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.     

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.     

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 systematic evaluation of protocols for a proteomics analysis of (lyophilized) fruit tissues

This study represents a systematic evaluation of protocols for protein extraction and cleanup for fruit proteomic analysis. Procedures were optimized using pooled lyophilized banana fruit pulp, which is known to be particularly tricky due to high concentrations of soluble polysaccharides, phenolics, and other substances that interfere with protein extraction and purification. A total of 18 combinations of three protein extraction procedures (SDS‐based, Triton X‐100‐based, and phenol‐based), three protein precipitating agents (ammonium acetate/methanol, TCA/acetone, and acetone), and two resolubilization buffers (classical Rabilloud and the so‐called R2D2) were compared for total protein yields and efficiency of recovery. The results demonstrate that while losses in total recovered protein are unavoidable, the degree of these losses depends on the method combinations used. Combinations based on buffer‐saturated phenol always gave the highest yields, and overall recovery and purity was highest when acetone was combined with the R2D2 buffer for protein purification and concentration. Comparative 2D‐PAGE analysis confirmed that this method combination produced high‐quality and reproducible gels and the largest numbers of spots per gel. The usefulness of this methodology was demonstrated on ripe fruits from several other species and shown to give excellent results.     

Influence of nonrubber components on properties of unvulcanized natural rubber

Low‐protein natural rubber (LPNR) and acetone‐extracted natural rubber (AENR) were prepared in solid form by alkaline treatment and acetone extraction to remove proteins and lipids. The content of proteins and lipids along with gel content were characterized by Fourier‐transform infrared spectroscopy (FTIR) and size exclusion chromatography with multiangle light scattering (SEC‐MALS) analysis. It was found that natural rubber (NR) treatment by alkaline hydrolysis or acetone extraction decreased proteins or lipids along with gel content. Also, having less proteins and lipids changed the network structure from macroaggregates to microaggregates. This resulted in inferior plasticity and poor mechanical, rheological, and dynamic properties. Furthermore, decreased strain‐induced crystallization and storage hardening were confirmed by temperature scanning stress relaxation (TSSR), after removal of proteins and lipids. Therefore, protein and lipid contents together with gel content play essential roles in controlling various properties of unvulcanized NR.     

Proteome profile of salt gland‐rich epidermis extracted from a salt‐tolerant tree species

Preparation of proteins from salt‐gland‐rich tissues of mangrove plant is necessary for a systematic study of proteins involved in the plant's unique desalination mechanism. Extraction of high‐quality proteins from the leaves of mangrove tree species, however, is difficult due to the presence of high levels of endogenous phenolic compounds. In our study, preparation of proteins from only a part of the leaf tissues (i.e. salt gland‐rich epidermal layers) was required, rendering extraction even more challenging. By comparing several extraction methods, we developed a reliable procedure for obtaining proteins from salt gland‐rich tissues of the mangrove species Avicennia officinalis. Protein extraction was markedly improved using a phenol‐based extraction method. Greater resolution 1D protein gel profiles could be obtained. More promising proteome profiles could be obtained through 1D‐LC‐MS/MS. The number of proteins detected was twice as much as compared to TUTS extraction method. Focusing on proteins that were solely present in each extraction method, phenol‐based extracts contained nearly ten times more proteins than those in the extracts without using phenol. The approach could thus be applied for downstream high‐throughput proteomic analyses involving LC‐MS/MS or equivalent. The proteomics data presented herein are available via ProteomeXchange with identifier PXD001691.     

