Optimization Of Protein Extraction Research Articles

Surfactant and Chaotropic Agent Assisted Sequential Extraction/On-Pellet Digestion (SCAD) for Enhanced Proteomics.

As a popular sample preparation approach, filter-aided sample preparation (FASP) has been widely used in proteomic analysis. However, several limitations have been noted, including sample loss during filtration, repetitive centrifugation steps, and the possibility of breakage of filtration membrane. Extraction bias among different sample preparation strategies presents another challenge. To overcome these limitations and address remaining challenges, we developed a novel surfactant and chaotropic agent assisted sequential extraction/on-pellet digestion (SCAD) protocol. The new strategy resulted in higher protein yield and improved peptide recovery and protein coverage compared to two conventional sample preparation methods (FASP and urea). In combination of three strategies, more than 10,000 distinct protein groups were identified with 1% FDR from MDA-MB-231 cells without any prefractionation. This in-depth proteome analysis was accomplished by optimization of protein extraction, enzymatic digestion, LC gradient, and peptide sequencing method. Ingenuity Pathways Analysis (IPA) of proteins exclusively identified in SCAD revealed several crucial signaling pathways that regulate breast cancer progression. SCAD also enabled an unbiased extraction of different categories of proteins (membrane, intracellular, nuclear) associated with tumorigenesis, which integrates the advantages of FASP and urea extraction. This novel strategy expedites comprehensive protein identification, which is applicable for biomarker discovery in various types of cancers.

Optimization of protein extraction from Gelidiella acerosa by carbohydrases using response surface methodology

SUMMARYIn this study, application of response surface methodology for enzymic pretreatment optimization of Gelidiella acerosa was investigated in order to improve the extraction of algal proteins using Viscozyme L and Celluclast 1.5L. The total protein, soluble proteins and reducing sugar recovery in the water‐soluble fraction were studied in relation to the hydrolysis time, type and concentration of the enzymes. Enzymatic digestion appeared to be an effective treatment for protein extraction. While enzyme hydrolysis by Celluclast 1.5L was able to facilitate the protein extraction, it was a relatively inefficient way to improve protein extraction yield, in comparison with Viscozyme L. The optimum conditions for protein extraction was found to be hydrolysis by 2.8 μL mL−1 of Viscozyme L for 12 h.

Optimization of protein extraction from tree peony (Paeonia suffruticosa Andr.) seed using response surface methodology

Optimization of extraction ratio (ER) of tree peony seed protein (TPSP) was investigated using response surface methodology (RSM). The second-degree equation for ER of TPSP had high coeffi cient (0.9625) of determination. The probability (P) value of regression model signifi cance was less than 0.001 by analysis of central composite rotatable design. Relationships of ER to pH, liquid/solid ratio, squares of all factors, and cross-product factors (x2x3, x2x4, x3x4) were signifi cant (P<0.05). Whereas, extraction time, temperature, and cross-product terms (x1x2, x1x3, x1x4) were not signifi cant factors (P>0.05). Optimum extraction conditions were 3.42 h, pH 9.50, 50.80 ºC, and 9.54 ml g–1 of liquid/solid ratio with the maximum ER (43.60%) . SDS-PAGE indicated TPSP had mainly four proteins (180, 100, 60, and 35 kDa) with four subunits of 60, 48, 38, and 23 kDa. TPSP had a good amino acid composition with abundant essential amino acids (39.76%) determined by amino acid analysis.

Optimization of Protein Extraction and Decoloration Conditions for Tea Residues

To optimize alkaline method for extracting proteins from tea residue (TR), the effect of extraction conditions on tea protein extraction rate (TPER) was investigated. Single factor experiment showed the extraction temperature 80 °C, extraction time 100 min, pH value 13 and liquid–solid ratio 40:1 as the optimal extraction conditions. The orthogonal test revealed that the maximum TPER reached 29.71% under the following optimal combination of conditions: extraction temperature 70 °C, extraction time 60 min, pH 12 and liquid–solid ratio 50:1. For optimizing the purification of tea residue proteins, isoelectric point precipitation (pI), ammonium sulfate precipitation (aS) and isoelectric point plus ammonium sulfate precipitation (iPAS) were compared. The result showed that the highest protein precipitation rate (PPR) was 89.70% which was generated by using iPAS. Furthermore, powdered activated carbon was chosen as the most suitable decolorant for the extracted proteins.

