The holm oak leaf proteome: analytical and biological variability in the protein expression level assessed by 2-DE and protein identification tandem mass spectrometry de novo sequencing and sequence similarity searching

As a first approach in establishing the holm oak leaf proteome, we have optimised a protocol for this plant and tissue which includes the following steps: trichloroacetic acid-acetone extraction, two-dimensional gel electrophoresis (2-DE) on pH 5 to 8 linear gradient immobilised pH gradient strips as the first dimension, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 13% polyacrylamide gels as the second one. Proteins were detected by Coomassie staining. Gel images were recorded and digitalized, and the protein spots quantified by using a linear regression equation of protein quantity on spot volume obtained against standard proteins. Analytical variance was calculated for one-hundred protein spots from three replicate 2-DE gels of the same protein extract. Biological variance was determined for the same protein spots from independent tissue extracts corresponding to leaves from different trees, or the same tree at different orientations or sampling times during a day. Values of 26% for the analytical variance and 58.6% for the biological variance among independent trees were obtained. These values provide a quantified and statistical basis for the evaluation of protein expression changes in comparative proteomic investigations with this species. A representative set of the major proteins, covering the isoelectric point range of 5 to 8 and the relative molecular mass(r) range of 14 to 78 kDa, were subjected to liquid chromatography-tandem mass spectrometry analysis. Due to the absence of Quercus DNA or protein sequence databases, a method based on the procedure reported by Liska and Shevchenko including de novo sequencing and BLAST similarity searching against other plant species databases was used for protein identification. Out of 43 analysed spots, 35 were positively identified. The identified proteins mainly corresponded to enzymes involved in photosynthesis and energetic metabolism, with a significant number corresponding to RubisCO.     

Analysis of porcine peripheral blood mononuclear cells proteome by 2-DE and MS: analytical and biological variability in the protein expression level and protein identification

In this paper, we present the protein map corresponding to the porcine peripheral blood mononuclear cells (PBMC) to better understand the role of these cells in the pig immune system. To conform the map, the proteins were separated by 2-DE using a 5-8 range pH gradient in IEF and approximately 800 spots were detected. Due to the high level of indeterminate variability associates to the 2-DE, analytical and biological variances were analyzed. The analytical variance was calculated for 50 proteins in three replicate 2-DE gels from the same protein extract whereas the biological variance was determined by comparison of the patterns obtained for the same 50 proteins in different animals. Values of 15.13 and 33.70% were determined for analytical and biological variances, respectively. These average variances will provide a quantified and statistical basis for future proteomic studies directed to evaluate relevant quantitative changes in the biological response. A representative set of the major proteins was subjected to MALDI-TOF analysis and over 75% of the proteins were identified on the basis of their similarity with its human homologue proteins. A large number of cytoskeletal and metabolic proteins were found as well as some proteins related to cell mobility and immunological functions. Finally, other proteins implicated in the cell signaling process, transport or apoptosis were also identified giving a wide overview of the porcine PBMC protein map.     

Sub-cellular proteomic analysis of a Medicago truncatula root microsomal fraction

Since the last decade, Medicago truncatula has emerged as one of the model plants particularly investigated in the field of plant-microbe interactions. Several genetic and molecular approaches including proteomics have been developed to increase knowledge about this plant species. To complement the proteomic data, which have mainly focused on the total root proteins from M. truncatula, we carried out a sub-cellular approach to gain access to the total membrane-associated proteins. Following the setting up of the purification process, microsomal proteins were separated on 2-DE. Ninety-six out of the 440 well-resolved proteins were identified by MALDI-TOF peptide mass fingerprinting. A high percent (83%) of successful protein identification was obtained when using M. truncatula clustered EST database for queries. During the purification process, the enrichment in membrane-associated proteins was monitored on 2-D gels. The membrane location of microsomal proteins was further confirmed using PMF identification. This study reports a fractionation process for characterizing microsomal root proteins of M. truncatula, which could be an interesting tool for investigating the molecular mechanisms involved in root symbioses.     

