Comparative Proteomic Analysis of Wild and Cultivated Amaranth Species Seeds by 2-DE and ESI-MS/MS.

Amaranth is a promising staple food that produces seeds with excellent nutritional quality. Although cultivated species intended for grain production have interesting agronomic traits, relatively little is known about wild species, which can prosper in diverse environments and could be a rich genetic source for crop improvement. This work focuses on the proteomic comparison between the seeds of wild and cultivated amaranth species using polarity-based protein extraction and two-dimensional gel electrophoresis. Differentially accumulated proteins (DAPs) showed changes in granule-bound starch synthases and a wide range of 11S globulin isoforms. The electrophoretic profile of these proteins suggests that they may contain significant phosphorylation as post-translational modifications (PTMs), which were confirmed via immunodetection. These PTMs may impact the physicochemical functionality of storage proteins, with potential implications for seed agronomic traits and food system applications. Low-abundant DAPs with highly variable accumulation patterns are also discussed; these were involved in diverse molecular processes, such as genic regulation, lipid storage, and stress response.

Comparative proteomic analysis of patients with acute myeloid leukemia before and after induction therapy.

BACKGROUND: Acute myeloid leukemia (AML) is a malignant disorder of hematopoietic stem and progenitor cells, characterized by accumulation of immature blasts in the bone marrow and peripheral blood of affected patients. Response to chemotherapy treatment in patients with AML is wide-ranging, and to date there are no adequate molecular biomarkers used to predict clinical outcome. OBJECTIVE: The aim of this study was to identify potential protein biomarkers which could help predict response to induction treatment in AML patients. METHODS: Peripheral blood samples were obtained from 15 AML patients both before and after treatment. A comparative proteomic analysis was performed using 2D gel electrophoresis followed by Mass Spectrometry. RESULTS: This comparative proteomic study, combined with a protein network analysis, revealed several proteins that could be considered potential biomarkers of poor prognosis in AML: GAPDH which favors increased glucose metabolism; eEF1A1 and Annexin A1 that promote proliferation and migration, cofilin 1 which plays a role in the activation of apoptosis; and GSTP1 which is involved in the processes of detoxification and chemoresistance. CONCLUSIONS: This study gives an insight into a panel of protein biomarkers with prognostic potential that should be further investigated.

Identification of Allergenic Proteins in Velvet Mesquite (Prosopis velutina) Pollen: An Immunoproteomics Approach.

Velvet mesquite (Prosopis velutina) is a native legume of the southwestern United States and northwestern Mexico, contributing significantly to the desert ecosystem and playing key ecological roles. It is also an important cause of allergic respiratory disease widely distributed in the Sonoran, Chihuahuan, and Mojave Deserts. However, no allergens from velvet mesquite pollen have been identified to date. Pollen proteins were extracted and analyzed by one- and two-dimensional electrophoresis and immunoblotting using a pool of 11 sera from mesquite-sensitive patients as the primary antibody. IgE-recognized protein spots were identified by mass spectrometry and bioinformatics analysis. Twenty-four unique proteins, including proteins well known as pollen, food, airway, or contact allergens and four proteins not previously reported as pollen allergens, were identified. This is the first report on allergenic proteins in velvet mesquite pollen. These findings will contribute to the development of specific diagnosis and treatment of mesquite pollen allergy.

Proteomic analysis of Burkholderia zhejiangensis CEIB S4-3 during the methyl parathion degradation process.

