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Royal jelly (RJ) is an important functional food, is used for many purposes. New methods are needed for the practical characterization of royal jelly. In this preliminary study, SDS-PAGE electrophoretic profiles of royal jelly proteins were investigated. Raw royal jelly samples were analyzed by classical SDS-PAGE electrophoresis using 5% and 10% gels. The major protein spot were obtained at the samples collected same regions. Compared the proteins with known marker proteins they were between 50 and 80 kDa. Classical SDS-PAGE electrophoresis alone is not sufficient to determine royal jelly proteins, but can be used to basic characterization of royal jelly. Furthers electrophoretic techniques are needed for a better separation.

Objective: Proteomics is the large-scale study of localization, identification, structure, and function of the proteome. A proteome is the complete set of proteins expressed and modified by an organism under a specific set of environmental conditions. This study was undertaken to investigate the novel protein biomarkers that play a role in breast cancer under inflammatory condition. Methods: The two-dimensional gel electrophoresis (2-DE) was applied in the context of the breast cancer model system to investigate the effect of interferon-gamma (IFN-γ) on the differential protein expression in breast cancer-derived cell lines CAMA-1 and 3,4-methylenedioxyamphetamine (MDA)-MB-231. Whole cell lysates were prepared from IFN-γ-stimulated and non-stimulated CAMA-1 and MDA-MB-231 cells for 2-DE to obtain information for potential differential protein expression. Protein spots in the gels were visualized by silver staining and analyzed by Progenesis SameSpot. Gels were then scanned using the Epson image scanner with LabScan 6.0 software. The ExPASy tool was used to identify and quantify breast cancer cell membrane proteins expressed in response to IFN-γ. Results: In the present proteomics study, a series of differentially expressed proteins were analyzed in IFN-γ-stimulated CAMA-1 and MDA-MB-231 cells. While results obtained from this analysis can be used as preliminary data to identify differences between untreated and IFN-γ-treated samples, they were not used for further mass spectrometry analysis. Conclusion: The data described and discussed here can be utilized for further data validation projects and could assist in the discovery of new breast cancer-related proteins and molecular pathways.

Background Two-dimensional electrophoresis (2DE) is one of the most widely applied techniques in comparative proteomics. The basic task of 2DE is to identify differential protein expression by quantitative analysis of 2DE images. To reduce the errors of spot quantification in 2DE images, a novel brightness correction method based on gradient interval histogram (GIH) is proposed in this paper. Results First, GIH equalization is proposed to enhance the protein spot edges, especially the weak protein spots in the 2DE image. Second, to eliminate the overall brightness shift, GIH matching is applied to the 2DE images that need to be compared. Finally, the proposed method is verified by subjective quality evaluation and quantitative analysis of protein spots in real 2DE images. Conclusions The experimental results show that the average error of the quantification of corresponding protein spots in the resulting image pairs is less than 3%, which is significantly superior to that of the existing methods.

Two-dimensional gel electrophoresis (2DE) is a proven high-resolution separation technology in the protein laboratory. Nevertheless, ever since its invention the technique has had the drawback of low reproducibility. For protein expression analysis based on replicates, differential gel electrophoresis (DIGE) has, therefore, been invented. This technique is however not applicable for the comparison of singular samples. With comparative fluorescence 2DE (CoFGE) a method was developed, which creates a protein reference net for gel matching on top of the gel-separated protein analyte. This internal standard enables both the correction of the protein spot coordinates and the estimation of the analyte concentration. It extends the use of 2DE and truly enables the use of searchable, browser-based 2DE databases.

Heat treatment of milk may cause whey proteins and caseins to form aggregates. These soluble and micellar aggregates and their other properties (size, composition, shape, etc.) can affect the techno-functionalities to the milk, conferring interesting or negative features depending on the application in dairy industries. In this study, we propose a new approach to characterise those protein aggregates. SDS-agarose electrophoresis is followed by the calculation of a retention factor (Rf) for each protein spot. Rf allows milk aggregates to be compared qualitatively under the same conditions. This method could be transposed to the dairy industry for a better knowledge of the milk subsequent to heat treatment.

Two-dimensional electrophoresis as a complementary approach to gel-free proteomic methods possesses the ability to separate physiologically important isoforms of proteins in an unbiased manner. Frequently, those isoforms are low-abundant regulators, and therefore, detection and identification of low-abundant proteins is highly necessary to exploit this advantage. We describe an experimental sequence of classical operations to process gels but optimized them, in order to identify each detectable protein spot on gel.

