玉米T型细胞质雄性不育系与相应保持系线粒体蛋白质组中的差异蛋白

利用固相pH3—10梯度双向凝胶电泳．对玉米T型细胞质雄性不育系（T—CMS）及其相应保持系叶片（苗期、拔节期、孕穗期）,胚轴,胚根和花药（花粉母细胞减数分裂期、花粉粒小孢子单-双核期）线粒体蛋白质组中的差异蛋白进行分析。PDQuest2D图像软件分析表明,苗期和拔节期叶片约有150个蛋白质斑点,胚轴和胚根中可识别出150个线粒体蛋白质斑点,花药中约有100个斑点。利用MALDI—TOF—MS方法．运用MASCOT软件于NCBI进行数据查询．对T—CMS与相应保持系中存在的差异蛋白进行归属鉴定,在T—CMS中存在,保持系中缺失的线粒体蛋白质有：r40cl protein（胚轴中）,mature anther—specific protein,DNA—directed RNA polymerase 23kD subunit．hexokinaseⅡ和T—CMS中缺失而在保持系中存在的有：glutathione S—transferase．putative protein。其中T—CMS与相应保持系间．线粒体蛋白质组呈现出差异的组织有胚轴、胚根和小孢子单-双核期的花药。叶片的不同发育时期线粒体蛋白质组呈现明显变化．但T—CMS与保持系间没有差异。在小孢子单-双核期（花粉败育期）的花药中,T—CMS与保持系间线粒体蛋白质组出现明显差异,线粒体蛋白质组出现变异的时期与花粉败育时期相一致。     

Comparative analysis of young panicle proteome in thermo-sensitive genic male-sterile rice Zhu-1S under sterile and fertile conditions

Proteome analysis was carried out to identify the young panicle proteins during different developmental stages under sterile and fertile conditions. Based on spot quantity and quality, 50 protein spots were analyzed by matrix associated laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and 20 spots were identified. Most of these proteins are closely associated with energy metabolism, protein biosynthesis, cell wall formation and stress responses, which are essential cell activities to the pollen development. Gene expression analysis of three different proteins by semi-quantitative RT-PCR showed that the mRNA level was not correlated exactly with the protein level.     

Proteomic analysis of protein expression profiles during Caenorhabditis elegans development using two-dimensional difference gel electrophoresis

Coordinated protein expression is critical for the normal execution of animal development. To obtain overall proteome profiles during animal development, a small free-living soil nematode, Caenorhabditis elegans, was used as a model and the developmental changes of protein expressions were analyzed using two-dimensional difference gel electrophoresis. Protein samples from six developmental stages were prelabeled with fluorescent cyanine dyes and separated on two-dimensional electrophoresis gels. Image-to-image analysis of protein abundances together with protein identification by peptide mass fingerprinting yielded the developmental expression profiles of 231 spots representing 165 proteins. About a quarter of the identified proteins were expressed in multiple spots with different isoelectric points, suggesting a certain proportion of proteins were variously modified. This notion was supported by the observation that about a third of the multispot proteins were stained positive for a phosphoprotein specific dye. While a fairly large number of the proteins showed little alteration in their expression profiles during development, about 40 proteins were found to be significantly either up- or down-regulated between the embryos and newly hatched L1 larvae. Down-regulated proteins included those related to the cell cycle such as MCM-7, PCN-1, and the mitotic checkpoint protein, while up-regulated proteins included structural proteins such as actins, LEV-11, DIM-1, VAB-21, metabolic enzymes such as ATP synthase, ALH-12, fluctose-1,6-bisphosphate aldolase and GPD-3, and galectins. A standard proteome map was obtained where the defects in the mutations of developmental genes and the effects of reagents on the development in C. elegans were analyzed.     

