Proteome analysis of the silkworm (Bombyx mori. L) colleterial gland during different development stages

The silkworm, Bombyx mori, colleterial gland developed very slowly until 2 days before emergence, then markedly enlarged due to the accumulation of a glue-like substances (mainly including 85% water and 11% proteins). However, the No glue (Ng) mutant female moth secreted only very little glue-like substance and laid loose eggs naturally. High-resolution two-dimensional polyacrylamide gel electrophoresis, followed by computer-assisted analysis, was used to screen the secretory region of colleterial gland protein patterns during different development stages to find quantitative and qualitative difference in protein expression during the pupae and moth stages. More than 700 protein spots were resolved in different developmental stages from the secretory region of the glands and most of the proteins were distributed in the mass range from 30 to 70 kD with pH 4-8. Through comparison and analysis, it was found that 3 proteins were only expressed in the later pupae stage (one or two days before emergence) and moth stage. Furthermore, these proteins were not expressed in the Ng mutant especially actin. There was a great variation of some protein expression volume during the development. Protein spots that changed more than 1.5-fold in expression level (relative to day 9), including 6 spots that were down-regulated and 2 spots that were up-regulated in expression were excised for identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Results indicated that actins that participated or regulated the exocytosis of colleterial gland and other differentially expressed proteins might be related to colleterial gland development or the secretion of a glue-like substance.     

Proteomic analysis of somatic embryogenesis in Medicago truncatula. Explant cultures grown under 6-benzylaminopurine and 1-naphthaleneacetic acid treatments

The Medicago truncatula line 2HA has a 500-fold greater capacity to regenerate plants in culture by somatic embryogenesis than wild-type Jemalong. We have compared proteomes of tissue cultures from leaf explants of these two lines. Both 2HA and Jemalong explants were grown on media containing the auxin 1-naphthaleneacetic acid and the cytokinin 6-benzylaminopurine. Proteins were extracted from the cultures at different time points (2, 5, and 8 weeks), separated by two-dimensional gel electrophoresis, and detected by silver staining. More than 2,000 proteins could be reproducibly resolved and detected on each gel. Statistical analysis showed that 54 protein spots were significantly (P < 0.05) changed in expression (accumulation) during the 8 weeks of culture, and most of these spots were extracted from colloidal Coomassie-stained two-dimensional gel electrophoresis gels and were subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry or liquid chromatography-tandem mass spectrometry analysis. Using a publicly available expressed sequence tag database and the Mascot search engine, we were able to identify 16 differentially expressed proteins. More than 60% of the differentially expressed protein spots had very different patterns of gene expression between 2HA and Jemalong during the 8 weeks of culture.     

Proteome analysis of Escherichia coli K-12 by two-dimensional native-state chromatography and MALDI-MS

To identify proteins expressed in Escherichia coli K-12 MG1655 during exponential growth in defined medium, we separated soluble proteins of E. coli over two dimensions of native-state high-performance liquid chromatography, and examined the components of the protein mixtures in each of 380 fractions by peptide mass fingerprinting. To date, we have identified the products of 310 genes covering a wide range of cellular functions. Validation of protein assignments was made by comparing the assignments of proteins to specific first-dimension fractions to proteins visualized by two-dimensional gel electrophoresis. Co-fractionation of proteins suggests the possible identities of components of multiprotein complexes. This approach yields high-throughput gel-independent identification of proteins. It can also be used to assign identities to spots visualized by two-dimensional gels, and should be useful to evaluate differences in expressed proteome content and protein complexes among strains or between different physiological states.     

Proteome analysis of wheat leaf under salt stress by two-dimensional difference gel electrophoresis (2D-DIGE)

Salt stress is a major abiotic stress that limits agricultural productivity in many regions of the world. To understand the molecular basis of the salt stress response in wheat (Triticum aestivum L.), a proteomic approach was used to identify the salt stress-responsive proteins in an elite Chinese wheat cultivar, Zhengmai 9023, which exhibits a high yield, superior gluten quality and better biotic resistance. Three-week-old seedlings were treated with NaCl of four different concentrations (1.0%, 1.5%, 2.0%, and 2.5%). The total proteins from the leaves of untreated and NaCl-treated plants were extracted and separated by two-dimensional difference gel electrophoresis (2D-DIGE). A total of 2358 protein spots were detected on the gels, among which 125 spots showed a significant change in protein abundance, and 83 differentially expressed spots were localised on preparative gels. Using Q-TOF mass spectrometry, 52 salt-responsive spots were identified, which were classified into six functional categories that included transport-associated proteins, detoxifying enzymes, ATP synthase, carbon metabolism, protein folding, and proteins with unknown biological functions. Of the 52 differentially expressed proteins, 26 were up-regulated, 21 were down-regulated, and five spots showed multi-expression patterns. In particular, some important proteins for salt tolerance were found to be up-regulated in Zhengmai 9023 under salt stress, such as H⁺-ATPases, glutathione S-transferase, ferritin and triosephosphate isomerase.     