In proteomic studies, a population of proteins are often examined on a gel using a technique called two-dimensional gel eletrophoresis. The technique separates the protein population into individual protein spots on a two-dimensional gel by isoelectric charge and molecular weight. The resulting gel images are then processed by a software system for spot detection and subsequent analysis. The performance of a spot-detection program is evaluated by the total number of spots that are detected. A popular spot-detection program uses the “master–slave” approach, where all spots on “slave images” are subsets of the spots on the “master image.” We argue that this approach potentially misses a large proportion of proteins and propose a model that quantifies the lack of performance. We provide nonparametric estimators for the protein population size and the expected number of proteins to be detected if a “fusion-gel” approach was used. Using the data from a rat liver proteome study, we estimate that more than half of the protein population is missed by the master–slave approach.

Amphoteric azo dyes were used for internal control of pI values in Comparative two-dimensional Fluorescence Gel Electrophoresis (CoFGE) [1]. The 2D-gel images of separated Escherichia coli proteins as well as those of colored amphoteric dyes separated by isoelectric focussing are presented. The latter were used to correct for variation in the first electrophoretic dimension and further improve protein coordinate assignment in 2D-gel electrophoresis. Data tables are supplied to demonstrate pI-value calibration and the effect on the assignment of protein spot coordinates.

In the present study, different expression of protein from Taekwang was revealed by 2-DE, and expressions of protein on each week after flowering was investigated. After analysis of expression of protein, MALDI-TOF was executed to identify expected protein function. Results revealed that there were three patterns of expression of protein during the maturing. The first pattern was that proteins were gradually expressed as up-regulation from 1 week to 6 week. The second pattern was that proteins were expressed gradually from 1 week to 5 week and then it started down-regulation in 6 week. The last pattern was that proteins were gradually as up-regulation from 1 week to 3 week and then down-regulation until 6 week. This phenomenon suggests that young stage has more protein related to correspondence mechanism against disease and growth and then maturing stage has more expression of protein related to storage protein. In MALDI-TOF analysis, p24 oleosin isoform A protein was identified that relates oleosin which is synthetic product in oil body. This protein spot increased gradually until 5 week and then decreased after 5 week. It explained that the protein is active until maturing stage to protect oil in seed and then its activity has gradually degraded. This result may be expected that a protein, related to growth of a seed has increased until maturing and then a seed fills up with a storage protein.

espanolLa lectina V-2 aislada de semillas de Pisum sativum L. ("arveja") fue purificada mediante cromatografia de exclusion molecular en Sephadex G-75 y cromatografia liquida de alta precision de fase reversa (HPLC). El analisis en SDS-PAGE de dos dimensiones demostro que la lectina es homogenea con un unico spot proteico de ~ 14 kDa y un punto isoelectrico de 7,5. Mediante espectrometria de masa (MALDI-TOF) fue confirmado el peso molecular de la lectina mostrando una masa de 14.662,0 Da. La secuencia completa de aminoacidos (estructura primaria) revelo que la lectina V-2 esta compuesta de 128 aminoacidos. El estudio comparativo con otras lectinas, mostro que la lectina V-2 tiene mayor homologia con la lectina de semillas de Cratylia mollis L. (91,4%), seguido por la lectina de semillas de Cratylia argentea (61,6%). De acuerdo al arbol filogenetico, la lectina V-2 presenta una aproximacion microevolutiva ~ 1.000 nucleotidos con la lectina de C. mollis. Asimismo, la lectina V-2 mostro accion antibacteriana sobre Escherichia coli y Staphylococcus aureus formando un halo inhibitorio de crecimiento en una concentracion de 1 mg. EnglishThe lectin V-2 from Pisum sativum L. ("arveja") seeds was purified by Sephadex G-75 molecular exclusion chromatography and reverse phase high performance liquid chromatography (RP-HPLC). Two dimensional SDS-PAGE analyses demonstrated that the purified lectin was homogeneous since it appeared as a single protein spot corresponding to ~14 kDa with an isoelectric point of 7.5. Its molecular weight was confirmed by mass spectrometry (MALDI-TOF) to be 14,662.0 Da. The complete amino acid sequence (primary structure) showed that the lectin V-2 contains 128 amino acids. Comparative studies with other lectins show that it has high homology to the lectin from Cratylia mollis L. (91.4%), seeds and continued by the lectin from Cratylia argentea (61.6%) seeds. According to aphylogenetic tree, the lectin V-2 showed an approximation microevolutionary of ~ 1,000 nucleotides with the lectin from C. mollis. Additionally, the lectin V-2 showed antibacterial action on Escherichia coli and Staphylococcus aureus makes an inhibition halo of growth with a concentration of 1 mg.