温敏核不育水稻花药蛋白质组初步分析

采用固相pH梯度 SDS聚丙烯酰胺双向凝胶电泳对温敏核不育水稻 96 4 2S可育与不育条件下减数分裂期花药总蛋白进行了分离 ,通过银染显色 ,获得了分辨率和重复性较好的双向电泳图谱 .PDQuest 2DE图像分析软件可识别约 10 0 0个蛋白质点 .蛋白质点在 2D胶上的重复性为 :沿等电聚焦方向偏差为 1 4 5± 0 2 3mm(n =8) ,沿SDS PAGE方向偏差为 :1 15± 0 17mm(n =8) .对两种育性不同样品的 2D胶上部分共有的蛋白质点 ,采用基质辅助激光解析电离飞行时间质谱 (matrixassistedlaserdesorption ionizationtimeofflightmassspctrometry ,MALDI TOF MS)进行了肽质谱指纹图分析 .通过采用PeptIdent软件对SWISS PROT数据库的查询 ,有 5 0个蛋白质点在数据库得到归属鉴定 .对育性不同的2种样品 2D较上明显差异的蛋白质点进行了分析鉴定 .在不育变化为可育的过程中 ,明显表达上调的蛋白质点包括几丁质酶 ,酸性磷酸酶 ,胞浆激酶 ,谷蛋白前体 ,以及ESTSC72 61蛋白 ,明显下调的蛋白质包括β expansin前体 ,谷氨酸氨甲酰转移酶和 1种未知功能的蛋白质     

Proteome analysis on an early transformed human bronchial epithelial cell line,BEP2D,after alpha-particle irradiation

To probe the mechanism of carcinogenesis of lung cancer at the molecular level and to find potential protein markers involved in the early phase of tumorgenesis, differential proteome analysis on primary passage cell line R15H, and early transformed cell line R15H20 derived from (238)Pu alpha-particle irradiation of human papillomavirus (HPV) 18-immortalized human bronchial epithelial cell line (BEP2D), was carried out using two-dimensional electrophoresis (2-DE) and peptide mass fingerprinting (PMF) with matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. Image analysis and Student's t-test (p < 0.05) showed that three protein spots were only expressed in R15H, intensities of 43 protein spots on the gels were altered between R15H and R15H20. Two of the three spots that were only expressed in R15H were identified as high mobility group protein 1. Two proteins decreased in abundance in R15H20 were identified as maspin precursor, a tumor suppressor and aminoacylase-1. Ornithine aminotransferase and peptidyl-prolyl cis-trans isomerase A that were increased in R15H20, were also identified. Relationships between these differentially expressed proteins and the carcinogenesis mechanism of lung cancer are discussed. The protein expression profile of the R15H cell line was also constructed during the study as a reference map for further comparative proteome analysis of the irradiation induced BEP2D cell line. Of the 90 spots analyzed with PMF in the 2-DE gel of R15H cell line, 50 proteins were identified by searching the nonredundant protein database SWISS-PROT/TrEMBL.     

The cytosolic, cell surface and extracellular proteomes of the biotechnologically important soil bacterium Corynebacterium efficiens YS-314 in comparison to those of Corynebacterium glutamicum ATCC 13032

Reference maps of the cytosolic, cell surface and extracellular proteome fractions of the amino acid-producing soil bacterium Corynebacterium efficiens YS-314 were established. The analysis window covers a pI range from 3 to 7 along with a molecular mass range from 10 to 130 kDa. After second-dimensional separation on SDS-PAGE and Coomassie staining, computational analysis detected 635 protein spots in the cytosolic proteome fraction, whereas 76 and 102 spots were detected in the cell surface and extracellular proteomes, respectively. By means of MALDI-TOF-MS and tryptic peptide mass fingerprinting, 164 cytosolic proteins, 49 proteins of the cell surface and 89 extracellular protein spots were identified, representing in total 177 different proteins. Additionally, reference maps of the three cellular proteome fractions of the close phylogenetic relative Corynebacterium glutamicum ATCC 13032 were generated and used for comparative proteomics. Classification according to the Clusters of Orthologous Groups of proteins scheme and abundance analysis of the identified proteins revealed species-specific differences. The high abundance of molecular chaperones and amino acid biosynthesis enzymes in C. efficiens points to environmental adaptations of this recently discovered amino acid-producing bacterium.     

