Kinetics for the Removal of Paraquat Dichloride from Aqueous Solution by Activated Date (Phoenix dactylifera) Stone Carbon

For the adsorption of Paraquat dichloride (1,1′-dimethyl-4,4′-bipyridyl dichloride) from synthetic aqueous solutions, batch experiments were carried out to study the kinetics of Paraquat dichloride onto calcium oxide activated date (Phoenix dactylifera) stone carbon powder (particle size dia. about 0.85 mm). Studies were conducted at various initial concentrations of 75 mg/L, 100 mg/L, and 125 mg/L of Paraquat dichloride with 8.0 g/L of adsorbent dose. The equilibrium time was found to be 140 minutes. Maximum removal was observed at pH 9.0. The regression analysis for the experimental data showed that the removal kinetic profiles followed a pseudo-second order kinetic models for all initial concentrations, pH, temperature, and salinity of sorbate. The experimental data were analyzed using Langmuir, Freundlich, and Dubinin-Radushkevich (D-R) isotherm models. Thermodynamic parameters, such as standard change in Gibbs free energy, standard change in enthalpy, and standard change in entropy, were evaluated, indicating that the overall adsorption process was exothermic and spontaneous in nature.     

适于双向电泳分析的苹果叶片蛋白质提取方法

为了探索适用于双向电泳(2-DE)分析的苹果叶片蛋白质提取方法,比较了三氯乙酸(TCA)/丙酮沉淀法、二硫苏糖醇(DTT)/丙酮法、Tris-HCl提取法和改良的Tris-HCl提取法等4种蛋白质提取方法。以7 cm、pH 3～10的线性固相pH梯度(immobilized pH gradient,IPG)胶条作为第一向电泳,以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)(12.5%的分离胶)作为第二向电泳,对提取物进行2-DE分离,采用银染显色。结果表明,上述4种方法在2-DE图谱上分别得到140,215,181和616个蛋白质点。其中以改良的Tris-HCl提取法得到的蛋白质点数最多,且背景清晰、图谱上没有明显的横纵条纹。为了进一步验证改良的Tris-HCl提取法的有效性,用18 cm、pH 3～10的线性IPG胶条和12.5%的分离胶对提取的苹果叶片蛋白质进行2-DE分离,考马斯亮蓝R-250染色,共检测到455个蛋白质点,其相对分子质量主要分布在14000～66000范围内,图谱背景清晰,再次证明应用该方法制备的样品适用于双向电泳分析,可用于苹果叶片的蛋白质组学分析。     

Protein extraction for 2-DE from the lamina of Ecklonia kurome (laminariales): recalcitrant tissue containing high levels of viscous polysaccharides

Extraction of proteins from the tissues of laminarialean algae, i.e. kelp, is difficult due to high levels of nonprotein interfering compounds, mainly viscous polysaccharides. To establish proteomic analysis of kelp species, an ethanol/phenol extraction method was developed and compared to other popular methods. Proteins were extracted with phenol from crude protein powder, obtained by homogenizing the kelp tissues in ice-cold ethanol. The ethanol/phenol method produced high-quality proteins of the highest purity from the lamina of Ecklonia kurome, one of the Japanese dominant laminarialean algae. This method gave well-resolved 1-D SDS-PAGE or 2-DE images with low background and the highest number of bands or spots. In particular, proteins with neutral to basic pI's were efficiently extracted. Furthermore, 27 spots on the 2-DE gel were extensively identified by MALDI-TOF/TOF analysis. To the best of our knowledge, this is the first report of a protocol for protein extraction from kelp tissues that gives satisfactory 2-D protein profiles. It is expected that the protocol can be applied to other algae tissues or other recalcitrant plant tissues containing high levels of nonprotein interfering compounds.     

Protein extraction for two-dimensional electrophoresis from olive leaf,a plant tissue containing high levels of interfering compounds

The purpose of this research is to establish a routine procedure for the application of proteomic analysis to olive tree. Olive leaf tissue is notoriously recalcitrant to common protein extraction methods due to high levels of interfering compounds. We developed a protocol for isolating proteins suitable for two-dimensional electrophoresis (2-DE) from olive leaf. The remarkable characteristics of the protocol include: (i) additional grinding dry acetone powder of leaf tissue to a finer extent, (ii) after extensive organic solvent washes to remove pigments, lipids etc., using aqueous tricholoroacetic acid washes to remove water-soluble contaminants, and (iii) phenol extraction of proteins in the presence of sodium dodecyl sulfate. The final protein preparation is free of interfering compounds based on its well-resolved 2-DE patterns. The protocol can be completed within 3 h, and protein yield is approximately 2.49 mg.g(-1) of aged leaf. We also evaluated the protocol by immunoblotting with anti-tyrosinate alpha-tubulin antibody. To our knowledge, this is the first time that a protocol for protein extraction from olive leaf appears to give satisfactory and reproducible results. The protocol is expected to be applicable to other recalcitrant plant tissues and could be of interest to laboratories involved in plant proteomics.     