Previously, we identified protein spots with differential intensity between normal and tumor tissues of renal cell carcinoma (RCC) using 2D-DIGE. Here, we further examined two proteins, raf-1 kinase inhibitory protein (RKIP) and manganese superoxide dismutase (MnSOD), identified in one protein spot. Western blotting demonstrated that RKIP and MnSOD exhibited opposite expression patterns in normal and tumor tissues. Immunohistochemisry showed that MnSOD level significantly correlated with shorter progression-free survival. Gene-silencing assay demonstrated that RKIP and MnSOD had suppressive and promotive effects on tumor cell proliferation and invasion, respectively. Our findings reveal biological and clinical significance of RKIP and MnSOD in RCC.

A common method for quantifying proteins is to separate them by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and quantify the intensity of the spots with image analysis software. Protein spot variability can be quite high averaging about 60 to 80% for three biological replicates. Gel to gel variability is quite high requiring the researcher to make many technical replicates and select from the best of the gels made. Our lab decided to determine where most of this variability came from. We designed an experiment to determine if the variability came from the biological replicates, protein extractions or the custom made 2D-PAGE gels. A total of 27 gels were run; there were 3 gels of 3 extractions of 3 biological replicates of ?Cabernet Sauvignon? shoot tips. Nearly 500 common protein spots were quantified on all 27 gels. Variability was quantified with the coefficient of variation (CV). The average CV was highest for the gels and lowest for the biological replicates, averaging 55, 33 and 25% for gels, extractions and biological replicates, respectively. The CV ranged from 5 to 122% for some protein spots indicating that for many proteins it is difficult to get good quantitative data for comparative purposes. The variability of this method limits the number of proteins that can be quantified adequately and inflates the cost and labor due to the replacement of bad gels. A similar analysis was performed on buds of Seyval and Vitis riparia. The average CV of 861 spots was 68%. Difference Gel Electrophoresis (DIGE) is designed to reduce this variability. We found that DIGE significantly reduced the CV to about 33% and the use of internal standards reduced gel-to-gel variability and increased the detection of differentially expressed proteins.

Francisella tularensis is the causative agent of tularemia. Although major contributors and the main mechanism of the virulence are well known, some of the molecular details are still missing. Proteomics studies regarding F. tularensis have provided snapshot pictures of the organism grown under different culture conditions to understand the mechanism of virulence. In general, such studies were carried out with standard strains e.g., LVS and did not involve comparisons of F. tularensis isolates from either clinical or environmental sources. In this study, we performed two-dimensional gel electrophoresis (2DE)-based proteomic analysis and compared the protein profiles of the F. tularensis subsp. holarctica strains isolated from the clinical and the environmental samples. Regulations were detected in 14 spots when twofold regulation criteria were applied. The regulated protein spots were subjected to MALDI-TOF/TOF analysis and identified. Classification of the identified proteins based on metabolic functions revealed that the translation machinery was the most varying metabolic processes among the isolates. Using normalized protein spot intensities, PCA analysis was also performed. The results indicated that the strain isolated from water source was different then the strains isolated from the patients. Most interestingly, the isolates were strikingly distinguishable from the standard NCTC 10857 strain.

The Binder of SPerm 1 (BSP1) protein is involved in the fertilization and semen cryopreservation processes and is described to be both beneficial and detrimental to sperm. Previously, the relationship of BSP1 with freezability events has not been completely understood. The objective of this work was to determine the differential abundance of the forms of the BSP1 protein in cryopreserved seminal plasma of Bos taurus indicus bulls with different patterns of semen freezability using proteomics. A wide cohort of adult bulls with high genetic value from an artificial insemination center was used as donors of high quality, fresh semen. Nine bulls presenting different patterns of semen freezability were selected. Two-dimensional gel electrophoresis showed differential abundance in a group of seven protein spots in the frozen/thawed seminal plasma from the bulls, ranging from 15 to 17 kDa, with pI values from 4.6 to 5.8. Four of these spots were confirmed to be BSP1 using mass spectrometry, proteomics, biochemical, and computational analysis (Tukey's test at P