光周期诱导光敏感核不育水稻花药蛋白变化的研究

采用双向电脉技术对不同光周期条件下,光敏感核不育水稻（农垦５８ｓ）的可育与不育花药蛋白的变化进行分析,发现花粉发育的不同阶段中,不育花药具有四个特异蛋白ｐＩ６．２／ｂＭＷ７０ＫＤ,ｐＩ６．２／ＭＷ６８ＫＤ,ｐＩ６．２／ＭＷ３８ＫＤ和ｐＩ７．４／ＭＷ３７ＫＤ．对游离组蛋白的分析表明．长日照诱导的不育花药中游离组蛋白的相对百分率均明显低于短日照下的可育花药．据此推测长日照诱导不百花药蛋白质组成和代谢变化．不育花药中游离组蛋白含量低,可能受ＤＮＡ合成数量少的影响．     

A low molecular weight proteome comparison of fertile and male sterile 8 anthers of Zea mays

During maize anther development, somatic locular cells differentiate to support meiosis in the pollen mother cells. Meiosis is an important event during anther growth and is essential for plant fertility as pollen contains the haploid sperm. A subset of maize male sterile mutants exhibit meiotic failure, including ms8 (male sterile 8) in which meiocytes arrest as dyads and the locular somatic cells exhibit multiple defects. Systematic proteomic profiles were analysed in biological triplicates plus technical triplicates comparing ms8 anthers with fertile sibling samples at both the premeiotic and meiotic stages; proteins from 3.5 to 20 kDa were fractionated by 1‐D PAGE, cleaved with Lys‐C and then sequenced using a LTQ Orbitrap Velos MS paradigm. Three hundred and 59proteins were identified with two or more assigned peptides in which each of those peptides were counted at least two or more times (0.4% peptide false discovery rate (FDR) and 0.2% protein FDR); 2761 proteins were identified with one or more assigned peptides (0.4% peptide FDR and 7.6% protein FDR). Stage‐specific protein expression provides candidate stage markers for early anther development, and proteins specifically expressed in fertile compared to sterile anthers provide important clues about the regulation of meiosis. 49% of the proteins detected by this study are new to an independent whole anther proteome, and many small proteins missed by automated maize genome annotation were validated; these outcomes indicate the value of focusing on low molecular weight proteins. The roles of distinctive expressed proteins and methods for mass spectrometry of low molecular weight proteins are discussed.     

Proteomic analysis of the human pathogen Trypanosoma cruzi

Trypanosoma cruzi, the protozoan that causes Chagas disease, possesses a complex life cycle involving different developmental stages. Experimental conditions for two-dimensional electrophoresis (2-DE) analysis of T. cruzi trypomastigote, amastigote and epimastigote proteomes were optimized. Comparative proteome analysis of the cell-cycle stages were carried out, revealing that few proteins included in the 2-DE maps displayed significant differential expression among the three developmental forms of the parasite. In order to identify landmark proteins, spots from the trypomastigote 2-DE map were subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry peptide mass fingerprinting, resulting in 26 identifications that corresponded to 19 different proteins. Among the identified polypeptides, there were heat shock proteins (HSP; chaperones, HSP 60, HSP 70 and HSP 90), elongation factors, glycolytic pathway enzymes (enolase, pyruvate kinase and 2,3 bisphosphoglycerate mutase) and structural proteins (KMP 11, tubulin and paraflagellar rod components). The relative expression of the identified proteins in the 2-DE maps of the T. cruzi developmental stages is also presented.     

Proteome reference maps of Medicago truncatula embryogenic cell cultures generated from single protoplasts

Using a combination of two-dimensional gel electrophoresis (2-DE) protein mapping and mass spectrometry (MS) analysis, we have established proteome reference maps of Medicago truncatula embryogenic tissue culture cells. The cultures were generated from single protoplasts, which provided a relatively homogeneous cell population. We used these to analyze protein expression at the globular stages of somatic embryogenesis, which is the earliest morphogenetic embryonic stage. Over 3000 proteins could reproducibly be resolved over a pI range of 4-11. Three hundred and twelve protein spots were extracted from colloidal Coomassie Blue-stained 2-DE gels and analyzed by matrix-assisted laser desorption/ionization-time of flight MS analysis and tandem MS sequencing. This enabled the identification of 169 protein spots representing 128 unique gene products using a publicly available expressed sequence tag database and the MASCOT search engine. These reference maps will be valuable for the investigation of the molecular events which occur during somatic embryogenesis in M. truncatula. The proteome reference maps and supplementary materials will be available and updated for public access at http://semele.anu.edu.au/.     