Methyl parathion is an organophosphorus pesticide widely employed worldwide to control pests in agricultural and domestic environments. However, due to its intensive use, high toxicity, and environmental persistence, methyl parathion is recognized as an important ecosystem and human health threat, causing severe environmental pollution events and numerous human poisoning and deaths each year. Therefore, identifying and characterizing microorganisms capable of fully degrading methyl parathion and its degradation metabolites is a crucial environmental task for the bioremediation of pesticide-polluted sites. Burkholderia zhejiangensis CEIB S4-3 is a bacterial strain isolated from agricultural soils capable of immediately hydrolyzing methyl parathion at a concentration of 50 mg/L and degrading the 100% of the released p-nitrophenol in a 12-hour lapse when cultured in minimal salt medium. In this study, a comparative proteomic analysis was conducted in the presence and absence of methyl parathion to evaluate the biological mechanisms implicated in the methyl parathion biodegradation and resistance by the strain B. zhejiangensis CEIB S4-3. In each treatment, the changes in the protein expression patterns were evaluated at three sampling times, zero, three, and nine hours through the use of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and the differentially expressed proteins were identified by mass spectrometry (MALDI-TOF). The proteomic analysis allowed the identification of 72 proteins with differential expression, 35 proteins in the absence of the pesticide, and 37 proteins in the experimental condition in the presence of methyl parathion. The identified proteins are involved in different metabolic processes such as the carbohydrate and amino acids metabolism, carbon metabolism and energy production, fatty acids ß-oxidation, and the aromatic compounds catabolism, including enzymes of the both p-nitrophenol degradation pathways (Hydroquinone dioxygenase and Hydroxyquinol 1,2 dioxygenase), as well as the overexpression of proteins implicated in cellular damage defense mechanisms such as the response and protection of the oxidative stress, reactive oxygen species defense, detoxification of xenobiotics, and DNA repair processes. According to these data, B. zhejiangensis CEIB S4-3 overexpress different proteins related to aromatic compounds catabolism and with the p-nitrophenol  degradation pathways, the higher expression levels observed in the two subunits of the enzyme Hydroquinone dioxygenase, suggest a preferential use of the Hydroquinone metabolic pathway in the p-nitrophenol degradation process. Moreover the overexpression of several proteins implicated in the oxidative stress response, xenobiotics detoxification, and DNA damage repair reveals the mechanisms employed by B. zhejiangensis CEIB S4-3 to counteract the adverse effects caused by the methyl parathion and p-nitrophenol exposure.

Immunoproteomic identification of allergenic proteins in pecan (Carya illinoinensis) pollen.

Pecan (C. illinoinensis) pollen is an important cause of allergic respiratory disease. Pecan is distributed worldwide as shade, ornamental or cultivation tree. To date three well known pecan food allergens have been reported, however, pollen allergens have not been identified. Here, we describe the first identification of IgE recognized pecan pollen proteins, for which proteins were analyzed by 2-DE and immunoblotting using a pool of 8 sera from pecan sensitive patients as primary antibody. IgE recognized protein spots were analyzed by LC-MS/MS and identified using a database of translated protein sequences obtained by the assembly of C. illinoinensis public transcriptomic information. This study has identified 17 IgE binding proteins from pecan pollen including proteins widely recognized as allergens and panallergens. These findings will contribute to develop specific diagnosis and treatment of pecan pollen allergy. SIGNIFICANCE: Pecan is a tree highly valued for its fruits that have a great commercial value. To date three pecan seed storage proteins have been officially recognized by the WHO/IUIS allergen nomenclature subcommittee as food allergens (Car i 1, Car i 2 and Car i 4). Pecan tree pollen is highly allergenic and a clinically relevant cause of allergies in North America (USA and Mexico) and regions where the tree is extensively cultivated (Israel, South Africa, Australia, Egypt, Peru, Argentina, and Brazil). Here, we describe the first identification of IgE recognized pollen proteins using an immunoproteomics approach and a protein database created by the assembly of pecan public transcriptomic information. The findings described here will allow the development of new diagnostic and therapeutic modalities for pecan pollen allergy.

Joint pre-processing framework for two-dimensional gel electrophoresis images based on nonlinear filtering, background correction and normalization techniques.