2D-gel electrophoresis (2DGE) is an important technique in proteomics for analyzing the protein expressions. However, the analysis of 2DGE images is still a cumbersome and tedious task. One of the main reasons is the presence of a large amount of the inhomogeneities in the foreground and the background intensities. In this paper, we have proposed a novel approach of segmentation of the protein spots in the non separable wavelet domain. It utilizes the inter-scale relationship among enhanced wavelet coefficients, which can easily distinguish the different features of the image the interior region of spots, the edges and the background. This technique is based on a single threshold and is independent of the gray value of the image. It copes with the inhomogeneities in the 2DGE images up to a great extent, which is helpful for finding the protein spots accurately. The artifacts are further removed using a non-threshold based method comprising a weighted Gaussian energy distribution model. Experimental results show that our method outperforms the available commercial software and previously reported works. (C) 2015 Elsevier Ltd. All rights reserved.

To identify the molecular target of diallyl trisulfide (DATS) in human leukemic cell line U937, we examined modification of thiol group(s) of cellular proteins by the redox 2D PAGE. A unique protein spot appeared by DATS treatment was identified to be heat shock protein 27 (HSP27). Hsp27 is suggested to be one of the molecular target of DATS in U937.

Straipsnyje nagrinėjama baltymų pėdsakų atpažinimo ir parametrizavimo problema dvimatės elektroforezės gelių vaizduose. Pristatomas naujas būdas baltymų persisotinimams dvimatės elektroforezės gelių vaizduose aptikti ir rekonstruoti. Siūlomą paieškos ir rekonstravimo būdą sudaro keli etapai: gelio vaizdo paruošimas taikant naują medianos filtro kaukės dydžio parinkimo algoritmą, baltymų persisotinimų paieška taikant autorių siūlomus iškraipymų modelius, automatinis persisotinusio baltymų pėdsako išskyrimas ir rekonstravimas. Straipsnyje pateikti eksperimentinio tyrimo rezultatai įrodo, kad siūlomas būdas leidžia atpažinti iki 96% baltymų persisotinimų gelių vaizduose taikant vieną iš dviejų autorių siūlomų iškraipymų modelių. Baltymų persisotinimų rekonstravimui straipsnyje siūlomi nauji baltymų pėdsakų modeliai, gebantys tiksliau ir sparčiau atstatyti baltymo pėdsako formų nei alternatyvusis difuzinis modelis.The paper addresses the over-saturated protein spot detection and extraction problem in two-dimensional electrophoresis gel images. The effective technique for detection and reconstruction of over-saturated protein spots is proposed. The paper presents. an algorithm of the median filter mask adaptation for initial filtering of gel image, the models of over-saturation used for gel image analysis; several models of protein spots used for reconstruction, technique of the automatic over-saturated protein spot search and reconstruction. Experimental investigation confirms that proposed search technique lets to find up to 96% of over-saturated protein spots Moreover the proposed flexible protein spot shape models for reconstruction are faster and more accurate in comparison to the flexible diffusion model.

The spot detection phase of the 2-DE image analysis program segments a gel image into spot regions by an image segmentation algorithm and fits the spot regions to a spot shape model and quantifies the spot informations for the next phases. Currently the watershed algorithm is generally used as the segmentation algorithm and there are the Gaussian model and the diffusion model for the shape model. The diffusion model is closer to real spot shapes than the Gaussian model however spots have very various shapes and especially an asymmetric formation in x-coordinate and y-coordinate. The reason for asymmetric formation of spots is known that a protein could not be diffused completely because the 2-DE could not be processed under the ideal environment usually. Accordingly we propose an asymmetric diffusion model in this paper. The asymmetric diffusion model assumes that a protein spot is diffused from a disc at initial time of diffusing process, but is diffused asymmetrically for x-axis and y-axis respectively as time goes on. In experiments we processed spot matching for 19 gel images by using three models respectively and evaluated averages of SNR for comparing three models. As averages of SNR we got 14.22dB for the Gaussian model, 20.72dB for the diffusion model and 22.85dB for the asymmetric diffusion model. By experimental results we could confirm the asymmetric diffusion model is more efficient and more adequate for spot matching than the Gaussian model and the diffusion model.