Protein extraction and 2-DE of water- and lipid-soluble proteins from bovine pericardium, a low-cellularity tissue

Bovine pericardium (BP) is an important biomaterial used in the production of glutaraldehyde-fixed heart valves and tissue-engineering applications. The ability to perform proteomic analysis on BP is useful for a range of studies, including investigation of immune rejection after implantation. However, proteomic analysis of fibrous tissues such as BP is challenging due to their relative low-cellularity and abundance of extracellular matrix. A variety of methods for tissue treatment, protein extraction, and fractionation were investigated with the aim of producing high-quality 2-DE gels for both water- and lipid-soluble BP proteins. Extraction of water-soluble proteins with 3-(benzyldimethylammonio)-propanesulfonate followed by n-dodecyl beta-D-maltoside extraction and ethanol precipitation for lipid-soluble proteins provided the best combination of yield, spot number, and resolution on 2-DE gels (Protocol E2). ESI-quadrupole/ion trap or MALDI-TOF/TOF MS protein identifications were performed to confirm bovine origin and appropriate subcellular prefractionation of resolved proteins. Twenty-five unique, predominantly cytoplasmic bovine proteins were identified from the water-soluble fraction. Thirty-two unique, predominantly membrane bovine proteins were identified from the lipid-soluble fraction. These results demonstrated that the final protocol produced high-quality proteomic data from this important tissue for both cytoplasmic and membrane proteins.     

A simple protocol for protein extraction of recalcitrant fruit tissues suitable for 2-DE and MS analysis

Fruit tissues are considered recalcitrant plant tissue for proteomic analysis. Three phenol-free protein extraction procedures for 2-DE were compared and evaluated on apple fruit proteins. Incorporation of hot SDS buffer, extraction with TCA/acetone precipitation was found to be the most effective protocol. The results from SDS-PAGE and 2-DE analysis showed high quality proteins. More than 500 apple polypeptides were separated on a small scale 2-DE gel. The successful protocol was further tested on banana fruit, in which 504 and 386 proteins were detected in peel and flesh tissues, respectively. To demonstrate the quality of the extracted proteins, several protein spots from apple and banana peels were cut from 2-DE gels, analyzed by MS and have been tentatively identified. The protocol described in this study is a simple procedure which could be routinely used in proteomic studies of many types of recalcitrant fruit tissues.     

A novel method of protein extraction from perennial Bupleurum root for 2-DE

The perennial Bupleurum root is thick and woody and contains high levels of interfering compounds. Common protein extraction methods have proved refractory towards the isolation of proteins suitable for 2-DE, due to the presence of interfering compounds. A novel method for extracting proteins suitable for 2-DE was established to overcome these problems. The main characteristic of this protocol is the partitioning of the proteins into the aqueous (fraction A-2), chloroform and isoamyl alcohol phases (A-3), and the interphase (A-1). The proteins are then extracted from each of these phases. From A-1, 85% (extracted protein against total proteins) proteins could be extracted and purified. For fraction A-2, a novel phenol extraction step is employed for the extraction of proteins. Based on the well-resolved 2-DE patterns, our protein preparation is free of interfering compounds. Using these methods (A-1, A-2, and A-3-3), a total of 3662 (1526 + 1128 + 1008) spots could be separated, and a protein yield of about 1.41 mg per 1.0 g fresh root material was obtained. To our knowledge, this is the first time that a protocol for protein extraction from perennial Bupleurum root has been reported that gives reproducible results. The protocol is expected to be applicable to other recalcitrant plant tissues as well.     

An improved pixel-based approach for analyzing images in two-dimensional gel electrophoresis

An improved pixel-based approach for analyzing 2-DE images is presented. The key feature of the method is to create a mask based on all gels in the experiment using image morphology, followed by multivariate analysis on the pixel level. The method reduces the impact of noise and background by identifying regions in the image where protein spots are present, but make no assumption on individual spot boundaries for isolated spots. This makes it possible to detect significant changes in complex regions, and visualize these changes over multiple gels in an easy way. False missing values and spot volumes caused by imposing erroneous spot boundaries are thus circumvented. The approach presented gives improved pixel-based information from the gels, and is also an alternative to existing methods for data-reduction, significance testing and visualization of 2-DE data. Results are compared with software using a common spot boundary approach on an experiment consisting of 35 full size gel images. Gel alignment is required before analysis.     