BACKGROUND: Two-dimensional gel electrophoresis (2-DGE) is a commonly used tool for proteomic analysis. This gel-based technique separates proteins in a sample according to their isoelectric point and molecular weight. 2-DGE images often present anomalies due to the acquisition process, such as: diffuse and overlapping spots, and background noise. This study proposes a joint pre-processing framework that combines the capabilities of nonlinear filtering, background correction and image normalization techniques for pre-processing 2-DGE images. Among the most important, joint nonlinear diffusion filtering, adaptive piecewise histogram equalization and multilevel thresholding were evaluated using both synthetic data and real 2-DGE images. RESULTS: An improvement of up to 46% in spot detection efficiency was achieved for synthetic data using the proposed framework compared to implementing a single technique of either normalization, background correction or filtering. Additionally, the proposed framework increased the detection of low abundance spots by 20% for synthetic data compared to a normalization technique, and increased the background estimation by 67% compared to a background correction technique. In terms of real data, the joint pre-processing framework reduced the false positives up to 93%. CONCLUSIONS: The proposed joint pre-processing framework outperforms results achieved with a single approach. The best structure was obtained with the ordered combination of adaptive piecewise histogram equalization for image normalization, geometric nonlinear diffusion (GNDF) for filtering, and multilevel thresholding for background correction.

Proteomic identification of allergenic proteins in red oak (Quercus rubra) pollen.

BACKGROUND: Red oak pollen is an important cause of allergic respiratory disease and it is widely distributed in North America and central Europe. To date, however, red oak pollen allergens have not been identified. Here, we describe the allergenic protein profile from red oak pollen. METHODS: Total proteins were extracted from red oak pollen using a modified phenolic extraction method, and, subsequently, proteins were separated by two-dimensional gel electrophoresis (2DE) for both total protein stain (Coomassie Blue) and immunoblotting. A pool of 8 sera from red oak sensitive patients was used to analyze blotted proteins. Protein spots were analyzed by Mass Spectrometry. RESULTS: Electrophoretic pattern of total soluble proteins showed higher intensity bands in the regions of 26-40 and 47-52 kDa. Two dimensional immunoblots using pool sera from patients revealed four allergenic proteins spots with molecular masses in the range from 50 to 55 kDa. Mass spectrometry analysis identified 8 proteins including Enolase 1 and Enolase 1 chloroplastic, Xylose isomerase (X1 isoform), mitochondrial Aldehyde dehydrogenase, UTP-Glusose-1-phosphate uridylyltransferase, Betaxylosidase/alpha-l-arabinofuranosidase and alpha- and beta subunits of ATP synthase. CONCLUSIONS: This study has identified for first time 8 IgE binding proteins from red oak pollen. These findings will pave the way towards the development of new diagnostic and therapeutic modalities for red oak allergy.

Molecular characterization and in vitro interaction analysis of Op14-3-3 µ protein from Opuntia ficus-indica: identification of a new client protein from shikimate pathway.

In plants, 14-3-3 proteins are important modulators of protein-protein interactions in response to environmental stresses. The aim of the present work was to characterize one Opuntia ficus-indica 14-3-3 and get information about its client proteins. To achieve this goal, O. ficus-indica 14-3-3 cDNA, named as Op14-3-3 µ, was amplified by 3'-RACE methodology. Op14-3-3 µ contains an Open Reading Frame of 786 bp encoding a 261 amino acids protein. Op14-3-3 µ cDNA was cloned into a bacterial expression system and recombinant protein was purified. Differential Scanning Fluorimetry, Dynamic Light Scattering, and Ion Mobility-Mass Spectrometry were used for Op14-3-3 µ protein characterization, and Affinity-Purification-Mass Spectrometry analysis approach was used to obtain information about their potential client proteins. Pyrophosphate-fructose 6-phosphate 1-phosphotransferase, ribulose bisphosphate carboxylase large subunit, and vacuolar-type H+-ATPase were identified. Interestingly chorismate mutase p-prephenate dehydratase was also identified. Op14-3-3 µ down-regulation was observed in Opuntia calluses when they were induced with Jasmonic Acid, while increased accumulation of Op14-3-3 µ protein was observed. The putative interaction of 14-3-3 µ with chorismate mutase, which have not been reported before, suggest that Op14-3-3 µ could be an important regulator of metabolites biosynthesis and responses to stress in Opuntia spp. SIGNIFICANCE: Opuntia species are important crops in arid and semiarid areas worldwide, but despite its relevance, little information about their tolerance mechanism to cope with harsh environmental conditions is reported. 14-3-3 proteins have gained attention due to its participation as protein-protein regulators and have been linked with primary metabolism and hormones responses. Here we present the characterization of the first Opuntia ficus-indica 14-3-3 (Op14-3-3) protein using affinity purification-mass spectrometry (AP-MS) strategy. Op14-3-3 has high homology with other 14-3-3 from Caryophyllales. A novel Op14-3-3 client protein has been identified; the chorismate mutase p-prephenate dehydratase, key enzyme that links the primary with secondary metabolism. The present results open new questions about the Opuntia spp. pathways mechanisms in response to environmental stress and the importance of 14-3-3 proteins in betalains biosynthesis.