An optimized method to extract poplar leaf proteins for two‐dimensional gel electrophoresis guided by analysis of polysaccharides and phenolic compounds

Commonly used methods for protein extraction from plant leaves, such as extraction with phenol or a combination of trichloroacetic acid and acetone, were ineffective for four tested cultivars of poplar. Moreover, multiple protocols for 2DE of the extracted proteins gave different results when protein profiles of relatively closely related plants were compared. Given that polycyclic compounds strongly hinder 2DE, we analyzed the impact of polyphenols and polysaccharides present in the plant tissues used for protein extraction, on the quality of 2DE protein profiles. Analysis of content of polyphenols and polysaccharides in leaves of poplar cultivars showed that even small differences in concentrations of analyzed metabolites accompany large differences between poplar cultivars when considering the susceptibility of samples to protein extraction for 2DE. High‐quality 2DE results were correlated with decreased amounts of polyphenols. Additional analysis using MS/MS suggested that only levels of total phenolics affected the results of 2DE. Soluble total nonstructural carbohydrates also had a negative effect, but the level of starch was not important. Finally, we present an optimized method for extraction of proteins from poplar leaves, which enables reliable comparative analysis of four different poplar cultivars, that is, “Eridano,” “Villafranca,” “NE‐42,” and “Luisa Avanzo,” which have not yet been used for the proteomic studies.     

Development of a rubber elongation factor, surface-imprinted polymer-quartz crystal microbalance sensor, for quantitative determination of Hev b1 rubber latex allergens present in natural rubber latex products

Molecularly imprinted polymers (MIPs) for screening to detect rubber latex allergens (Hev b1) in natural rubber based products were designed as artificial recognition polymeric materials coated onto a quartz crystal microbalance (QCM). The polymers were prepared using a stamp imprinting procedure after mixing optimum amounts of methacrylic acid–vinylpyrrolidone–dihydroxyethylene bisacrylamide and Hev b1 latex allergen proteins, obtained from rubber gloves. QCM measurements showed that the resulting polymer layers after removal of the proteins used in their preparation could incorporate structures and features down to nanometer scale of protein templates into the imprinted polymer much better than a non-specific control polymer under controlled sensor conditions and an optimized polymerization process. This selective polymer but not the non-selective polymer clearly distinguished between the latex allergen Hev b1 and proteins such as lysozyme, ovalbumin and bovine serum albumin, with a selectivity factor of from 2 to 4, and the response of the rubber elongation factors by an astonishing factor of 12. The imprinted cavities recognized specific binding sites and could distinguish among related hevein latex allergenic proteins isolated from fresh natural rubber latex; Hev b1, Hev b2, and Hev b3 with a selectivity factor of from 4 to 6. The different QCM measurements obtained presumably reflected slightly different conformations and affinities to the MIP binding sites. The sensor layers selectively adsorbed Hev b1 within minutes in amounts ranging from 10 to 1500 μg L⁻¹ and with a detection limit of 1 μg L⁻¹. This work has demonstrated that this new sensor provides a fast and reliable response to natural rubber latex protein, even after being extracted from the matrix of rubber gloves.     

Sugarcane proteomics: Establishment of a protein extraction method for 2‐DE in stalk tissues and initiation of sugarcane proteome reference map

Sugarcane is an important commercial crop cultivated for its stalks and sugar is a prized commodity essential in human nutrition. Proteomics of sugarcane is in its infancy, especially when dealing with the stalk tissues, where there is no study to date. A systematic proteome analysis of stalk tissue yet remains to be investigated in sugarcane, wherein the stalk tissue is well known for its rigidity, fibrous nature, and the presence of oxidative enzymes, phenolic compounds and extreme levels of carbohydrates, thus making the protein extraction complicated. Here, we evaluated five different protein extraction methods in sugarcane stalk tissues. These methods are as follows: direct extraction using lysis buffer (LB), TCA/acetone precipitation followed by solubilization in LB, LB containing thiourea (LBT), and LBT containing tris, and phenol extraction. Both quantitative and qualitative protein analyses were performed for each method. 2‐DE analysis of extracted total proteins revealed distinct differences in protein patterns among the methods, which might be due to their physicochemical limitations. Based on the 2‐D gel protein profiles, TCA/acetone precipitation‐LBT and phenol extraction methods showed good results. The phenol method showed a shift in pI values of proteins on 2‐D gel, which was mostly overcome by the use of 2‐D cleanup kit after protein extraction. Among all the methods tested, 2‐D cleanup‐phenol method was found to be the most suitable for producing high number of good‐quality spots and reproducibility. In total, 30 and 12 protein spots commonly present in LB, LBT and phenol methods, and LBT method were selected and subjected to eLD‐IT‐TOF‐MS/MS and nESI‐LC‐MS/MS analyses, respectively, and a reference map has been established for sugarcane stalk tissue proteome. A total of 36 nonredundant proteins were identified. This is a very first basic study on sugarcane stalk proteome analysis and will promote the unexplored areas of sugarcane proteome research.     