Two-dimensional electrophoresis protein profile of the phytopathogenic fungus Botrytis cinerea

Botrytis cinerea is a phytopathogenic fungi causing disease in a number of important crops. It is considered a very complex species in which different populations seem to be adapted to different hosts. In order to characterize fungal virulence factors, a proteomic research was started. A protocol for protein extraction from mycelium tissue, with protein separation by 2-DE and MS analysis, was optimised as a first approach to defining the B. cinerea proteome. Around 400 spots were detected in 2-DE CBB-stained gels, covering the 5.4-7.7 pH and 14-85 kDa ranges. The averages of analytical and biological coefficients of variance for 64 independent spots were 16.1% and 37.5%, respectively. Twenty-two protein spots were identified by MALDI-TOF or ESI IT MS/MS, with some of them corresponding to forms of malate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Two more spots matched a cyclophilin and a protein with an unknown function.     

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/.     

Comparing SILAC and two-dimensional gel electrophoresis image analysis for profiling urokinase plasminogen activator signaling in ovarian cancer cells

Two-dimensional gel electrophoresis (2-DE) image analysis is conventionally used for comparative proteomics. However, there are a number of technical difficulties associated with 2-DE protein separation that limit the depth of proteome coverage, and the image analysis steps are typically labor-intensive and low-throughput. Recently, mass spectrometry-based quantitation strategies have been described as alternative differential proteome analysis techniques. In this study, we investigated changes in protein expression using an ovarian cancer cell line, OVMZ6, 24 h post-stimulation with the relatively weak agonist, urokinase-type plasminogen activator (uPA). Quantitative protein profiles were obtained by MALDI-TOF/TOF from stable isotope-labeled cells in culture (SILAC), and these results were compared to the quantitative ratios obtained using 2-DE gel image analysis. MALDI-TOF/TOF mass spectrometry showed that differential quantitation using SILAC was highly reproducible (approximately 8% coefficient of variation (CV)), and this variance was considerably lower than that achieved using automated 2-DE image analysis strategies (CV approximately 25%). Both techniques revealed subtle alterations in cellular protein expression following uPA stimulation. However, due to the lower variances associated with the SILAC technique, smaller changes in expression of uPA-inducible proteins could be found with greater certainty.     

Proteomics of Medicago sativa cell walls

A method for the sequential extraction and profiling by two-dimensional gel electrophoresis (2-DE) of Medicago sativa (alfalfa) stem cell wall proteins is described. Protein extraction included freezing, grinding in a sodium acetate buffer, separation by filtration of cell walls from cytosolic contents, and extensive washing. Cell wall proteins were then extracted sequentially with a solution containing 200 mM CaCl2 and 50 mM sodium acetate, followed by extraction with 3.0 M LiCl and 50 mM sodium acetate. Cell wall proteins from both the CaCl2 and LiCl fractions were profiled by 2-DE. Approximately 150 protein spots were extracted from these two gels, digested with trypsin, and analyzed using nanoscale HPLC coupled to a hybrid quadrupole time-of-flight (Q-tof) tandem mass spectrometer (LC/MS/MS). More than 100 proteins were identified and used in conjunction with the 2-DE profiles to generate proteomic reference maps for cell walls of this important legume. Identified proteins include classical cell wall proteins as well as proteins traditionally considered as non-secreted. Two unique extracellular proteins were also identified.     

果实蛋白质组学研究的实验方法

双向电泳技术是蛋白质组学研究的基本方法之一。果实由于富含糖、多酚、单宁和有机酸等物质,蛋白质的提取比其它植物组织更加困难。本文主要介绍不同果实蛋白质的提取、等电聚焦系统和凝胶染色技术,并建立了一套适用于桃、樱桃、苹果、芒果和冬枣等多种果实蛋白质组学的研究方法。结果表明,采用匀浆法和酚抽提法提取果实的蛋白质,裂解缓冲液2溶解蛋白质,并用固相pH梯度进行等电聚焦,可以获得背景清晰和分辨率高的凝胶图谱,具有较好的重复性,可用于果实蛋白质组学的研究。我们的研究结果显示,固相干胶条与IEF管胶相比,具有更加明显的优势。而不同的染色方法,对结果影响不大。     

Proteomic approach to study leaf proteins in a fast-growing tree species, Gmelina arborea Linn. Roxb

Foliar proteome studies have become highly significant for a comprehensive understanding of complex processes associated with plant growth and development. In the present study, we present a proteomic approach to analyze leaf proteins in an important timber-yielding and fast-growing forest tree species, Gmelina arborea Linn. Roxb. (Verbanaceae). Foliar protein analysis involved protein extraction, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight (MALDI–TOF–TOF). From the 2-DE protein profile of Gmelina leaves, we identified and isolated 150 well-separated protein spots; among these, 64 protein spots were identified by mass spectrometric (MS/MS) analysis. These proteins were classified according to their involvement in basic biological functions, such as photosynthesis, amino acid metabolism, cytoskeleton, cell wall metabolism, stress-related proteins, redox maintenance, electron transport chain, phytohormone metabolism and protein translation and folding. Analytical variance was determined for the protein spots of samples from different plants. The present study is believed to provide a foundation for the use of leaf proteomics in addressing fundamental physiological and biochemical processes associated with growth and productivity of tree species such as Gmelina arborea.     