Proteome comparison for discrimination between honeydew and floral honeys from botanical species Mimosa scabrella Bentham by principal component analysis.

BACKGROUND: Due to the increasing valuation and appreciation of honeydew honey in many European countries and also to existing contamination among different types of honeys, authentication is an important aspect of quality control with regard to guaranteeing the origin in terms of source (honeydew or floral) and needs to be determined. Furthermore, proteins are minor components of the honey, despite the importance of their physiological effects, and can differ according to the source of the honey. In this context, the aims of this study were to carry out protein extraction from honeydew and floral honeys and to discriminate these honeys from the same botanical species, Mimosa scabrella Bentham, through proteome comparison using two-dimensional gel electrophoresis and principal component analysis. RESULTS: The results showed that the proteome profile and principal component analysis can be a useful tool for discrimination between these types of honey using matched proteins (45 matched spots). Also, the proteome profile showed 160 protein spots in honeydew honey and 84 spots in the floral honey. CONCLUSION: The protein profile can be a differential characteristic of this type of honey, in view of the importance of proteins as bioactive compounds in honey. © 2017 Society of Chemical Industry.

Two-Dimensional Gel Electrophoresis: A Reference Protocol.

Two-dimensional gel electrophoresis (2DE) has been a mainstay of proteomic techniques for more than four decades. It was even in use for several years before the term proteomics was actually coined in the early 1990s. Over this time, it has been used in the study of many diseases including cancer, diabetes, heart disease, and psychiatric disorders through the proteomic analysis of body fluids and tissues. This chapter presents a general protocol which can be applied in the study of biological samples such as blood serum or plasma and multiple tissues including the brain.

Comparative proteomic analysis of female and male venoms from the Mexican scorpion Centruroides limpidus: Novel components found.

Venom from male and female scorpions of the species Centruroides limpidus were separated by HPLC and their molecular masses determined by mass spectrometry. The relative concentration of components eluting in equivalent retention times from the HPLC column shows some differences. A new peptide with 29 amino acids, cross-linked by three disulfide bonds was found in male scorpions and its structure determined. Another unknown peptide present in female venom, with sequence identity similar to K+-channel blocking peptide was isolated. This peptide contains 39 amino acid residues linked by three disulfide bonds. Due to sequence similarities, a systematic number (αKTx2.18) was assigned. Venom from male and female scorpions was separated by Sephadex G-50 gel filtration. Components of fraction I of this chromatogram were analyzed by two-dimensional gel electrophoresis and 41 spots were selected (20 from female and 21 from male). The spots were excised from the gel, enzymatically digested and sequenced by LC-MS/MS. This procedure allowed the identification of several proteins containing similar amino acid sequence of other known proteins registered on UniProt database. Among these proteins the presence of metalloproteinases (proteolytic enzymes), hyaluronidases and phosphatases were experimentally determined and shown to be present in both venom samples. The results shown here should help further work aimed at fully identification of the structure and function of venom components form C. limpidus male and female scorpions.