Mapping and comprehensive analysis of the extracellular and cell surface proteome of the human pathogen Corynebacterium diphtheriae

Secreted proteins of the human pathogen Corynebacterium diphtheriae might be involved in important pathogen-host cell interactions. Here, we present the first systematic reference map of the extracellular and cell surface proteome fractions of the type strain C. diphtheriae C7s(-)tox-. The analysis window of 2-DE covered the pI range from 3 to 10 along with a MW range from 8 to 150 kDa. Computational analysis of the 2-D gels detected almost 150 protein spots in the extracellular proteome fraction and about 80 protein spots of the cell surface proteome. MALDI-TOF-MS and PMF with trypsin unambiguously identified 107 extracellular protein spots and 53 protein spots of the cell surface, representing in total 85 different proteins of C. diphtheriae C7s(-)tox-. Several of the identified proteins are encoded by pathogenicity islands and might represent virulence factors of C. diphtheriae. Additionally, four solute-binding proteins (HmuT, Irp6A, CiuA, and FrgD) of different iron ABC transporters were identified, with the hitherto uncharacterized FrgD protein being the most abundant one of the cell surface proteome of C. diphtheriae C7s(-)tox-.     

HUPO BPP pilot study: a proteomics analysis of the mouse brain of different developmental stages

This study is a part of the HUPO Brain Proteome Project (BPP) pilot study, which aims at obtaining a reliable database of mouse brain proteome, at the comparison of techniques, laboratories, and approaches as well as at preparing subsequent proteome studies of neurologic diseases. The C57/Bl6 mouse brains of three developmental stages at embryonic day 16 (E16), postnatal day 7 (P7), and 8 wk (P56) (n = 5 in each group) were provided by the HUPO BPP executive committee. The whole brain proteins of each animal were individually prepared using 2-DE coupled with PDQuest software analysis. The protein spots representing developmentally related or stably expressed proteins were then prepared with in-gel digestion followed with MALDI-TOF/TOF MS/MS and analyzed using the MASCOT search engines to search the Swiss-Prot or NCBInr database. The 2-DE gel maps of the mouse brains of all of the developmental stages were obtained and submitted to the Data Collection Centre (DCC). The proteins alpha-enolase, stathmin, actin, C14orf166 homolog, 28,000 kDa heat- and acid-stable phosphoprotein, 3-mercaptopyruvate sulfurtransferase and 40 S ribosomal protein S3a were successfully identified. A further Western blotting analysis demonstrated that enolase is a protein up-regulated in the mouse brain from embryonic stage to adult stage. These data are helpful for understanding the proteome changes in the development of the mouse brain.     

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.     

Distribution of insoluble polysaccharides, neutral lipids, and proteins in the developing anthers of Campsis radicans (L.) Seem. (Bignoniaceae)

In this study, distribution of polysaccharides, lipids, and proteins in the developing anthers of Campsis radicans (L.) Seem. was examined from sporogenous cell stage to mature pollen, using cytochemical methods. To detect the distribution and dynamic changes of insoluble polysaccharides, lipid bodies, and proteins in the anthers through progressive developmental stages, semi-thin sections of anthers at different developmental stages were stained with periodic-acid-Schiff (PAS) reagent, Sudan black B, and Coomassie brilliant blue, respectively, and examined under light microscope. Ultrastructural observations with TEM were also carried out to determine the storage form of starch in the connective tissue, and storage form of lipids in the tapetal cells. In sporogenous cell stage, anther wall contains numerous insoluble polysaccharides. However, from the sporogenous cell stage to the vacuolated microspore stage, the amount of insoluble polysaccharides in the anther wall decreases gradually. At bicellular pollen stage, tapetum degenerates completely and polysaccharides are not seen in the anther wall. Lipid bodies are observed in the cytoplasm of both middle layer and tapetal cells at tetrad stage, whereas they disappear in the vacuolated microspore stage. Compared with polysaccharides, proteins are limited in the anther wall at early stages of development. During pollen development, polysaccharides, proteins, and lipid bodies are scarce in the cytoplasm of sporogenous cells, but their amount increases at premeiotic stage. From tetrad stage to bicellular pollen stage, microspore cytoplasm contains variable amount of insoluble polysaccharide grains, lipid and protein bodies. At bicellular pollen stage, plentiful amount of starch granules are stored in the cytoplasm of the pollen grains. Proteins and lipid bodies are also present in the cytoplasm.     