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.     

Studies on middle and posterior silk glands of silkworm (Bombyx mori) using two-dimensional electrophoresis and mass spectrometry

Silk glands, present in the larval stage of the silkworm, produce threads of silky material to form the cocoon and are mainly composed of three parts: the anterior, the middle, and the posterior silk glands, each playing different roles in silk secretion. High-resolution two-dimensional polyacrylamide gel electrophoresis and computer-assisted analysis were used to investigate quantitative and qualitative differences between the middle and posterior silk glands. Silver staining revealed over 600 spots for each sample, mostly distributed from 15 to 100 kDa with pH 4-7. Computer-assisted image analysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and post-source decay technology suggested that there were significant differences in spot distribution and expression between the middle and posterior silk glands. In addition, 98 spots from the posterior silk gland were excised and further investigated following trypsin digestion. The results suggested that more than 20% of the 88 proteins identified were related to heat-shock proteins and chaperones. Redox system and DNA replication proteins involved in silk protein synthesis were also detected in the posterior silk gland. Interestingly, two novel serpin proteins were identified in the middle silk gland, and to a lesser extent in the posterior gland, which were presumed to be involved in regulation of proteolytic activity and protection of silk proteins from degradation.     

Comparative analysis of proteome changes induced by the two spotted spider mite Tetranychus urticae and methyl jasmonate in citrus leaves

Citrus plants are currently facing biotic and abiotic stresses. Therefore, the characterization of molecular traits involved in the response mechanisms to stress could facilitate selection of resistant varieties. Although large cDNA microarray profiling has been generated in citrus tissues, the available protein expression data are scarce. In this study, to identify differentially expressed proteins in Citrus clementina leaves after infestation by the two-spotted spider mite Tetranychus urticae, a proteome comparison was undertaken using two-dimensional gel electrophoresis. The citrus leaf proteome profile was also compared with that of leaves treated over 0-72 h with methyl jasmonate, a compound playing a key role in the defense mechanisms of plants to insect/arthropod attack. Significant variations were observed for 110 protein spots after spider mite infestation and 67 protein spots after MeJA treatments. Of these, 50 proteins were successfully identified by liquid chromatography-mass spectrometry-tandem mass spectrometry. The majority constituted photosynthesis- and metabolism-related proteins. Five were oxidative stress associated enzymes, including phospholipid glutathione peroxidase, a salt stressed associated protein, ascorbate peroxidase and Mn-superoxide dismutase. Seven were defense-related proteins, such as the pathogenesis-related acidic chitinase, the protease inhibitor miraculin-like protein, and a lectin-like protein. This is the first report of differentially regulated proteins after T. urticae attack and exogenous MeJA application in citrus leaves.     

Fractionation of soluble proteins in Escherichia coli using DEAE-, SP-, and phenyl sepharose chromatographies.

In an effort to simplify a complex mixture of soluble proteins from Escherichia coli, methods to fractionate the samples prior to two-dimensional (2D) gel electrophoresis were developed. These methods involve the use of DEAE-Sepharose, SP-Sepharose, and phenyl Sepharose chromatographic columns and the fractionation of the protein mixtures based on differential anionic, cationic, and hydrophobic properties of the proteins, respectively. Fractionation of the soluble proteins from an E. coli extract with DEAE-Sepharose resulted in a threefold increase in the number of detectable 2D gel spots. These gel spots were amenable to protein identification by using in-gel trypsin digestions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and peptide mass fingerprinting. Significantly, the DEAE-Sepharose column fractionation effectively partitioned the soluble proteins from the cell extracts. Similarly, an SP-Sepharose column was used to fractionate the soluble proteins from E. coli and resulted in over a twofold increase in the number of detectable gel spots. Lastly, fractionation of the cell extract with the phenyl Sepharose column resulted in a threefold increase in the number of detectable 2D gel spots. This work describes an easy, inexpensive way to fractionate the soluble proteins in E. coli and a way to better profile the E. coli proteome.     