Proteomic analysis of Rhodotorula mucilaginosa: dealing with the issues of a non-conventional yeast.

Red yeasts ascribed to the species Rhodotorula mucilaginosa are gaining increasing attention, due to their numerous biotechnological applications, spanning carotenoid production, liquid bioremediation, heavy metal biotransformation and antifungal and plant growth-promoting actions, but also for their role as opportunistic pathogens. Nevertheless, their characterization at the 'omic' level is still scarce. Here, we applied different proteomic workflows to R. mucilaginosa with the aim of assessing their potential in generating information on proteins and functions of biotechnological interest, with a particular focus on the carotenogenic pathway. After optimization of protein extraction, we tested several gel-based (including 2D-DIGE) and gel-free sample preparation techniques, followed by tandem mass spectrometry analysis. Contextually, we evaluated different bioinformatic strategies for protein identification and interpretation of the biological significance of the dataset. When 2D-DIGE analysis was applied, not all spots returned a unambiguous identification and no carotenogenic enzymes were identified, even upon the application of different database search strategies. Then, the application of shotgun proteomic workflows with varying levels of sensitivity provided a picture of the information depth that can be reached with different analytical resources, and resulted in a plethora of information on R. mucilaginosa metabolism. However, also in these cases no proteins related to the carotenogenic pathway were identified, thus indicating that further improvements in sequence databases and functional annotations are strictly needed for increasing the outcome of proteomic analysis of this and other non-conventional yeasts. Copyright © 2016 John Wiley & Sons, Ltd.

Optimization of Protein Extraction and Two-Dimensional Electrophoresis Protocols for Oil Palm Leaf.

Oil palm (Elaeis guineensis) is an important economic crop cultivated for its nutritional palm oil. A significant amount of effort has been undertaken to understand oil palm growth and physiology at the molecular level, particularly in genomics and transcriptomics. Recently, proteomics studies have begun to garner interest. However, this effort is impeded by technical challenges. Plant sample preparation for proteomics analysis is plagued with technical challenges due to the presence of polysaccharides, secondary metabolites and other interfering compounds. Although protein extraction methods for plant tissues exist, none work universally on all sample types. Therefore, this study aims to compare and optimize different protein extraction protocols for use with two-dimensional gel electrophoresis of young and mature leaves from the oil palm. Four protein extraction methods were evaluated: phenol-guanidine isothiocyanate, trichloroacetic acid-acetone precipitation, sucrose and trichloroacetic acid-acetone-phenol. Of these four protocols, the trichloroacetic acid-acetone-phenol method was found to give the highest resolution and most reproducible gel. The results from this study can be used in sample preparations of oil palm tissue for proteomics work.

Optimization of Protein Extraction from Spirulina platensis to Generate a Potential Co-Product and a Biofuel Feedstock with Reduced Nitrogen Content

The current work reports protein extraction from Spirulina platensis cyanobacterial biomass in order to simultaneously generate a potential co-product and a biofuel feedstock with reduced nitrogen content. S. platensis cells were subjected to cell disruption by high pressure homogenization and subsequent protein isolation by solubilisation at alkaline pH followed by precipitation at acidic pH. Response surface methodology (RSM) was used to optimize the process parameters - pH, extraction (solubilisation/precipitation) time and biomass concentration for obtaining maximum protein yield. The optimized process conditions were found to be pH 11.38, solubilisation time of 35 min and biomass concentration of 3.6 % (w/w) solids for the solubilisation step, and pH 4.01 and precipitation time of 60 min for the precipitation step. At the optimized conditions, a high protein yield of 60.7 % (w/w) was obtained. The protein isolate (co-product) had a higher protein content (80.6 % (w/w)), lower ash (1.9 % (w/w)) and mineral content and was enriched in essential amino acids, the nutritious γ-lenolenic acid and other high-value unsaturated fatty acids compared to the original biomass. The residual biomass obtained after protein extraction had lower nitrogen content and higher total non-protein content than the original biomass. The loss of about 50 % of the total lipids from this fraction did not impact its composition significantly owing to the low lipid content of S.platensis (8.03 %).