Análisis proteómico del veneno de la abeja africanizada: comparación de métodos de extracción / Proteomic Analysis of Africanized Bee Venom: a Comparison of Protein Extraction Methods

La abeja africanizada es la más común en la apicultura colombiana y a su veneno (apitoxina) se le han atribuido propiedades terapéuticas para diferentes enfermedades, sin mayor soporte científico. Al revisar en la literatura los reportes publicados sobre el análisis proteómico de la apitoxina, se encontraron cuatro métodos distintos para la extracción de proteínas de la apitoxina. El primer método consiste en resuspender la apitoxina en Urea 7 M, precipitar con acetona y finalmente resuspender en Urea 7 M y CHAPS 4 %. Para el segundo método se resuspende la apitoxina en buffer de lisis, se precipita con ácido tricloroacético, y luego se resuspende en Urea 7 M y CHAPS 4 %. El tercer método es igual al anterior, excepto que la precipitación se realiza con acetona en vez de ácido tricloroacético. Finalmente, el cuarto método consiste en resuspender la apitoxina en agua destilada, precipitar con acetona y resuspender en Urea 7 M y CHAPS 4 %. Este trabajo se enfocó en comparar el desempeño de estos cuatro métodos de extracción y determinar el método con el mejor resultado en cuanto a la concentración e integridad obtenida de las proteínas. De los distintos métodos evaluados, se encontró que los mejores resultados en cuanto a concentración de proteínas se obtuvieron con la resuspensión de apitoxina en buffer de lisis y precipitación con acetona (método 3) y con el método de resuspensión de apitoxina en agua destilada y precipitación con acetona (método 4). De estos, el mejor método de extracción en cuanto a integridad de las proteínas y perfil proteómico fue el de resuspensión de apitoxina en buffer de lisis seguido de precipitación con acetona (método 3).

The Africanised bee is the most common type of bee in Colombia, and therapeutic properties for different diseases have been attributed to its venom, without much scientific support. A literature search of reports on the proteomic analysis of honeybee venom yielded four different methods for extracting proteins from bee venom. The first method consists in resuspending the venom in 7 M Urea, followed by precipitation with acetone and finally resuspending the pellet in 7 M Urea and 4 % CHAPS. For the second method, the venom is resuspended in lysis buffer, precipitated with trichloroacetic acid, and then resuspended in 7 M Urea and 4 % CHAPS. The third method is similar to the previous one, except that the precipitation step is performed with acetone instead of trichloroacetic acid. Finally, the fourth method is to resuspend the venom in distilled water, precipitate with acetone and resuspend in 7 M Urea and 4 % CHAPS. This work focused on comparing the performance of these four extraction methods, in order to determine the method with the best results in terms of concentration and integrity of the proteins obtained. Of the four methods evaluated, the best results in terms of protein concentration and yield were obtained by resuspending the bee venom in lysis buffer followed by precipitation with acetone (method 3), and by resuspending in distilled water followed by precipitation with acetone (method 4). Of these, the method that maintained protein integrity and yielded the best proteomic profile was that in which the bee venom was resuspended in lysis buffer followed by precipitation with acetone (method 3).

Effects of (-)-6,6'-dinitrohinokinin on adult worms of Schistosoma mansoni: a proteomic analyses

Abstract Schistosomiasis, a chronic disease that affects million people worldwide, is caused by trematode flukes of the genus Schistosoma. The lack of an anti-schistosomiasis vaccine and massive monotherapy with praziquantel reinforces the need for search and development of new therapeutic drugs. Recently, we demonstrated that the essential oil of Piper cubeba L., Piperaceae, and their derivative dibenzylbutyrolactolic (-)-6,6'-dinitrohinokinin, presents in vitro and in vivo activities against Schistosoma mansoni. Here, we identified changes in the protein expression after exposure to dibenzylbutyrolactolic (-)-6,6'-dinitrohinokinin. We applied two-dimensional gel electrophoresis (2-DE) to S. mansoni soluble protein extracts and observed at least 38 spots to be affected by dibenzylbutyrolactolic (-)-6,6'-dinitrohinokinin. We further identified 25 differentially expressed proteins by mass spectrometry. Enrichment for biological processes and predictive analyses of protein-protein interactions suggest that dibenzylbutyrolactolic (-)-6,6'-dinitrohinokinin targets proteins involved mainly in metabolic processes, especially carbohydrate metabolism. In summary, this study provides an interesting approach to understand the anti-parasitic activity of semi-synthetic (-)-6,6'-dinitrohinokinin a derivative compound from lignan and for the development of new therapy strategies.