Proteome analysis of the responses of Panax ginseng C. A. Meyer leaves to high light: use of electrospray ionization quadrupole-time of flight mass spectrometry and expressed sequence tag data

We performed comparative proteomic analyses in order to understand the physiological responses of ginseng (Panax ginseng C. A. Meyer) to high light (HL). As a first step, we analyzed the proteins expressed in ginseng leaves. Proteins extracted from leaves were separated by two-dimensional polyacrylamide gel electrophoresis. Protein spots were identified by tandem mass spectra analysis using electrospray ionization quadrupole-time of flight mass spectrometry (ESI Q-TOF MS). We used a ginseng expressed sequence tag (EST) database as well as a nonredundant protein database from NCBI to identify proteins. Eighty-one proteins were identified using the nr protein database, 51 of which were also verified from the ginseng EST database. An additional 66 proteins were identified only from the ginseng EST database. Proteins that function in energy metabolism, protein stabilization, and protection against oxidative stress were abundant. To understand the light responses of ginseng leaves, we studied time dependent changes in expressed proteins produced by 0-4 h of HL exposure. Six HL-responsive proteins were identified: three proteins were up-regulated (cytosolic small heat-shock protein, cytosolic ascorbate peroxidase, and putative major latex-like protein) and three proteins were down-regulated (Rieske Fe/S protein, putative 3-beta hydroxysteroid dehydrogenase/isomerase-like protein, and oxygen-evolving enhancer-like protein). Our results show that the ginseng EST database combined with ESI Q-TOF MS analysis can be used to identify ginseng proteins and to elucidate the protective mechanism of ginseng against HL induced damage.     

Development of narrow immobilized pH gradients covering one pH unit for human seminal plasma proteomic analysis

Micropreparatively loaded two-dimensional (2-D) electrophoresis gels were used to identify human seminal plasma polypeptides by using narrow immobilized pH gradients (IPGs) covering one pH unit as first dimension. This investigation was restricted to IPG 4.5-5.5 and 5-6 because of the main spot distribution in the acidic part of the 2-D map performed with IPG 3-10, a zone presumed to be rich in spermatogenic markers. Both highly expressed and minor spots of the 2-D map were analyzed by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectroscopy analysis. Identification was obtained by a combination of mass spectrometry and database searching. Identified proteins were of different origin from the male genital tract and some proteolysis was observed. They appeared as either isolated molecules or isoforms. At the analytical level, narrow IPGs allowed a two-fold increase in the number of spots, improved resolution of minor spots particularly in surrounding of highly expressed spots and sensitivity level. Some of these faint spots were differently expressed in azoospermic patients as compared to normospermic men. Therefore, zooming-in on the proteome of human seminal plasma allowed more accurate differential expression analysis of impaired spermatogenesis associated markers.     

左肾动脉缩窄诱导心肌肥大大鼠心肌蛋白差异表达研究

目的：应用蛋白质组学方法分析病理性心肌肥大心肌细胞蛋白表达特征。方法：雄性SD大鼠16只,分为2组（n=8）：两肾一夹组（2K1C）和假手术组（SO）,术后饲养8周,使用普通多普勒和组织多普勒鉴定动物模型,然后提取心肌总蛋白,应用二维凝胶电泳技术,建立分辨率高和重复性良好的凝胶图像,质谱（MALDI—TOF-MS）鉴定差异蛋白点,与网络数据库进行匹配,并对鉴定蛋白进行分类。结果：两肾一夹大鼠心肌细胞有21个蛋白点表现出显著增加或减少,经数据库匹配,获得14种差异表达的蛋白质。结论：两肾一夹大鼠心肌出现一些差异表达蛋白席官们可能在病理性心肌肥大的发生中起重要作用。     

Analysis of proteomic changes in roots of soybean seedlings during recovery after flooding