Effective protein extraction protocol for proteomics studies of Jerusalem artichoke leaves

Protein extraction is a crucial step for proteomics studies. To establish an effective protein extraction protocol suitable for two‐dimensional electrophoresis (2DE) analysis in Jerusalem artichoke (Helianthus tuberosus L.), three different protein extraction methods—trichloroacetic acid/acetone, Mg/NP‐40, and phenol/ammonium acetate—were evaluated using Jerusalem artichoke leaves as source materials. Of the three methods, trichloroacetic acid/acetone yielded the best protein separation pattern and highest number of protein spots in 2DE analysis. Proteins highly abundant in leaves, such as Rubisco, are typically problematic during leaf 2DE analysis, however, and this disadvantage was evident using trichloroacetic acid/acetone. To reduce the influence of abundant proteins on the detection of low‐abundance proteins, we optimized the trichloroacetic acid/acetone method by incorporating a PEG fractionation approach. After optimization, 363 additional (36.2%) protein spots were detected on the 2DE gel. Our results suggest that trichloroacetic acid/acetone method is a better protein extraction technique than Mg/NP‐40 and phenol/ammonium acetate in Jerusalem artichoke leaf 2DE analysis, and that trichloroacetic acid/acetone method combined with PEG fractionation procedure is the most effective approach for leaf 2DE analysis of Jerusalem artichoke.     

A new method for assigning common spot boundaries for multiple gels in two-dimensional gel electrophoresis

The benefits of defining common spot boundaries when several gels from 2-DE are compared and analyzed have lately been stressed by both commercial software producers and users of this software. Though the importance of common spot boundaries is clearly stated, few reports exist that target this issue explicitly. In this study a method for defining common spots boundaries is developed, called the spot density method. The method consists of the following steps: segmentation and spot identification on each individual gel, transferring the spot-center coordinates for all gels onto a single new gel, collecting spot centers clustered together in the new gel and finally assigning pixels and new spot boundaries based on the spots in each cluster. The method is compared to a synthetic gel approach, and validated by visual inspection of three representative areas in the gels. The gel images need to be aligned prior to segmentation and spot identification, but the method can be used regardless of the choice of segmentation procedure. This makes the method an easy extension to existing methods for spot identification and matching. Conclusions based on the visual inspection are that the spot density method identifies partly overlapping spots and low-intensity spots better than the synthetic gel approach.     

Efficient extraction of proteins from recalcitrant plant tissue for subsequent analysis by two‐dimensional gel electrophoresis

Protein extraction for two‐dimensional electrophoresis from tissues of recalcitrant species is quite problematic and challenging due to the low protein content and high abundance of contaminants. Proteomics in Shorea robusta is scarcely conducted due to the lack of a suitable protein preparation procedure. To establish an effective protein extraction protocol suitable for two‐dimensional electrophoresis in Shorea robusta, four procedures (borate buffer/trichloroacetic acid extraction, organic solvent/trichloroacetic acid precipitation, sucrose/Tris/phenol, and organic solvent/phenol/sodium dodecyl sulfate) were evaluated. Following these, proteins were isolated from mature leaves and were analyzed for proteomics, and also for potential contaminants, widely reported to hinder proteomics. The borate buffer/trichloroacetic acid extraction had the lowest protein yield and did not result in any banding even in one‐dimensional electrophoresis. In contrast, organic solvent/phenol/sodium dodecyl sulfate extraction allowed the highest protein yield. Moreover, during proteomics, organic solvent/phenol/sodium dodecyl sulfate extracted protein resolved the maximum number (144) of spots. Further, when proteins were evaluated for contaminants, significant (77–95%) reductions in the nucleic acids, phenol, and sugars were discernible with refinement in extraction procedure. Accumulated data suggested that the organic solvent/phenol/sodium dodecyl sulfate extraction was the most effective protocol for protein isolation for proteomics of Shorea robusta and can be used for plants that have a similar set of contaminants.     