Optimization of Protein Extraction from Fermented and Non Fermented Perah Seed by using Response Surface Methodology

The optimum protein extraction from fermented and non-fermented Perah seed (Elateriospermumtapos) was investigated using the response surface methodology (RSM). A box-behnken design with three independent variables which are NaOH concentration (6, 8 and 10%), extraction time (10, 20 and 30 minutes) and solvent/meal ratio (50:1, 100:1 and 150:1, v/w) was used to study the response of protein yield. A second-degree equation for independent and response variables was produced from simulation to obtain the contour plot graphs. The best protein extraction procedure from both fermented and non-fermented Perah seed was obtained at 5.5% of solvent extraction, 40:1 ratio of solvent/meal and at 32 minutes of reaction time. Surface concentration and ratio of solvent/meal were found to influence the protein yield from fermented seed; meanwhile only the solvent concentration influenced protein yield from non-fermented seed. The maximum protein yields for both fermented and non-fermented Perah seeds were 18.0 g/100g and 5.0 g/100g seed meal respectively.

English

Optimization of protein extraction using silk cocoon was carried out by the design of experiment (DOE) to obtain the response surface methodology ( RSM ). Box-Behnken rotatable design was used to create an experimental program to provide data to model the effects of various factors on protein extraction efficiency. The variables chosen were sodium carbonate concentration ( 1 X ), Lithum bromide concentration ( 2 X ) and temperature ( 3 X ). The mathematical relationship between protein extraction efficiency and three significant independent variables can be approximated by a second order quadratic model. RSM was used to describe the individual and interactive effects of three variables at three levels, combined according to a Box-Behnken Design. The coefficient of determination ( 2 R ) for the model is 0.9761. Probability value ( P < 0.0001) demonstrates a very high significance for the regression model indicating that Box-Behnken Design can be applied to the protein extraction from silk cocoon, and it is an economical way of obtaining the maximum amount of information with the fewest experiments.

The optimization of protein extraction during electrophysical whey processing

The properties of milk whey protein fractions are analyzed. The need for obtaining them is out-lined; the possible mechanisms of complexing and coagulation during the electrophysical processing of secondary raw milk products are explained. The possible methods for optimizing the conversion of the protein fractions into a protein-mineral concentrate, as well as the fractional analysis of the minerals, are described, and their role in the complexing of the concentrate is estimated. The electrophysical parameters under the conditions of the optimization, the different compositions of the anode liquid, and the types of separating of the elements are analyzed. The fields of future investigations aimed at obtaining high quality products during developing nonwaste electrotechnologies of milk whey processing are identified.

Otimização da extração enzimática da proteína do feijão

Optimization of enzymatic protein extraction from beans. Considering the importance of beans in the Brazilian diet and the fact that it is forbidden in phenylketonuric feeding, it is relevant to promote the extraction of its proteins, for phenylalanine removal. Aiming for the optimization of protein extraction from beans, two enzymatic methods were initially employed. For the one that produced the highest protein extract yield (PEY), a study was conducted to assess de effect of several parameters, such as centrifugation velocity, reaction time, initial pH and type of enzyme (one of Bacillus licheniformis, two of Bacillus subtilis, one of Aspergillus sojae and one of Papaya carica). The results showed that the best method was the one that employed a concentration of raw material of 1:10 (w v -1 ), initial pH of 10.5, temperature of 50°C, in the absence of ultra- turrax treatment. With regard to the parameters tested, the largest PEY (93.1%) was obtained by using the enzyme from Bacillus licheniformis, the centrifugation velocity of 10,640 x g, the reaction time of 3h and initial pH of 10.5.