Preparation and characterization of protein‐free natural rubber

Protein‐free natural rubber was prepared by incubation of natural rubber latex with urea and polar organic solvent in the presence of surfactant. Effect of the polar organic solvent on the removal of the proteins was investigated with respect to chemical affinity and concentration of the solvents. Under a suitable condition, nitrogen content of the deproteinized natural rubber (DPNR) was 0.000 wt%, which was less than that of natural rubber deproteinized with proteolytic enzyme or urea in the presence of surfactant. The removal of all proteins from natural rubber was proved through FT‐IR spectroscopy. Changes in morphology of the DPNR were also investigated by transmission electron microscopy. Copyright © 2011 John Wiley & Sons, Ltd.     

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.     

Removal of proteins from natural rubber with urea

Removal of proteins from natural rubber was achieved by incubation of the rubber latex with urea in the presence of a surfactant to prevent the latex‐allergy caused with thin film products. Temperature, pH and time for the incubation were investigated to remove the proteins effectively, in which nitrogen content of the rubber was reduced to 0.02 from 0.38 wt% under the optimum condition. To remove further the proteins, deproteinization of natural rubber was made by incubation of the latex with proteolytic enzyme in the presence of a surfactant followed by incubation with urea. Amount of allergen decreased through the procedure to less than 0.7 μg/ml, which is a small amount of allergen compared to that for the commercial, deproteinized natural rubber. Copyright © 2004 John Wiley & Sons, Ltd.     

An efficient protein preparation for proteomic analysis of developing cotton fibers by 2-DE

Preparation of high-quality proteins from cotton fiber tissues is difficult due to high endogenous levels of polysaccharides, polyphenols, and other interfering compounds. To establish a routine procedure for the application of proteomic analysis to cotton fiber tissues, a new protocol for protein extraction was developed by optimizing a phenol extraction method combined with methanol/ammonium acetate precipitation. The protein extraction for 2-DE was remarkably improved by the combination of chemically and physically modified processes including polyvinylpolypyrrolidone (PVPP) addition, acetone cleaning, and SDS replacement. The protocol gave a higher protein yield and vastly greater resolution and spot intensity. The efficiency of this protocol and its feasibility in fiber proteomic study were demonstrated by comparison of the cotton fiber proteomes at two growth stages. Furthermore, ten protein spots changed significantly were identified by MS/tandem MS and their potential relationships to fiber development were discussed. To the best of our knowledge, this is the first time that a protocol for protein extraction from cotton fiber tissues appears to give satisfactory and reproductive 2-D protein profiles. The protocol is expected to accelerate the process of the proteomic study of cotton fibers and also to be applicable to other recalcitrant plant tissues.     

Mechanisms of antioxidant action: The reaction of hindered phenols with rubber in the presence of free radicals

Simple hindered phenols, containing ortho or para methyl groups, react with natural rubber latex in the presence of oxidizing free radicals (alkoxy or acyloxy), giving up to 20% yields of bound antioxidant. This reaction is most efficient when the concentration of the phenolic antioxidant in the rubber is less than 1%. As the concentration is increased, side reactions involving both the phenol (stilbenequinone formation) and the rubber (cross-linking) supervenes. The efficiency of the bound antioxidants is much higher than conventional antioxidants under aggressive conditions of air oven ageing and solvent or detergent extraction. This effect is due primarily to the non-removal of the bound antioxidants under these conditions whereas the conventional antioxidants are removed completely.     