果实蛋白质组学研究的实验方法

双向电泳技术是蛋白质组学研究的基本方法之一。果实由于富含糖、多酚、单宁和有机酸等物质,蛋白质的提取比其它植物组织更加困难。本文主要介绍不同果实蛋白质的提取、等电聚焦系统和凝胶染色技术,并建立了一套适用于桃、樱桃、苹果、芒果和冬枣等多种果实蛋白质组学的研究方法。结果表明,采用匀浆法和酚抽提法提取果实的蛋白质,裂解缓冲液2溶解蛋白质,并用固相pH梯度进行等电聚焦,可以获得背景清晰和分辨率高的凝胶图谱,具有较好的重复性,可用于果实蛋白质组学的研究。我们的研究结果显示,固相干胶条与IEF管胶相比,具有更加明显的优势。而不同的染色方法,对结果影响不大。     

Molecular cloning and characterisation of a Rab-binding GDP-dissociation inhibitor from Medicago truncatula.

We have isolated and sequenced the full-length cDNA of a GDP-dissociation inhibitor (GDI) from the model legume Medicago truncatula L. The cDNA (MtGDI) contains an open reading frame of 1335 bp, coding for a protein of 444 amino acids with a calculated molecular mass of 49,785 kDa. The deduced amino acid sequence shows significant homology to other plant GDIs, the highest homology being found to GDI from the legume Cicer arietinum (96% identity). The MtGDI was expressed as a N-terminal FLAG-fusion protein in Escherichia coli BL21 (DE3). Its direct interaction with a small G protein of Rab type from Medicago sativa, MsRab11f, was demonstrated in vitro by co-immunoprecipitation using a peptide-specific antibody raised against MtGDI. The dissociation constant of the MtGDI-MsRab11f complex (4 muM) was determined by a surface plasmon resonance (SPR) assay. Real-time RT-PCR and Western blot analyses suggested that MtGDI is ubiquitously expressed in M. truncatula. High levels of MtGDI mRNA were detected in uninfected roots, leaves and root nodules. In etiolated seedlings and cell cultures, the amount of MtGDI mRNA was much lower. In all tissues tested, the peptide-specific anti-MtGDI antibody detected the expected 50 kDa protein in the total protein extracts. MtGDI was found in the cytosol; however, a significant fraction was associated with the intracellular membranes in seedlings and roots indicating a membrane localisation of the protein. A second immunoreactive band was detected in leaves suggesting that more than one GDI isoform exist in M. truncatula.     

Proteomics of ionically bound and soluble extracellular proteins in Medicago truncatula leaves

A large proportion of the apoplast proteome resides in the intercellular fluid (IF) or is ionically bound (IB) to the wall matrix. A combined analysis of IF and IB proteins of the Medicago truncatula leaf apoplast was performed. 2-DE analyses demonstrated the reproducible presence of 220 IF and 84 IB proteins in the apoplast. These two protein populations were largely distinct; 22 proteins could be spatially matched, but MALDI-TOF/TOF analyses suggested a considerably smaller number had common identities. MALDI-TOF/TOF characterisation identified 81 distinct proteins. Analyses of selected IF proteins (45) indicated 17 distinct proteins with mainly defence-related functions, whereas analyses of IB proteins (70) identified 63 distinct proteins of diverse natures, including proteins of non-canonical natures. The presence of non-canonical proteins in IB extracts is discussed in the light of evidence supporting a low level of contamination of purified walls from symplastic proteins. This work indicates that IB and IF proteins are functionally distinct fractions of the apoplast. The data obtained complements earlier studies of the Medicago proteome and therefore will be useful in future studies investigating the role of apoplastic proteins in plant processes.     