Quantification, 2DE analysis and identification of enriched glycosylated proteins from mouse muscles: Difficulties and alternatives.

One of the problems with 2DE is that proteins present in low amounts in a sample are usually not detected, since their signals are masked by the predominant proteins. The elimination of these abundant proteins is not a guaranteed solution to achieve the desired results. The main objective of this study was the comparison of common and simple methodologies employed for 2DE analysis followed by MS identification, focusing on a pre-purified sample using a wheat germ agglutinin (WGA) column. Adult male C57Black/Crj6 (C57BL/6) mice were chosen as the model animal in this study; the gastrocnemius muscles were collected and processed for the experiments. The initial fractionation with succinylated WGA was successful for the elimination of the most abundant proteins. Two quantification methods were employed for the purified samples, and bicinchoninic acid (BCA) was proven to be most reliable for the quantification of glycoproteins. The gel staining method, however, was found to be decisive for the detection of specific proteins, since their structures affect the interaction of the dye with the peptide backbone. The Coomassie Blue R-250 dye very weakly stained the gel with the WGA purified sample. When the same gel was stained with silver nitrate, however, MS could positively assign 12 new spots. The structure of the referred proteins was not found to be prone to interaction with Coomassie blue.

Identification of Ligustrum lucidum pollen allergens using a proteomics approach.

BACKGROUND: Ligustrum spp. are members of the Oleaceae family, one of the most prominent allergic families worldwide. The genus Ligustrum contains approximately fifty species, including Ligustrum lucidum, which have been widely cultivated as ornamental plants, and its pollen is a source of inhalant allergens associated with respiratory allergic diseases. Little is known about the presence of allergenic proteins in L. lucidum. METHODS: The L. lucidum pollen proteins were extracted by a modified phenolic extraction method. A pool of four sera from mono sensitive patients was analyzed by 2DE immunoblotting and mass spectrometric analysis was performed on 6 immunoreactive protein spots. RESULTS: SDS-PAGE of L. lucidum pollen extract revealed proteins in ranges of 15-150 kDa. The 2DE gel profile of the L. lucidum pollen protein extract showed approximately 180 spots, and the 2DE immunoblots obtained using sera from Ligustrum monosensitive patients as the source of IgE antibodies revealed six allergen protein spots, corresponding to Profilin, Enolase, Fra e 9.01 (ß-1,3-glucanase), Pollen-specific Polygalacturonases, Alanine aminotransferase, and two ATP synthase beta subunits. CONCLUSION: We report for the first time the identification of IgE-reactive proteins from L. lucidum.

Proteomics analysis after traumatic brain injury in rats: the search for potential biomarkers / Análise proteômica após lesão cerebral traumática em ratos: a busca por biomarcadores

Many studies of protein expression after traumatic brain injury (TBI) have identified biomarkers for diagnosing or determining the prognosis of TBI. In this study, we searched for additional protein markers of TBI using a fluid perfusion impact device to model TBI in S-D rats. Two-dimensional gel electrophoresis and mass spectrometry were used to identify differentially expressed proteins. After proteomic analysis, we detected 405 and 371 protein spots within a pH range of 3-10 from sham-treated and contused brain cortex, respectively. Eighty protein spots were differentially expressed in the two groups and 20 of these proteins were identified. This study validated the established biomarkers of TBI and identified potential biomarkers that could be examined in future work.