Brassinolide insensitive associated receptor kinase 1(BAK1) is a critical component that play an important roles in signaling of brassinosteroid biosynthesis. Brassinosteroid-deficient and -insensitive mutants showed the characteristic of dwarf symptom. The nutrient deficient tomato showing stunt phenomenon was selected from soiless cultivation system using modified Sonneveld hydroponic solution. Twenty eight protein spots showing different expression levels compared to the control were isolated from extracts of stunted tomato leaves by 2D PAGE analyses. Significantly down-regulated 6 protein spots out of 28 protein spots were analyzed and sequenced by MALDI-TOF mass spectrometry. The protein spot having pI

We consider a characterization of proteomics maps based on an alternative kind of neighborhood graphs for the protein spots on 2-D gel. The novel approach considers for every protein spot only the nearest neighborhood consisting of protein spots of higher abundance. The approach has the simplicity and advantages of the recently introduced characterization of proteome maps based on considering the nearest neighborhoods of protein spots, but it also has important additional desirable computational features. The characterization of the nearest neighborhood graphs of 2-D gel proteomics maps is sensitive to the number of spots considered and may lead to changes in the degree of similarity of different maps when the number of points has been changed, thus imposing restrictions on the protocol used for comparison of maps. The novel approach presented in this work is less sensitive to the number of points used in the analysis because graphs are constructed in a stepwise process in which the role of more distant neighbors has been diminished by linking a new spot to the nearest spot that has been already part of the neighborhood graph. In this way a graph with N + 1 spots is obtained from the graph on N spots by adding a single new link, while in the case of the nearest neighborhood graphs adding a new spot introduces novel neighborhoods and generally results in a graph that may differ significantly from the neighborhood graph on N points.

Methyl mercaptan is derived from l -methionine by the action of l -methionine-α-deamino-γ-mercaptomethane lyase (METase) and is a major component of oral malodor. This compound is highly toxic and is thought to play an important role in periodontal disease. We found that Treponema denticola , a member of the subgingival biofilm at periodontal disease sites, produced a large amount of methyl mercaptan even at low concentration of l -methionine. METase activity in a cell-free extract from T. denticola was detected by two-dimensional electrophoresis under non-denaturing conditions, and the protein spot that exhibited high METase activity was identified using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. The identified gene produced a METase with a K m value for l -methionine (0.55 mM) that is much lower than those of METases previously identified in the other organisms. This result suggests that T. denticola is an important producer of methyl mercaptan in the subgingival biofilm.

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis remains the core of proteomic technology because it is currently the most powerful method to analyze large collections of proteins. Advances in electrophoresis equipment are making this technique more accessible but effective computer assisted protein spot detection remains a very labor-intensive endeavor. Protein spot analysis is still time consuming, requires human intervention and is in need of further development. This study explores a technique of recursively applying a Support Vector Machine (SVM) in identifying protein. An SVM is a powerful learner capable of optimizing differences between classes. In this context the different classes correspond to the presence/absence of a protein. Different experiments are conducted to assess these differences in class formation in the context of a normal image and a highly saturated image.

Strawberry plants (Fragaria×ananassa Duch.) cvs. Nyoho and Toyonoka were exposed to temperatures of 20, 33, and 42 °C for 4 h, and protein patterns in leaves and flowers was analyzed by 2-dimensional polyacrylamide gel electrophoresis and immunoblotting. In leaves and flowers of both cultivars, the content of most proteins decreased, but a few new proteins appeared in response to heat stress. These heat shock proteins (Hsps) were detected in the range of 19 – 29 kDa in leaves, and 16 – 26 kDa in flowers. The intensity of a 43 kDa protein spot increased in response to heat stress in Nyoho flowers, but not in Toyonoka flowers. The peaHsp17.7 antibody recognized one band at approximately 26 kDa in leaves, and two bands at approximately 16 and 17 kDa in flowers of both cultivars. These results show that the effects of heat stress on Hsp synthesis in strawberry plants differ between plant organs and between cultivars.

MRSA-ST9-t4358 from pigs and pig handlers were analysed for exoproteins profiles. Similar protein patterns with variation in protein spot intensity were observed. No significant variation among the protein profiles indicates that transmission of MRSA-ST9 from pig to human or vice versa, do not strongly alter the gene expression patterns. Indirectly, virulence factors that acquired from pig environment might have chance to be expressed in human host.