Optimization of Protein Extraction from Hypericum perforatum Tissues and Immunoblotting Detection of Hyp-1 at Different Stages of Leaf Development

Sample preparation is crucial for obtaining high-quality proteins for the purpose of electrophoretic separation and further analysis from tissues that contain high levels of interfering compounds. Hypericum perforatum is a medicinal plant that contains high amounts of phenolic compounds, of which hypericins, hyperforins, and flavonoids contribute to the antidepressant activities of the plant. This study focuses on obtaining optimized amounts of high-quality proteins from H. perforatum, which are suitable for electrophoretic analyses. From the tested protein extraction solutions, sodium borate buffers at pH 9 and 10 gave the best protein yields from mature H. perforatum leaves. With these buffers, relatively high protein yields could also be obtained from roots, stems, and flower buds. The protein extracts of all organs were well resolved in SDS-PAGE after an efficient removal of non-protein contaminants with PVPP, phenol extraction, and methanolic ammonium acetate precipitation. The method was suitable for high-quality protein extraction also from other tested species of genus Hypericum. The applicability of the protocol for immunoblotting was demonstrated by detecting Hyp-1 in H. perforatum leaves at different stages of development. Hyp-1, which has been suggested to attend to the biosynthesis of hypericin, accumulated in high amounts in H. perforatum leaves at mature stage.

Optimisation of protein extraction and 2‐DE for metaproteomics of microbial communities from anaerobic wastewater treatment biofilms

The feasibility of metaproteomic analysis of the microbial communities underpinning anaerobic wastewater treatment relies on efficient protein extraction and separation techniques. The microorganisms involved in anaerobic digestion (AD) of wastewater typically aggregate to form tightly organised, spherical granules, from which protein extraction is challenging. Here, we compare two methods of protein extraction, one using a French press previously used successfully to analyse the proteome of an activated sludge [Wilmes, P., Bond, P. L., Environ. Microbiol. 2004, 6, 911-920.] and one using sonication developed in the context of pure culture [Abram, F., Wan-Lin, S., Wiedmann, M., Boor, K. J., Coote, P., Botting, C., Karatzas, K. A. G., O'Byrne, C. P., Appl. Environ. Microbiol. 2008, 74, 594-604.]. We used 2-DE to carry out this comparison. The protein extraction using the sonication method resulted in a significant increase in the number of protein spots and higher quality 2-D gels.

Optimization of Protein Extraction from Fish Waste using Response Surface Methodology

The aim of present study was to optimize protein extraction from Fish Waste (FW), sardine (Sardina pilchardus) using enzymatic method. In this study, enzymatic method involved two combinations of enzymes which were alcalase and protamex. Response Surface Methodology (RSM) was used to study the effect of independent variables, namely temperature (35-55°C), rotation speed (100-300 rpm), time (60-1440 min) and enzyme:substrate ratio (0.5-1.5) on protein extraction from FW. From RSM-generated model, the optimum conditions for extraction of protein from FW were identified to be at temperature 35°C in 1429 min reaction time, with rotation speed of 171 rpm and enzyme:substrate ratio of 1.50. At the optimum conditions, predicted protein yield in the extraction process was 80.75 mg mL-1.

The Optimization of Protocols for Proteome Difference Gel Electrophoresis (DiGE) Analysis of Preneoplastic Skin

Difference gel electrophoresis (DiGE) allows the reliable comparison of proteome differences between two or three samples within a single gel, by way of a CyDye fluorescent labeling system. This facilitates identification of protein differences avoiding the difficulties associated with gel-to-gel variation. A drawback of this approach is the necessity for high-purity protein samples, since contaminants can interfere with the labeling process, affecting subsequent analysis. Thus far, DiGE has been applied to the study of various sample types derived from relatively simple starting materials such as serum, cell lines, or primary cells. Herein, we describe optimization of protein extraction and purification from a complex tissue (the murine ear) of which a major component is skin, which is compatible with the CyDye labeling system and DiGE. Protein samples obtained by this method from preneoplastic, transgenic tissue have been effectively compared to normal tissue samples to reveal bona fide differences, verifiable by Western blotting. In total, 41 protein differences (21 up- and 20 down-regulated in the pathological samples) were identified by mass spectrometry (MS). This method can therefore form a guide for those wishing to perform DiGE on complex tissues, and is especially useful for samples with relatively insoluble components such as skin.