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.     

基于不同提取方法的水稻叶片蛋白质组的二维液相色谱分离及高分辨质谱分析

建立了酚法提取-二维液相色谱分离-高分辨质谱分析水稻叶片蛋白质组的方法。水稻叶片蛋白质经过酚法提取,酶解肽段脱盐后用离线反相-反相二维液相色谱分离,然后用线性离子阱/静电场轨道阱组合式高分辨质谱分析,共鉴定到2712种蛋白质。比较了液相色谱分离系统（一维液相色谱与二维液相色谱）和水稻叶片蛋白质提取方法（酚法、十二烷基硫酸钠法（SDS法）和三氯乙酸/丙酮法（TCA/丙酮法））对鉴定蛋白质数量的影响,结果表明：在二维液相色谱条件下,酚法、SDS法和TCA/丙酮法鉴定到的蛋白质数目为2712、2415和1914,分别是一维液相色谱条件下鉴定到的蛋白质数目的2.7、2.5和1.9倍。二维液相色谱条件下,酚法鉴定到的蛋白质数目比SDS法和TCA/丙酮法分别多297和798。与SDS法和TCA/丙酮法相比,酚法不但鉴定到的蛋白质数量多,而且能够鉴定到一些极端蛋白质,如酸性、碱性及高等电点的蛋白质。此外,对二维液相色谱条件下3种蛋白质提取方法提取到的蛋白质进行生物学功能分类,发现3种方法鉴定到的蛋白质的功能存在互补性,但酚法鉴定到的蛋白质功能种类最多。该法为水稻蛋白质组学研究提供了技术支撑,同时也为其他作物的蛋白质组学研究技术提供重要的借鉴。     

18O同位素标记定量肽段串联体蛋白质结合同位素稀释-多反应监测质谱的蛋白质绝对定量新方法

建立了定量肽段串联体蛋白质(concatamers of Q peptides, QconCATs)结合¹⁸O同位素标记-多反应监测质谱的蛋白质绝对定量新方法。首先对QconCAT重组蛋白质进行了纯度表征,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)表征结果表明重组蛋白质的纯度在99%以上,相对分子质量约为63.4 kDa。对QconCAT重组蛋白质酶切后的肽段混合物进行质谱分析,并经pFind和pLabel软件处理,验证了目标肽段。还考察了QconCAT重组蛋白质的酶切效率和¹⁸O标记效率,并对QconCAT蛋白质结合¹⁸O标记-同位素稀释-多反应监测质谱方法进行了评价。实验结果表明,采用该方法对腾冲嗜热厌氧菌(Thermoanaerobacter tengcongensis, TTE)中选定蛋白质的肽段进行绝对含量测定时,相对标准偏差小于20%,准确度较高,说明该方法可用于复杂生物样本中蛋白质的绝对定量。更重要的是所建方法不仅解决了细胞培养氨基酸稳定同位素标记(SILAC)技术的重标试剂价格昂贵的问题,也为定量蛋白质组学提供了一种新的方法。     

Enhanced Protein Extraction from Tobacco Roots for Proteomic Analysis

Plant roots contain low protein concentrations and many interferences for protein extraction and two-dimensional electrophoresis analysis. Therefore, the extraction of high-quality protein from tobacco roots for proteomic analysis is a challenge. Three protein extraction methods (the trichloroacetic acid-acetone, phenol extraction, and trichloroacetic acid-acetone-phenol methods) for tobacco root proteins were compared using protein yields, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and two-dimensional electrophoresis. The trichloroacetic acid-acetone-phenol method provided a higher spot resolution (505 ± 18 spots), the least streaking, and larger protein yields (2200 ± 20 µg/g fresh weight) on two-dimensional electrophoresis gels for tobacco roots, and hence is the most suitable method for the characterization of tobacco roots.     

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.