A comparative survey of proteins from recalcitrant tissues of a non-model gymnosperm, Douglas-fir

Most research in plants and other organisms has, for the sake of convenience, focused on the use of model species to identify mechanisms that are conserved throughout the whole kingdom. Nevertheless, unique features and processes such as those related to plant cell wall and fiber formation, and to wood quality, sometimes need to be studied directly in the non-model organism of interest. Such organisms, like the economically and ecologically important gymnosperm Douglas-fir (Pseudotsuga menziesii), which is one of the crucial softwood timber species in Northern America, are often difficult to investigate. High phenolic, resin, and tannin contents in the woody tissues, as well as an incompletely sequenced genome, have contributed greatly to the species' recalcitrance for molecular biology investigations. In this study, we present a complete procedure detailing protein sample preparation, separation, and proteomic analysis based on cross-species identification of Douglas-fir. Proteins from the cambial zone, mature needles, and in vitro callus were extracted, purified, and separated via 1D and 2D SDS-PAGE. One-dimensional electrophoresis coupled with ESI-MS/MS was used for cross-species protein identification in order to evaluate the potential of this approach and reveal major differences in protein profiles among tested tissues. Identified proteins were functionally and developmentally compared. The likely contribution of these proteins to the properties of the cell wall and wood is indicated and discussed.     

Protein extraction for proteome analysis from cacao leaves and meristems, organs infected by Moniliophthora perniciosa, the causal agent of the witches' broom disease

Preparation of high-quality proteins from cacao vegetative organs is difficult due to very high endogenous levels of polysaccharides and polyphenols. In order to establish a routine procedure for the application of proteomic and biochemical analysis to cacao tissues, three new protocols were developed; one for apoplastic washing fluid (AWF) extraction, and two for protein extraction--under denaturing and nondenaturing conditions. The first described method allows a quick and easy collection of AWF--using infiltration-centrifugation procedure--that is representative of its composition in intact leaves according to the smaller symplastic contamination detected by the use of the hexose phosphate isomerase marker. Protein extraction under denaturing conditions for 2-DE was remarkably improved by the combination of chemically and physically modified processes including phenol, SDS dense buffer and sonication steps. With this protocol, high-quality proteins from cacao leaves and meristems were isolated, and for the first time well-resolved 1-DE and 2-DE protein patterns of cacao vegetative organs are shown. It also appears that sonication associated with polysaccharide precipitation using tert-butanol was a crucial step for the nondenaturing protein extraction and subsequent enzymatic activity detection. It is expected that the protocols described here could help to develop high-level proteomic and biochemical studies in cacao also being applicable to other recalcitrant plant tissues.     

Evaluation of extraction procedures for 2‐DE analysis of aphid proteins

Protein sample preparation is a crucial step in a 2‐DE proteomics approach. In order to establish a routine protocol for the application of proteomics analysis to aphids, this study focuses on the specific protein extraction problems in insect tissues and evaluates four methods to bypass them. The approaches of phenol extraction methanol/ammonium acetate precipitation (PA), TCA/acetone precipitation, PEG precipitation, and no precipitation were evaluated for proteins isolation and purification from apterous adult aphids, Sitobion avenae. For 2‐DE, the PA protocol was optimal, resulting in good IEF and clear spots. PA method yielded the greatest amount of protein and displayed most protein spots in 2‐DE gels, as compared with the TCA/acetone precipitation, PEG precipitation and no precipitation protocols. Analysis of protein yield, image quality and spot numbers demonstrate that the TCA/acetone precipitation protocol is a reproducible and reliable method for extracting proteins from aphids. The PEG precipitation approach is a newly developed protein extraction protocol for aphids, from which more unique protein spots can be detected, especially for detection of acid proteins. These protocols are expected to be applicable to other insects or could be of interest to laboratories involved in insect proteomics, despite the amounts and types of interfering compounds vary considerably in different insects.     

Optimizing soluble protein extraction and two-dimensional polyacrylamide gel electrophoresis quality for extremophile ciliates

An efficient protein extraction methodology is quite important for sample preparation and subsequent 2-D PAGE and MS analysis. Cell lysis is the first step in protein extraction and purification. Many techniques are available for cell disruption, including physical and detergent-based methods. Here, we report on a very fast and efficient detergent-free Tris-based method to extract the soluble fraction proteins of extremophile ciliates, comparing it with a detergent-based protocol. This comparison has been carried out by means of 2-D PAGE and subsequent MALDI-compatible silver staining of protein samples obtained from the intensely pigmented hypersaline ciliate Fabrea salina and the Antarctic hypotrich ciliate Euplotes focardii. Our results indicate that this fast and easy extraction method allows to obtain more clear crude extracts and more spot-abundant polyacrylamide gels.     