International audience; Cotyledonary somatic embryos (SEs) of maritime pine are routinely matured for 12 weeks before being germinated and converted to plantlets. Although regeneration success is highly dependent on SEs quality, the date of harvesting is currently determined mainly on the basis of morphological features. This empirical method does not provide any accurate information about embryo quality with respect to storage compounds (proteins, carbohydrates). We first analyzed SEs matured for 10, 12 and 14 weeks by carrying out biological (dry weight, water content) and biochemical measurements (total protein and carbohydrate contents). No difference could be found between collection dates, suggesting that harvesting SEs after 12 weeks is appropriate. Cotyledonary SEs were then compared to various stages, from fresh to fully desiccated, in the development of cotyledonary zygotic embryos (ZEs). We identified profiles that were similar using hierarchical ascendant cluster analysis (HCA). Fresh and dehydrated ZEs could be distinguished, and SEs clustered with fresh ZEs. Both types of embryo exhibited similar carbohydrate and protein contents and signatures. This high level of similarity (94.5 %) was further supported by proteome profiling. Highly expressed proteins included storage, stress-related, late embryogenesis abundant and energy metabolism proteins. By comparing overexpressed proteins in developing and cotyledonary SEs or ZEs, some (23 proteins) could be identified as candidate biomarkers for the late, cotyledonary stage. This is the first report of useful generic protein markers for monitoring embryo development in maritime pine. Our results also suggest that improvements of SEs quality may be achieved if the current maturation conditions are refined.

Two species of microcolonial fungi – Cryomyces antarcticus and Knufia perforans - and a species of black yeasts–Exophiala jeanselmei - were exposed to thermo-physical Mars-like conditions in the simulation chamber of the German Aerospace Center. In this study the alterations at the protein expression level from various fungi species under Mars-like conditions were analyzed for the first time using 2D gel electrophoresis. Despite of the expectations, the fungi did not express any additional proteins under Mars simulation that could be interpreted as stress induced HSPs. However, up-regulation of some proteins and significant decreasing of protein number were detected within the first 24 hours of the treatment. After 4 and 7 days of the experiment protein spot number was increased again and the protein patterns resemble the protein patterns of biomass from normal conditions. It indicates the recovery of the metabolic activity under Martian environmental conditions after one week of exposure.

Improvements in DNA-sequencing technology have produced a plethora of sequenced fungal genomes. As a result, the genomic era has opened the door for the dissection of phytopathogenicity via systems biology using sophisticated ‘-omics’ technologies. Proteomic and metabolomic are key analytical techniques that have been used at an increasing frequency in phytopathology. These high-throughput global techniques can provide a detailed metabolic ‘photograph’ of the fungal phytopathogen or pathosystem. This has greatly contributed to the understanding of fungal phytopathogenicity. Here, we provide a comprehensive review of recent literature that significantly contributed to the impact of proteomics and metabolomics on fungal phytopathology.

Despite an enormous amount of knowledge in biomedical science, we still do not know much about the mechanisms of our body in response to environmental toxins. No doubt these need to be investigated in order to Wnd a treatment and cure. Thus, in addition to the testing of chemicals for their toxicity, in which investigations use and need to use a broad spectrum of concentrations and doses, the question of explaining the basic mechanisms of observed eVects play an important role. Unlike in the Wrst case, in the latter case, it is of prime importance that reality-relevant concentrations and doses are used. The aim of preliminary/introductory epidemiological investigations, which are associated with or are the basis for toxicological studies, is the inquiry into possible exposures with adverse health eVects and the identiWcation of the causing mechanisms. While epidemiological investigations allow observations of possible associations, with a certain design-dependent probability, they do not ascertain the causal relationship nor do they explain the mechanisms. This can be achieved with follow-up toxicological studies, which will put the epidemiological Wndings into a causal perspective contributing to its understanding. Nevertheless, it has to be expressively pointed out that the premiss for conducting human toxicological investigations is not infrequently the results of epidemiological studies. Problem

Ehrlichia ruminantium (ER) is an obligate intracellular bacterium, from the order Rickettsiales, which causes Heartwater, a fatal tick-borne disease in ruminants. This disease is a major limitation to livestock production in sub-Saharan Africa and in some Caribbean islands. Recent studies showed that key proteins such as the Major Antigenic Protein 1 (MAP1) are glycosylated (Postigo et al., 2008) and that about 25 % of ER proteome account for isoforms, indicating the importance of post-translational modifications (PTMs) in the ER infection process (Marcelino et al., 2012).