A proteomic approach was used to identify proteins involved in post-flooding recovery in soybean roots. Two-day-old soybean seedlings were flooded with water for up to 3 days. After the flooding treatment, seedlings were grown until 7 days after sowing and root proteins were then extracted and separated using two-dimensional polyacrylamide gel electrophoresis (2-DE). Comparative analysis of 2-D gels of control and 3 day flooding-experienced soybean root samples revealed 70 differentially expressed protein spots, from which 80 proteins were identified. Many of the differentially expressed proteins are involved in protein destination/storage and metabolic processes. Clustering analysis based on the expression profiles of the 70 differentially expressed protein spots revealed that 3 days of flooding causes significant changes in protein expression, even during post-flooding recovery. Three days of flooding resulted in downregulation of ion transport-related proteins and upregulation of proteins involved in cytoskeletal reorganization, cell expansion, and programmed cell death. Furthermore, 7 proteins involved in cell wall modification and S-adenosylmethionine synthesis were identified in roots from seedlings recovering from 1 day of flooding. These results suggest that alteration of cell structure through changes in cell wall metabolism and cytoskeletal organization may be involved in post-flooding recovery processes in soybean seedlings.     

Identification of avocado (Persea americana) root proteins induced by infection with the oomycete Phytophthora cinnamomi using a proteomic approach

Avocado root rot, caused by Phytophthora cinnamomi, is the most important disease that limits avocado production. A proteomic approach was employed to identify proteins that are upregulated by infection with P. cinnamomi. Different proteins were shown to be differentially expressed after challenge with the pathogen by two-dimensional (2-D) gel electrophoresis. A densitometric evaluation of protein expression indicated differential regulation during the time-course analyzed. Some proteins induced in response to the infection were identified by standard peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and sequencing by MALDI LIFT-TOF/TOF tandem mass spectrometry. Of the 400 protein spots detected on 2-D gels, 21 seemed to change in abundance by 3 hours after infection. Sixteen proteins were upregulated, 5 of these were only detected in infected roots and 11 showed an increased abundance. Among the differentially expressed proteins identified are homologs to isoflavone reductase, glutathione S-transferase, several abscisic acid stress-ripening proteins, cinnamyl alcohol dehydrogenase, cinnamoyl-CoA reductase, cysteine synthase and quinone reductase. A 17.3-kDa small heat-shock protein and a glycine-rich RNA-binding protein were identified as downregulated. Our group is the first to report on gene induction in response to oomycete infection in roots from avocado, using proteomic techniques.     

Study of proteins associated with the Eimeria tenella refractile body by a proteomic approach

Refractile bodies (RB), whose function is still unknown, are specific structures of Eimeriidae parasites. In order to study their proteome, RB were purified from Eimeria tenella sporozoites by a new procedure using a reversible fixation followed by centrifugation. RB proteins were resolved by two-dimensional electrophoresis. Around 76 and 89 spots were detected on RB two-dimensional gels using gradients in the 3-10 and 4-7 range, respectively. RB proteins were located mainly between pH 5 and 7. RB gels were then compared with previously established maps of the entire sporozoite proteome. Proteins appearing in new spots were identified by mass spectrometry. Thirty protein isoforms were located in RB. Added to the already known RB proteins such as Eimepsin and SO7', the new RB proteins were defined as haloacid dehalogenase, hydrolase, subtilase, lactacte dehydrogenase or ubiquitin family proteins. The RB proteome analysis confirmed the hypothesis that this structure is a reservoir for proteins necessary to invasion but also suggests that RB have energetic and metabolic functions.     

A comprehensive proteomic analysis of the accessory sex gland fluid from mature Holstein bulls

The expression of proteins in accessory sex gland fluid (AGF) of proven, high use mature Holstein bulls was evaluated. Thirty-seven bulls with documented fertility based on their non-return rates were studied. AGF was obtained by artificial vagina after bulls were surgically equipped with cannulae in the vasa deferentia. Samples of AGF were evaluated by two-dimensional SDS-PAGE, gels stained with Coomassie blue and polypeptide maps analyzed by PDQuest software. A master gel generated by the software representing the best pattern of spots in the AGF polypeptide maps was used as a reference for protein identification. Proteins were identified by Western blots and capillary liquid chromatography-nanoelectrospray ionization tandem-mass spectrometry (CapLC-MS/MS). The product ion spectra were processed using Protein Lynx Global Server 2.1 prior to database search with both PLGS and MASCOT (Matrix Science) software. The entire NCBI database was considered for mass fingerprint matching. An average of 52+/-5 spots was detected in the AGF 2D gels, which corresponded to proteins potentially involved in capacitation (bovine seminal plasma protein-BSP-A1/A2 and A3, BSP 30 kDa, albumin); sperm membrane protection, prevention of oxidative stress, complement-mediated sperm destruction and anti-microbial activity (albumin, clusterin, acidic seminal fluid protein--aSFP, 5'-nucleotidase--5'-NT, phospholipase A2--PLA2); acrosome reaction and sperm-oocyte interaction (PLA2, osteopontin); interaction with the extracellular matrix (tissue inhibitor of metalloproteinase 2, clusterin) and sperm motility (aSFP, spermadhesin Z13, 5'-NT). The 20 spots distinguished in all gels were matched to proteins associated with these functions. Proteins identified by tandem mass spectrometry as ecto-ADP-ribosyltransferase 5 and nucleobindin, never described before in the accessory sex gland secretions, were also detected. In summary, we identified a diverse range of components in the accessory sex gland fluid of a select group of Holstein bulls with documented fertility. Known characteristics of these proteins suggest that they play important roles in sperm physiology after ejaculation.     