Comparative evaluation of five protocols for protein extraction from stony corals (Scleractinia) for proteomics

Corals especially the reef‐building species are very important to marine ecosystems. Proteomics has been used for researches on coral diseases, bleaching and responses to the environment change. A robust and versatile protein extraction protocol is required for coral proteomics. However, a comparative evaluation of different protein extraction protocols is still not available for proteomic analysis of stony corals. In the present study, five protocols were compared for protein extraction from stony corals. The five protocols were TRIzol, phenol‐based extraction (PBE), trichloroacetic acid (TCA)‐acetone, glass bead‐assisted extraction (GBAE) and a commercially available kit. PBE, TRIzol and the commercial kit were more robust for extracting proteins from stony corals. The protein extraction efficiency and repeatability, two dimensional electrophoresis (2‐DE) and matrix‐assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF MS) were employed to evaluate the protocols. The results indicated that PBE protocol had the better protein extraction efficiency than the others. Protein extraction coverage varied among the procedures. Each protocol favored for certain proteins. Therefore, it is very important for coral proteomic analysis to select a suitable protein protocol upon the experimental design. In general, PBE protocol can be the first choice for extracting proteins from stony corals.     

A protein extraction method compatible with proteomic analysis for the euhalophyte Salicornia europaea

Protein extraction from plants like the halophyte Salicornia europaea has been problematic using standard protocols due to high concentrations of salt ions in their cells. We have developed an improved method for protein extraction from S. europaea, which allowed us to remove interfering compounds and salt ions by including the chemicals borax, polyvinylpolypyrrolidone, and phenol. The comparative study of this method with several other protocols using NaCl-treated S. europaea shoots demonstrated that this method gave the best distinction of proteins on 2-DE gels. This protocol had a wide range of applications as high yields and good distinction of 1-DE gels for proteins isolated from twelve other plants were rendered. In addition, we reported results of 2-DE using the recalcitrant tissue of the S. europaea roots. We also demonstrated that this protocol is compatible with proteomic analysis as eight specific proteins generated by this method have been identified by MS. In conclusion, our newly developed protein extraction protocol is expected to have excellent applications in proteomic studies of halophytes.     

Alkaline extraction of human plasma proteins from nondenaturing micro-2-D gels for protein/polypeptide mass measurement and peptide mass fingerprinting using MALDI-TOF MS

Previously, we have reported a high-efficiency method of protein extraction from CBB-stained polyacrylamide gels for molecular mass measurement with MALDI-TOF MS [1]. In the present work, the alkaline extraction method was applied to CBB-stained 2-DE gels on which human plasma proteins were separated in the absence of denaturant. In order to examine the performance of the method, ten spots with apparent molecular masses (MMapp) in the range of 65 to 1000 kDa were selected and the proteins were extracted from the gel pieces. The extracts were subjected to whole-mass measurement by MALDI-TOF MS, with and without DTT treatment. In addition, the extracts were subjected to in-solution trypsin digestion followed by MALDI-TOF MS and PMF analysis. Successful extraction of proteins from the ten spots, up to MMapp 1000 kDa, has been ascertained by the significant PMF assignment (MASCOT) with high sequence coverage of the respective proteins or polypeptides. When direct mass measurement of the extracted proteins was attempted, three spots in MMapp range 65-100 kDa provided mass peaks. Five spots in MMapp range 150-400 kDa did not give mass peaks of the intact proteins, but showed those of the constituent polypeptides after the DTT treatment. Extraction of proteins prior to trypsin digestion enabled the procedure of PMF analysis to be much simpler than the conventional in-gel digestion method, providing comparable protein scores and sequence coverage. The technique presented here suggests a new strategy for the characterization of proteins separated by nondenaturing 2-DE.     

Comparison of microwave‐assisted and conventional extraction of mangiferin from mango (Mangifera indica L.) leaves

Mangiferin is the main bioactive component in mango leaves, which possesses anti‐inflammatory, antioxidative, antidiabetic, immunomodulatory, and antitumor activities. In the present study, a microwave‐assisted extraction method was developed for the extraction of mangiferin from mango leaves. Some parameters such as ethanol concentration, liquid‐to‐solid ratio, microwave power, and extraction time were optimized by single‐factor experiments and response surface methodology. The optimal extraction conditions were 45% ethanol, liquid‐to‐solid ratio of 30:1 (mL/g), and extraction time of 123 s under microwave irradiation of 474 W. Under optimal conditions, the yield of mangiferin was 36.10 ± 0.72 mg/g, significantly higher than that of conventional extraction. The results obtained are beneficial for the full utilization of mango leaves and also indicate that microwave‐assisted extraction is a very useful method for extracting mangiferin from plant materials.