90 It is known that jasmonic acid, a stress phytohor� mone, is a key factor in inducing the immunity of plants to necrotrophic pathogens and some phytopha� gous insects. Plant infection, action of elicitors, and tissue injury induce a rapid accumulation of jasmonic acid, triggering the jasmonateinducible signal trans� duction pathway and activating synthesis of several proteins involved in the plant's defense responses. Methyl jasmonate (MeJA), a derivative of jasmonic acid able to migrate over the plant, inducing systemic immunity, also displays signaling functions. The majority of the studies assessing the effect of jasmonates on the set and content of proteins in plants have involved the aboveground organs (1) rather than the plant toots; however, the changes in proteomes caused by different impacts, in particular, stress phy� tohormones, may significantly differ in these organs (2, 3). The available published data are limited to the effects of jasmonates on rice (2, 4) and arabidopsis (5) root proteomes.

Circannual hibernation is characterized by two cycles—the annual cycle between summer homeothermy and winter heterothermy as well as the individual torpor-arousal cycles within winter heterothermy. We hypothesized that proteins and pathways that promote or enhance survival throughout hibernation cycles are concordantly regulated in organs throughout the body. In this study, we applied a meta-analysis to previously described proteomic changes in six identical seasonal and physiological states of heart, skeletal muscle, and kidney in order to identify common protective mechanisms underlying resistance to challenges of hibernation. Unexpectedly, we detected little overlap among organs. In heart, few protein spots changed in abundance among the six states, while in kidney more than half of the spots detected differed among these same states. Only two significantly changing proteins were shared among all three datasets, and of proteins common between any two organs, many were discordant for abundance pattern changes among states. In sum, most proteomic changes accompanying hibernation appear to be organ-specific.

In this review, we explore the fish metalloproteins that have been discovered by ‘omic techniques, and their application as fish biomarkers of environmental contamination.

Profiling techniques have been suggested as a nontargeted approach to detect unintended effects in genetically modified (GM) plants. Seedlings from eight Brazilian maize varieties, four MON810 GM varieties and four non-GM isogenic varieties, were grown under controlled environmental conditions. Physiological parameters (aerial part weight, main leaf length, and chlorophyll and total protein contents) were compared, and some differences were observed. Nevertheless, these differences were not constant among all GM and non-GM counterparts. Leaf proteomic profiles were analyzed using two-dimensional gel electrophoresis (2DE) coupled to mass spectrometry, using six 2DE gels per variety. The comparison between MON810 and its counterpart was limited to qualitative differences of fully reproducible protein spot patterns. Twelve exclusive proteins were observed in two of four maize variety pairs; all of these leaf proteins were variety specific. In this study, MON810 leaf proteomes of four varieties were similar to non-GM counterpart leaf proteomes.

Refereed/Peer-reviewed

Two-dimensional gel electrophoresis (2-DE) is the most established protein separation method used in expression proteomics. Despite the existence of sophisticated software tools, 2-DE gel image analysis still remains a serious bottleneck. The low accuracies of commercial software packages and the extensive manual calibration that they often require for acceptable results show that we are far from achieving the goal of a fully automated and reliable, high-throughput gel processing system. We present a novel spot detection and quantification methodology which draws heavily from unsupervised machine-learning methods. Using the proposed hierarchical machine learning-based segmentation methodology reduces both the number of faint spots missed (improves sensitivity) and the number of extraneous spots introduced (improves precision). The detection and quantification performance has been thoroughly evaluated and is shown to compare favorably (higher F-measure) to a commercially available software package (PDQuest). The whole image analysis pipeline that we have developed is fully automated and can be used for high-throughput proteomics analysis since it does not require any manual intervention for recalibration every time a new 2-DE gel image is to be analyzed. Furthermore, it can be easily parallelized for high performance and also applied without any modification to prealigned group average gels.

Two-dimensional gel electrophoresis (2D-GE) is the powerful technique used by biochemists to resolve and visualize protein samples. The 2D-GE images depict protein signals as spots of various intensities and sizes. Currently, the most popular technique for spot detection in 2D-GE image is the watershed transform (WST) due to its robustness. However presence of noise in 2D-GE images forces WST to oversegment them. In this paper, we present a two-stage protein spot detection approach, which is achieved by improve marker controlled watershed transform (MCWST) followed by spot verification. Experiments results show that the proposed method is robust in noised image and results in low spurious spot detection rate.

