling of aspergilli. To obtain a comprehensive overview of proteins and their expression, a proteomic approach based on 2D gel electrophoresis and MALDI-TOF/TOF mass spectrometry was used to investigate A. ochraceus. The cell walls of fungi are exceptionally resistant to destruction, therefore two lysis protocols were tested: (1) lysis via manual grinding using liquid nitrogen, and (2) mechanical lysis via rapid agitation with glass beads using MagNalyser. Mechanical grinding with mortar and pestle using liquid nitrogen was found to be a more efficient extraction method for our purpose, resulting in extracts with higher protein content and a clear band pattern in SDS-PAGE. Two-dimensional electrophoresis gave a complex spot pattern comprising proteins of a broad range of isoelectric points and molecular masses. The most abundant spots were subjected to mass spectrometric analysis. We could identify 31 spots representing 26 proteins, most of them involved in metabolic processes and response to stress. Seventeen spots were identified by de novo sequencing due to a lack of DNA and protein database sequences of A. ochraceus. The proteins identified in our study have been reported for the first time in A. ochraceus and this represents the first proteomic approach with identification of major proteins, when the fungus was grown under submerged culture.     

Alteration of DBP Levels in CSF of Patients with MS by Proteomics Analysis

Objective The diagnosis of multiple sclerosis (MS) is still challenging recently due to the lack of a specific diagnostic test. Proteomics analysis was applied to biomarkers discovery and their pathways study. Methods First, the proteins of CSF from MS patients and control group were analyzed individually with 2D-DIGE technology (two-dimensional difference gel electrophoresis). Then, protein spots were found out with DeCyder6.0 software which showed different expression levels in the gel images between the two groups. The information regarding these proteins was collected based on MALDI-TOF/MS and related database searches. Lastly, interaction between these proteins was further analyzed by using Metacore software. Results There were 13 proteins that showed more than 1.5-fold difference in expression levels between the two groups. Furthermore, the identification made by MALDI-TOF/MS revealed that one of the most significant differential proteins was DBP (vitamin D-binding protein), which decreased in the experimental group. This result was confirmed by ELISA (P < 0.01). Moreover, network between the 13 proteins were partially got, which showed some biological interactions. Conclusion These results support a correlation between the level of DBP and MS. DBP may be a potential useful biomarker for diagnosis or a medicine target for treatment of MS.     

A proteomics approach for the analysis of hemolymph proteins involved in the immediate defense response of the soft tick, <Emphasis Type="Italic">Ornithodoros savignyi</Emphasis>, when challenged with <Emphasis Type="Italic">Candida albicans</Emphasis>

A proteomics approach was employed to identify proteins secreted into the hemolymph of Ornithodorus savignyi ticks 2 h after immune-challenge with the yeast, Candida albicans. Profiling of the proteins present in hemolymph of unchallenged ticks versus ticks challenged with heat-killed yeast revealed five proteins to be differentially expressed. The modulated protein spots were subjected to tandem mass spectrometry (MS/MS) analysis, but could not be positively identified. These proteins can be assigned to the immune response as they were not induced after aseptic injury. In an attempt to identify hemolymph proteins that recognize and bind to yeast cells, hemolymph obtained from both unchallenged and challenged ticks was incubated with C. albicans. Elution of the bound proteins followed by SDS–PAGE analysis indicated that three proteins (97, 88 and 26 kDa) present in both unchallenged and challenged hemolymph samples bind to yeast cells. The constant presence of these three proteins in tick hemolymph leads us to believe that they may be involved in non-self recognition and participate in yeast clearance from tick plasma. The analyzed yeast-binding proteins could also not be positively identified, suggesting that all the tick immune proteins investigated in this study are novel.     

Proteome analysis of the medicinal plant <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

A proteomic approach is undertaken aiming at the identification of novel proteins involved in the alkaloid biosynthesis of Catharanthus roseus. The C. roseus cell suspension culture A11 accumulates the terpenoid indole alkaloids strictosidine, ajmalicine and vindolinine. Cells were grown for 21 days, and alkaloid accumulation was monitored during this period. After a rapid increase between day 3 and day 6, the alkaloid content reached a maximum on day 16. Systematic analysis of the proteome was performed by two-dimensional polyacrylamide gel electrophoresis. After day 3, the proteome started to change with an increasing number of protein spots. On day 13, the proteome changed back to roughly the same as at the start of the growth cycle. 88 protein spots were selected for identification by mass spectrometry (MALDI-MS/MS). Of these, 58 were identified, including two isoforms of strictosidine synthase (EC 4.3.3.2), which catalyzes the formation of strictosidine in the alkaloid biosynthesis; tryptophan synthase (EC 4.1.1.28), which is needed for the supply of the alkaloid precursor tryptamine; 12-oxophytodienoate reductase, which is indirectly involved in the alkaloid biosynthesis as it catalyzes the last step in the biosynthesis of the regulator jasmonic acid. Unique sequences were found, which may also relate to unidentified biosynthetic proteins.     