Extraction and Separation of Water-Soluble Proteins from Different Wheat Species by Acidic Capillary Electrophoresis

Optimization of protein extraction and a capillary zone electrophoresis method for water-soluble protein analysis in wheat is described. The optimal separation was obtained with a 50 microm i.d. x 27 cm (20 cm to detector) uncoated capillary filled with 0.1 M phosphoric acid/beta-alanine, pH 2.5, buffer containing urea (1 M), 0.05% (w/v) hydroxypropylmethylcellulose, and 20% (v/v) acetonitrile. Separation was carried out at 15 kV and 35 degrees C for 9 min. Extract stability was also investigated within 2 h from the extraction. Good visual peak parameters and a higher sensitivity can be obtained when 30% ethanol is used as an extraction medium. The method was successfully used to analyze extracts obtained from whole and refined meals of six Triticum spp. Moreover, the described methodology could be applied to the discrimination of species with different ploidy levels and to the detection of durum wheat adulteration, as well as to screen wheat collections for enzymes involved with the quality of wheat derivatives.

Optimization of protein extraction and solubilization for mature grape berry clusters

Protein extraction from grape berries has been challenging, particularly in mature berries, which can have sugar concentrations as high as 26%. Grape skins and seeds contain large amounts of polyphenols, which can also interfere with efficient protein extraction. In plants, two extraction protocols, TCA/acetone-based and phenol-based methods, have been mainly used to extract proteins from different organs or tissues on many species. However, few results have been reported for grape berry clusters. We wanted to determine which of these protocols was optimal for berry clusters in order to achieve both efficient protein extraction and high spot resolution on 2-D gels. Four protocols, derived from either TCA/acetone or phenol procedures, were tested on mature Cabernet Sauvignon whole berry clusters. The phenol-based protocols were superior to the TCA/acetone methods, showing larger protein yields and greater spot resolution on 2-D gels. One method was clearly superior to the rest, a phenol-based extraction method combined with resuspension in the presence of both urea and thiourea as chaotropes. A total of 81 spots were excised and identified following MALDI-TOF/TOF MS analyses. Their identification helped further characterize the specificity of each extraction procedure.

Optimization of protein extraction from Aspergillus nidulans for gel electrophoresis

Efficient extraction of proteins from filamentous fungi is difficult both as a result of high endogenous protease activity, the existence of a hard cell wall, and the tendency of hyphae to clump. We have had difficulty using published protocols in extracting high molecular weight proteins (>150 kDa), and in extraction from germlings grown on minimal media with a poor carbon source such as glycerol. In order to address these problems, we have compared several different extraction protocols and found the protocol utilizing 9M Urea-sample buffer to be the fastest and most efficient with sample variation being kept to a minimum.

Optimization of protein extraction from flaxseed meal

Response surface methodology was used to determine optimum conditions for extraction and solubilization of protein from defatted flaxseed meal. Factors investigated included the ratio of extracting solvent to meal (10–40 l/kg), ionic strength (sodium chloride concentration 0–2 m), and solvent pH (6·0–10·0). Solvent pH was not a significant factor affecting protein solubility and recovery but solvent-to-meal ratio and ionic strength were highly significant. A simultaneous increase in ionic strength and decrease in solvent-to-meal ratio resulted in increased protein solubility. The maximum value of protein extracted from defatted flaxseed meal was 97% at a solvent-to-meal ratio of 10 l/kg, 0·8 m NaCl and pH 8·0. At constant ionic strength, pH and solvent-to-meal ratio, the concentration of soluble protein was a linear function of the initial protein content of defatted flaxseed meal.