Characterisation of global protein expression by two-dimensional electrophoresis and mass spectrometry: proteomics of Toxoplasma gondii.

The development of tools for the analysis of global gene expression is vital for the optimal exploitation of the data on parasite genomes that are now being generated in abundance. Recent advances in two-dimensional electrophoresis (2-DE), mass spectrometry and bioinformatics have greatly enhanced the possibilities for mapping and characterisation of protein populations. We have employed these developments in a proteomics approach for the analysis of proteins expressed in the tachyzoite stage of Toxoplasma gondii. Over 1000 polypeptides were reproducibly separated by high-resolution 2-DE using the pH ranges 4-7 and 6-11. Further separations using narrow range gels suggest that at least 3000-4000 polypeptides should be resolvable by 2-DE using multiple single pH unit gels. Mass spectrometry was used to characterise a variety of protein spots on the 2-DE gels. Peptide mass fingerprints, acquired by matrix-assisted laser desorption/ionisation-(MALDI) mass spectrometry, enabled unambiguous protein identifications to be made where full gene sequence information was available. However, interpretation of peptide mass fingerprint data using the T. gondii expressed sequence tag (EST) database was less reliable. Peptide fragmentation data, acquired by post-source decay mass spectrometry, proved a more successful strategy for the putative identification of proteins using the T. gondii EST database and protein databases from other organisms. In some instances, several protein spots appeared to be encoded by the same gene, indicating that post-translational modification and/or alternative splicing events may be a common feature of functional gene expression in T. gondii. The data demonstrate that proteomic analyses are now viable for T. gondii and other protozoa for which there are good EST databases, even in the absence of complete genome sequence. Moreover, proteomics is of great value in interpreting and annotating EST databases.     

Proteome analysis of hairy root from Panax ginseng C.A. Meyer using peptide fingerprinting, internal sequencing and expressed sequence tag data

As an initial step to the comprehensive proteomic analysis of Panax ginseng C. A. Meyer, protein mixtures extracted from the cultured hairy root of Panax ginseng were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). The protein spots were analyzed and identified by peptide finger printing and internal amino acid sequencing by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and electrospray ionization quadrupole-time of flight mass spectrometry (ESI Q-TOF MS), respectively. More than 300 protein spots were detected on silver stained two-dimensional (2-D) gels using pH 3-10, 4-7, and 4.5-5.5 gradients. Major protein spots (159) were analyzed by peptide fingerprinting or de novo sequencing and the functions of 91 of these proteins were identified. Protein identification was achieved using the expressed sequence tag (EST) database from Panax ginseng and the protein database of plants like Arabidopsis thaliana and Oryza sativa. However, peptide mass fingerprinting by MALDI-TOF MS alone was insufficient for protein identification because of the lack of a genome database for Panax ginseng. Only 17 of the 159 protein spots were verified by peptide mass fingerprinting using MALDI-TOF MS whereas 87 out of 102 protein spots, which included 13 of the 17 proteins identified by MALDI-TOF MS, were identified by internal amino acid sequencing using tandem mass spectrometry analysis by ESI Q-TOF MS. When the internal amino acid sequences were used as identification markers, the identification rate exceeded 85.3%, suggesting that a combination of internal sequencing and EST data analysis was an efficient identification method for proteome analysis of plants having incomplete genome data like ginseng. The 2-D patterns of the main root and leaves of Panax ginseng differed from that of the cultured hairy root, suggesting that some proteins are exclusively expressed by different tissues for specific cellular functions. Proteome analysis will undoubtedly be helpful for understanding the physiology of Panax ginseng.