Comparative proteomic analysis of apomictic monosomic addition line of <Emphasis Type="Italic">Beta corolliflora</Emphasis> and <Emphasis Type="Italic">Beta vulgaris</Emphasis> L. in sugar beet

Apomixis refers to a process in which plants produce seed without fertilization through female syngamy that produces embryos genetically identical to the maternal parent. In sugar beet, interspecific hybrids between diploid Beta vulgaris and tetraploid Beta corolliflora were established and monosomic addition line M14 was selected because of the apomictic phenotype. By using two-dimensional electrophoresis gels we identified the proteins which were differently expressed between the M14 and B. vulgaris. A total of 27 protein spots which varied expressed between lines were isolated and successfully identified with MALDI-TOF MS. Among them five protein spots were found to be only presented in M14 and two protein spots only expressed in Beta. According to their functional annotations described in Swissprot database, these proteins were, respectively, involved in important biological pathways, such as cell division, functionally classified using the KEGG functional classification system. The result may be useful for us to better understand the genetic mechanism of apomixes.     

Protoplast Preparation from <Emphasis Type="Italic">Laminaria japonica</Emphasis> with Recombinant Alginate Lyase and Cellulase

Functional recombinant abalone alginate lyase (rHdAly) and β-1,4-endoglucanase (rHdEG66) were expressed as secreted proteins with baculoviral expression systems. The specific activity of each recombinant enzyme, 2,490 and 18.2 U/mg for rHdAly and rHdEG66, respectively, was comparable to its native form at 30°C. Purified rHdAly and rHdEG66 showed the highest specific activity both at 35°C and optimum pH 8.7 and 5.9, respectively. These properties were also comparable to those of the native enzymes. Protoplast isolation was attempted from Laminaria japonica using both rHdAly and rHdEG66. When L. japonica blades were incubated in artificial seawater containing rHdAly and rHdEG66, very low numbers of protoplasts (<1 × 10³ protoplasts/g fresh weight) resulted. However, using blades pretreated with proteinase K, the protoplast was increased up to 5 × 10⁶ protoplasts/g fresh weight. Since the average diameter of isolated protoplasts was 11.6 μm, these cells were mostly derived from the epidermal layer rather than the cortical layer. Our results suggest that at least three enzymes, alginate lyase, cellulase, and protease, are essential for effective protoplast isolation from L. japonica. The protoplast isolation method in this study is more useful than earlier methods because it preferentially yielded protoplasts of the epidermal layer, which are known to be able to be regenerated.     

Purification and characterization of arsenite oxidase from Arthrobacter sp.

The chemolithoautotroph, Arthrobacter sp.15b oxidizes arsenite to arsenate using a membrane bound arsenite oxidase. The enzyme arsenite oxidase is purified to its homogeneity and identified using MALDI-TOF MS analysis. Upon further characterization, it was observed that the enzyme is a heterodimer showing native molecular mass as ~100 kDa and appeared as two subunits of ~85 kDa LSU and 14 kDa SSU on SDS–PAGE. The V _max and K _m values of the enzyme was found to be 2.45 μM (AsIII)/min/mg) and 26 μM, respectively. The purified enzyme could withstand wide range of pH and temperature changes. The enzyme, however, gets deactivated in the presence of 1 mM of DEPC suggesting the involvement of histidine at the binding site of the enzyme. The peptide analysis of large sub unit of the enzyme showed close match with the arsenite oxidases of Burkholderia sp. YI019A and arsenite oxidase, Mo-pterin containing subunit of Alcaligenes faecalis. The small subunit, however, differed from other arsenite oxidases and matched only with 2Fe–2S binding protein of Anaplasma phagocytophilum. This indicates that Rieske subunits containing the iron–sulfur clusters present in the large as well as small subunits of the enzyme are integral part of the protein.     

Mapping and proteomic analysis of albumin and globulin proteins in hexaploid wheat kernels (Triticum aestivum L.)

Albumins and globulins of wheat endosperm represent 20% of total kernel protein. They are soluble proteins, mainly enzymes and proteins involved in cell functions. Two-dimensional gel immobiline electrophoresis (2DE) (pH 4-7) × SDS-Page revealed around 2,250 spots. Ninety percent of the spots were common between the very distantly related cultivars ‘Opata 85’ and ‘Synthetic W7984’, the two parents of the International Triticeae Mapping Initiative (ITMI) progeny. ‘Opata’ had 130 specific spots while ‘Synthetic’ had 96. 2DE and image analysis of the soluble proteins present in 112 recombinant inbred lines of the F9-mapped ITMI progeny enabled 120 unbiased segregating spots to be mapped on 21 wheat (Triticum aestivum L. em. Thell) chromosomes. After trypsic digestion, mapped spots were subjected to MALDI-Tof or tandem mass spectrometry for protein identification by database mining. Among the ‘Opata’ and ‘Synthetic’ spots identified, many enzymes have already been mapped in the barley and rice genomes. Multigene families of Heat Shock Proteins, beta-amylases, UDP-glucose pyrophosphorylases, peroxydases and thioredoxins were successfully identified. Although other proteins remain to be identified, some differences were found in the number of segregating proteins involved in response to stress: 11 proteins found in the modern selected cultivar ‘Opata 85’ as compared to 4 in the new hexaploid `Synthetic W7984’. In addition, ‘Opata’ and ‘Synthetic’ differed in the number of proteins involved in protein folding (2 and 10, respectively). The usefulness of the mapped enzymes for future research on seed composition and characteristics is discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Protein profile analysis of salt-responsive proteins in leaves and roots in two cultivars of creeping bentgrass differing in salinity tolerance