Bt Cry1Ac毒素筛选的粉纹夜蛾离体抗性细胞与敏感细胞蛋白质组的差异分析

采用双向电泳技术和肽质量指纹图谱技术分析了Bt Cry1Ac毒素筛选的粉纹夜蛾Trichoplusia ni抗性BTI-Tn-5B1-4细胞与同源敏感细胞的蛋白质组的差。用Melanie ViewerⅡ软件在抗性细胞的双向电泳图谱上检测到的平均蛋白质点数为707±25个（n=3）,在敏感细胞的双向电泳图谱上检测到的平均蛋白质点数为637±19个（n=3）,其中分辨率高、重复性良好的显著差异点有10余个。对其中的一个敏感细胞特有的显著差异斑点进行了肽质量指纹图谱分析,经数据库 查询表明该蛋白质与胞质外周蛋白具有同源性。     

Comparison of preparative and analytical two-dimensional electrophoresis for isolation and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric analysis of transthyretin in cerebrospinal fluid

The variety of posttranslational modifications and the relative abundance of transthyretin (TTR) in cerebrospinal fluid (CSF) make TTR a suitable model molecule when comparing the performance of different combinations of methods for characterization of CSF proteins. We have compared three different electrophoretic methods: conventional two-dimensional gel electrophoresis (2-DE), liquid-phase isoelectric focusing (IEF) as a prefractionation step in combination with analytical 2-DE, and preparative 2-DE for isolation and mass spectrometric analysis of TTR in CSF. Using liquid-phase IEF in combination with 2-DE compared with conventional 2-DE improved the sequence coverage of TTR. Only the mass spectrum from the preparative 2-DE fraction contained a tryptic peptide from the first nine amino acids, thereby yielding 100% sequence coverage. Our results show that the use of a prefractionation step before 2-DE or the use of preparative 2-DE increases the sequence coverage and provide low abundant proteins in complex biological systems in sufficient quantities for protein characterization with mass spectrometry.     

Artificial neural networks and decision tree model analysis of liver cancer proteomes

Hepatocellular carcinoma (HCC) is a heterogeneous cancer and usually diagnosed at late advanced tumor stages of high lethality. The present study attempted to obtain a proteome-wide analysis of HCC in comparison with adjacent non-tumor liver tissues, in order to facilitate biomarkers' discovery and to investigate the mechanisms of HCC development. A cohort of 66 Chinese patients with HCC was included for proteomic profiling study by two-dimensional gel electrophoresis (2-DE) analysis. Artificial neural network (ANN) and decision tree (CART) data-mining methods were employed to analyze the profiling data and to delineate significant patterns and trends for discriminating HCC from non-malignant liver tissues. Protein markers were identified by tandem MS/MS. A total of 132 proteome datasets were generated by 2-DE expression profiling analysis, and each with 230 consolidated protein expression intensities. Both the data-mining algorithms successfully distinguished the HCC phenotype from other non-malignant liver samples. The detection sensitivity and specificity of ANN were 96.97% and 87.88%, while those of CART were 81.82% and 78.79%, respectively. The three biological classifiers in the CART model were identified as cytochrome b5, heat shock 70 kDa protein 8 isoform 2, and cathepsin B. The 2-DE-based proteomic profiling approach combined with the ANN or CART algorithm yielded satisfactory performance on identifying HCC and revealed potential candidate cancer biomarkers.     

Root responses of Medicago truncatula plants grown in two different iron deficiency conditions: changes in root protein profile and riboflavin biosynthesis

Iron deficiency is a yield-limiting factor with major implications for field crop production in one-third of the world's agricultural areas, especially those with high soil CaCO(3). In the present work, a two-dimensional gel electrophoresis proteomic approach was combined with a study on the riboflavin synthesis pathway, including qPCR and riboflavin determination, to investigate Fe-deficiency responses in Medicago truncatula plants grown with and without CaCO(3). Iron deficiency caused a de novo accumulation of DMRLs and GTPcII, proteins involved in riboflavin biosynthesis, as well as marked increases in root riboflavin concentrations and in the expression of four genes from the riboflavin biosynthetic pathway. Two novel changes found were the increased accumulation of proteins related to N recycling and protein catabolism. Other identified changes were consistent with previously found increases in glycolysis, TCA cycle, and stress-related processes. All effects were more marked in the presence of CaCO(3). Our results show that the riboflavin biosynthesis pathway was up-regulated at the genomic, proteomic, and metabolomic levels under both Fe-deficiency treatments, especially in the presence of CaCO(3). Results also indicate that N recycling occurs in M. truncatula upon Fe deficiency, possibly constituting an additional anaplerotic N and C source for the synthesis of secondary metabolites, carboxylates, and others.