Muitos estudos de expressão proteica após lesão cerebral traumática (LCT) identificam biomarcadores para determinação diagnóstica ou prognóstica do LCT. No presente estudo, foram investigados marcadores proteicos adicionais de LCT, através de um aparelho de impacto no fluxo e perfusão em ratos S-D. Eletroforese bidimensional em gel e espectrometria de massa foram utilizadas para identificar diferentes proteínas expressas. Após a análise proteômica, detectamos marcas de proteínas 405 e 371, com pH variando entre 3-10 no córtex de ratos sham e naqueles com contusão cerebral, respectivamente. Oitenta marcas proteicas foram expressas nos dois grupos e 20 destas proteínas foram identificadas. Este estudo validou o estabelecimento de biomarcadores de LCT e identificou potencial biomarcadores que poderão ser estudados em estudos futuros.

Changes in protein expression of pacific oyster Crassostrea gigas exposed in situ to urban sewage.

The composition and concentration of substances in urban effluents are complex and difficult to measure. These contaminants elicit biological responses in the exposed organisms. Proteomic analysis is a powerful tool in environmental toxicology by evidencing alterations in protein expression due to exposure to contaminants and by providing a useful framework for the development of new potential biomarkers. The aim of this study was to determine changes in protein expression signatures (PES) in the digestive gland of oysters Crassostrea gigas transplanted to two farming areas (LIS and RIB) and to one area contaminated by sanitary sewage (BUC) after 14 days of exposure. This species is one of the most cultivated molluscs in the world. The identified proteins are related to the cytoskeleton (CKAP5 and ACT2), ubiquitination pathway conjugation (UBE3C), G protein-coupled receptor and signal transduction (SVEP1), and cell cycle/division (CCNB3). CKAP5 showed higher expression in oysters kept at BUC in comparison with those kept at the farming areas, while ACT2, UBE3C, SVEP1, and CCNB3 were suppressed. The results suggest that these changes might lead to DNA damage, apoptosis, and interference with the immune system in oyster C. gigas exposed to sewage and give initial information on PES of C. gigas exposed to sanitary sewage, which can subsequently be useful in the development of more sensitive tools for biomonitoring coastal areas, particularly those devoted mainly to oyster farming activities.

2DE: the phoenix of proteomics.

Given the rapid developments in mass spectrometry (MS) in terms of sensitivity, mass accuracy, and throughput, some have suggested that two-dimensional gel electrophoresis (2DE) may no longer be a method of choice for proteomic analyses. However, as recognition of issues with these newer shotgun-MS approaches grows, there is a fresh and growing regard for the maturity of 2DE-MS as a genuine top-down analytical approach, particularly as it resolves thousands of intact protein species in a single run, enabling the simultaneous analysis of total protein complement, including isoforms and post-translational modifications. Given the strengths of both, it is most appropriate to view these as complementary or at least parallel approaches: as proteins encompass a myriad of physico-chemical properties, and the real aim is to explore proteomes as deeply as possible, all available resolving strategies must be considered in terms of the complexity encountered. It is time to critically and constructively focus on the optimization and integration of existing techniques rather than simplistically suggesting that one should replace the other. Our intention here is thus to present an overview of protein resolving techniques, focusing on milestones associated with 2DE, including pros, cons, advances and variations, in particular relative to shotgun proteomic approaches. BIOLOGICAL SIGNIFICANCE: Proteomic researchers recognize the importance of 2DE in the history of proteomics. But the latest developments in mass spectrometry-based techniques have led some researchers to retire 2DE in their labs. However, we argue here that 2DE-MS is a genuine top-down analytical approach. The significance of this discussion is to make proteomic researchers aware of the importance of this technique in a proteomic pipeline. This article is part of a Special Issue entitled: Environmental and structural proteomics.

Comparative analysis of the venom proteome of four important Malaysian snake species

Naja kaouthia, Ophiophagus hannah, Bungarus fasciatus and Calloselasma rhodostoma are four venomous snakes indigenous to Malaysia. In the present study, their proteomic profile by two-dimensional gel electrophoresis (2-DE) have been separated and compared.

Comparative analysis of the venom proteome of four important Malaysian snake species

Naja kaouthia, Ophiophagus hannah, Bungarus fasciatus and Calloselasma rhodostoma are four venomous snakes indigenous to Malaysia. In the present study, their proteomic profile by two-dimensional gel electrophoresis (2-DE) have been separated and compared.