Knowledge of stress-responsive proteins is critical for further understanding the molecular mechanisms of stress tolerance. The objectives of this study were to establish a proteomic map for a perennial grass species, creeping bentgrass (A. stolonifera L.), and to identify differentially expressed, salt-responsive proteins in two cultivars differing in salinity tolerance. Plants of two cultivars (‘Penncross’ and ‘Penn-A4’) were irrigated daily with water (control) or NaCl solution to induce salinity stress in a growth chamber. Salinity stress was obtained by adding NaCl solution of 2, 4, 6, and 8 dS m⁻¹ in the soil daily for 2-day intervals at each concentration, and then by watering soil with 10 dS m⁻¹ solution daily for 28 days. For proteomic map, using two-dimensional electrophoresis (2-DE), approximately 420 and 300 protein spots were detected in leaves and roots, respectively. A total of 148 leaf protein spots and 40 root protein spots were excised from the 2-DE gels and subjected to mass spectrometry analysis. In total, 106 leaf protein spots and 24 root protein spots were successfully identified. Leaves had more salt-responsive proteins than roots in both cultivars. The superior salt tolerance in ‘Penn-A4’, indicated by shoot extension rate, relative water content, and cell membrane stability during the 28-day salinity stress could be mainly associated with its higher level of vacuolar H⁺-ATPase in roots and UDP-sulfoquinovose synthase, methionine synthase, and glucan exohydrolase in leaves, as well as increased accumulation of catalase and glutathione S-transferase in leaves. Our results suggest that salinity tolerance in creeping bentgrass could be in part controlled by an alteration of ion transport through vacuolar H⁺-ATPase in roots, maintenance of the functionality and integrity of thylakoid membranes, sustained polyamine biosynthesis, and by the activation of cell wall loosening proteins and antioxidant defense mechanisms.     

Mitochondrial DNA sequence variation of four <Emphasis Type="Italic">Saccharina</Emphasis> species (Laminariales,Phaeophyceae) growing in Japan

The complete mitochondrial DNA (mtDNA) was sequenced for four Saccharina species including three varieties of Saccharina japonica in Hokkaido: S. japonica; S. japonica var. religiosa; S. japonica var. ochotensis; S. japonica var. diabolica; Saccharina longipedalis; Saccharina angustata; and Saccharina coriacea. Furthermore, the structure and the sequence were compared among them. The total nucleotide length was 37,500–37,657 bp. All mtDNAs were mapped and no differences in the organization of the coding region were found. From the total alignment of S. japonica including the three varieties and S. longipedalis, nucleotide substitutions were detected at 68 sites, and a nucleotide insertion/deletion was detected at one site on rps19 for the whole genome. Variable regions useful for varieties distinction of S. japonica were trnI, trnM, rps19, ORF41, and seven spacers. The pairwise distance between S. japonica, S. angustata, and S. coraiacea was 0.000–0.116. Sequencing comparisons of rps7, rps11, rpl5, and tatC were thought to be useful tools for discrimination and phylogenetic analysis of Saccharina species having similar morphologies.     

Proteome expression patterns in the stress tolerant evergreen <Emphasis Type="Italic">Ammopiptanthus nanus</Emphasis> under conditions of extreme cold

Low temperature is one of the important environmental changes that affect plant growth. The cold resistance capabilities of evergreen plants are the result of long-term adaptation to extreme environmental conditions. To investigate the responses of Ammopiptanthus nanus, a rare stress-tolerant evergreen plant, to extreme cold stress, we analyzed the proteome expression patterns of stressed plants; this is the first study to report these patterns for A. nanus. We collected adult A. nanus leaves under two conditions of cold stress: extreme cold (−29°C) and relatively less extreme cold (−5°C). Total crude proteins were extracted from leaf blades, separated by two-dimensional gel electrophoresis, and stained with Coomassie brilliant blue. Of the 500 protein spots detected in each of the samples, eight of the spots that exhibited clear changes under the different conditions were identified by MALDI-TOF analyses. Our results suggest that cold stress-related proteins may play diverse roles in the resistance to multiple environmental stresses.