Background The process of rod photoreceptor genesis, cell fate determination and differentiation is complex and multi-factorial. Previous studies have defined a model of photoreceptor differentiation that relies on intrinsic changes within the presumptive photoreceptor cells as well as changes in surrounding tissue that are extrinsic to the cell. We have used a proteomics approach to identify proteins that are dynamically expressed in the mouse retina during rod genesis and differentiation. Findings A series of six developmental ages from E13 to P5 were used to define changes in retinal protein expression during rod photoreceptor genesis and early differentiation. Retinal proteins were separated by isoelectric focus point and molecular weight. Gels were analyzed for changes in protein spot intensity across developmental time. Protein spots that peaked in expression at E17, P0 and P5 were picked from gels for identification. There were 239 spots that were picked for identification based on their dynamic expression during the developmental period of maximal rod photoreceptor genesis and differentiation. Of the 239 spots, 60 of them were reliably identified and represented a single protein. Ten proteins were represented by multiple spots, suggesting they were post-translationally modified. Of the 42 unique dynamically expressed proteins identified, 16 had been previously reported to be associated with the developing retina. Conclusions Our results represent the first proteomics study of the developing mouse retina that includes prenatal development. We identified 26 dynamically expressed proteins in the developing mouse retina whose expression had not been previously associated with retinal development.

Proteins of the piroplasms ofTheileria sergenti, T. buffeliandT. orientaliswere analysed by two-dimensional polyacrylamide gel electrophoresis. Protein spot patterns ofT. buffeliandT. orientaliswere identical except for a few minor proteins, whereas spot patterns of twoT. sergentistocks were differentiated from those ofT. buffeliandT. orientalisby a characteristic set of proteins including a major protein of molecular weight 33–34 kDa. This result indicates that JapaneseT. sergentican be phenotypically distinguishable from European and AustraliaTheileriaspecies;T. orientalisandT. buffeli.

As an initial screen for novel markers of renal cancer and to minimise background heterogeneity, we have compared the within-patient profiles of serum samples from seven patients pre- and post-nephrectomy. Samples were depleted of six of the most abundant proteins using Agilent's multiple affinity removal system (MARS) followed by solution-phase IEF prior to separation by 2-DE using narrow range IPG Strips, with a total of 84 gels. The reproducibility of the various steps was demonstrated and an approximate two-fold increase (from 374 to 779) in the number of protein spots observed in the pH region 4.6-7.0 was obtained. However, the majority of additional proteins seen were further isoforms of existing proteins due to the higher resolution and the majority of protein spots identified were still moderate to highly abundant species. Only one protein spot (as yet unidentified) was found to change significantly in the same direction in at least four patients. Although this powerful prefractionation and analysis strategy allows the visualisation of multiple protein isoforms, it is insufficient to allow detection of lower abundance proteins in serum without the implementation of further strategies.

Proteome is defined as the total set of proteins expressed in a given cell or biological sample at a given time. The study of proteome is termed “proteomics”. Proteomics research includes the separation, identification, qualitative, quantitative, and functional characterization of the entire protein profile of a given cell, tissue, and/or organism. In this chapter, we describe the processes of commonly used proteomics techniques like gel-based separation and mass-spectrometry.

Differential in-gel electrophoresis (DIGE) technique has been used for differential protein analysis to improve the reproducibility of comparative 2D gel experiments. Because the sample size in 2D DIGE experiments is usually very small, the traditional statistical methods such as student t-test could not provide an accurate and reliable detection/identification of differentially expressed protein spots. This paper proposed a ranking based approach for DE protein spot identification, in which the information of comparative ranks of the protein spot volume ratio rather than their distribution were used in selection. Two methods were designed to integrate the ranking information from a set of pair-wise comparisons. The evaluation results demonstrated that the proposed methods are effective in DE protein spot identification when sample size is small.

Proteomics has evolved greatly in the past few years and the number of researchers utilizing proteomics to investigate plant biology has substantially increased. The predominant aim of this chapter is to aid Medicago researchers in initiating proteomics experiments. The chapter consists of a brief literature review of Medicago proteomics, followed by protocols used in the authors’ labs. Further optimization of the protocols to suit ones individual needs may be required.

Assumptions that need to be considered prior to statistical analysis of protein spot volumes from two-dimensional gel electrophoresis (2-DE) data are studied using replicate gels of the same sample. The most important observation is that the data tables of protein spot volumes from 2-DE images contain a large number of missing values, which are not consistent with the presence or absence of the proteins. This implies both loss of information and problems for the subsequent statistical analysis. Challenges with 2-DE protein spot volumes are viewed in light of multiple gel comparisons and multivariate data analysis.