红莲型水稻细胞质雄性不育花药蛋白质组学初步分析

采用固相pH梯度－SDS PAGE 双向电泳对红莲型细胞质雄性不育水稻(YTA)的不育系和保持系(YTB)单核期花粉总蛋白质进行了分离,通过银染显色,获得了分辨率和重复性较好的双向电泳图谱。Image Master 2D V5.0 软件可识别约1800个蛋白质点,其中差异表达的蛋白质点数为85。将其中16个差异点采用基质辅助激光解析电离飞行时间质谱（matrix assisted laser desorption/ionizaton time of flight mass spectrometry, MALDI-TOF-MS）进行了肽质指纹图分析,通过采用Mascot 软件对MSDB数据库查询,其中9个蛋白质点得到了鉴定。YTA相对于YTB有部分参与碳代谢和淀粉合成的酶缺失或表达量降低,这些蛋白质分别是ADP-葡萄糖磷酸转移酶（AGPase）,UDP-葡萄糖醛酸脱羧酶,乙酰辅酶A合成酶和二氢硫辛酸脱氢酶等。其中AGPase是参与淀粉合成的蛋白,与花粉发育密切相关。乙酰辅酶A合成酶和二氢硫辛酸脱氢酶是细胞内合成乙酰辅酶A的重要酶,而乙酰辅酶A是进入TCA循环的重要底物,乙酰辅酶A的缺乏可以导致TCA循环不能顺利进行,从而不能提供小孢子发育所需要的大量能量。YTA相对于YTB部分参与碳水化合物代谢的重要酶缺失或表达量降低,有可能导致因线粒体提供的能量不足,淀粉合成受阻,因而花粉不能正常发育。      

Comparative analyses of the proteomes of leaves and flowers at various stages of development reveal organ‐specific functional differentiation of proteins in soybean

The functional differentiation of protein networks in individual organs and tissues of soybean at various developmental stages was investigated by proteomic approach. Protein extraction by Mg/NP‐40 buffer followed by alkaline phenol‐based method was optimized for proteomic analysis. Proteome analyses of leaves at various developmental stages showed 26 differentially expressed proteins, wherein proteins in translocon at the outer/inner envelope membrane of chloroplast protein‐transport machineries increased significantly at the first trifoliate. Immunoblot analysis showed chaperonin‐60 expressed abundantly in young leaves, whereas HSP 70 and ATP‐synthase β were constitutively expressed in all tissues. The net photosynthesis rate and chlorophyll content showed an age‐dependent correlation in leaves. These results suggest that proteins involved in carbon assimilation, folding and assembly, and energy may work synchronously and show a linear correlation to photosynthesis at developmental stages of leaves. Comparison of flower bud and flower proteome reveals 29 differentially expressed proteins, wherein proteins involved in mitochondrial protein transport and assembly, secondary metabolism, and pollen‐tube growth were up‐regulated during flower development. Together, these results suggest that during developmental stages, each type of tissue is associated with a specific group of proteins; wherein proteins involved in energy, sugar metabolism, and folding, assembly, and destination may play pivotal roles in the maturation process of each organ or tissue.     

Complexity of the human whole saliva proteome

Recent characterization of the whole saliva proteome led to contradictory pictures concerning the complexity of its proteome. In this work, 110 proteins were analysed by mass spectrometry allowing the identification of 10 accessions previously not detected on protein two-dimensional maps, including myosin heavy chain (fast skeletal muscle, IIA and IIB), phosphatidylethanolamine binding protein, secretory actin-binding protein precursor and triosephosphate isomerase. Further comparison with available data demonstrated simultaneously a low diversity in terms of variety of accessions and a high complexity in terms of number of protein spots identifying the same accession, the two thirds of identified spots corresponding to amylases, cystatins and immunoglobulins. This diversity may be of interest in the development of non invasive diagnostic tool for several disease.