Acetone Precipitation Method Research Articles

Comparison of Extraction Assays and Quantification of Protein from Ulva anandii (Cholorophycota)

Seaweeds contain many macronutrients including protein, therefore they can be utilized to fulfil the protein requirements of human beings. This research focused on extracting total protein in green seaweed Ulva anandii (Amjad et Shameel 1993), from the crude extracts, by using the trichloroacetic acid (TCA) and acetone precipitation methods, and the estimation of crude extract (water-soluble proteins), and those obtained from the two above-mentioned methods. The results indicate that the water-soluble proteins had the highest quantity (949.75µg/mL) followed by the TCA precipitation method (831µg/mL), while the acetone precipitation method had the least concentration of total protein (100 µg/mL). The study concludes that treatment with organic solvents lowers the quantity of protein extracted from U. anandii.

Occurrence of Streptomyces tauricus in mangrove soil of Mangalore region in Dakshina Kannada as a source for antimicrobial peptide.

Microbial resistance and deprivation of the effective drugs have become the foremost problem that propels to seek out for advanced approach. This concept initiated a need to search for more effective antimicrobial compounds from reliable sources. The Streptomyces is grouped under phylum Actinobacteria and are considered prolific producers of antibiotics, around 70% of presently available antibiotics are contributed by Streptomyces alone. In this study, Mangroves of the Mangalore Coast offered a unique source for screening Actinomyces group of microorganisms. We investigated on the four soil samples collected from Mangrove swamps of Mangalore, Karnataka, India. Based on their culture traits, the 18 distinct Actinomyces isolates were analyzed through a series of morphological and biochemical tests on starch casein nitrate (SCN) media. Culture biomasses were subjected for intracellular protein extraction through acetone precipitation method; the extracted proteins from each Actinomyces isolate were examined for antimicrobial activity against test organisms. The isolate ANTB-YKMU4 showed potential antimicrobial activity against significant number of test organisms; Bacillus cereus, Proteus vulgaris, Staphylococcus aureus, Salmonella typhimurium, and Pseudomonas aeruginosa. The isolate ANTB-YKMU4 through 16 s rRNA gene sequence analysis was identified as Streptomyces tauricus strain with GenBank accession no. MW785875.1. The S. tauricus further cultivated for efficient biomass growth on SCN media for subsequent protein extraction and purification by a series of Electrophoretic and chromatographic techniques. Thus, by intracellular extractions from S. tauricus resulted in the identification of peptide with a molecular weight of 266 Da that was characterized by LC-MS.

Production, Purification and Characterization of Β-D-Fructofuranosidase from Fusarium oxysporum Isolated from Grape Juice and Soil Sample from Sugar Cane Dumpsite

β-D-fructofuranosidase is an important enzyme needed in various industrial setup for the production of invert sugars. Production, purification and characterization of β-D-fructofuranosidase by Fusarium oxysporum under submerged fermentation were investigated. β-D-fructofuranosidase production by Fusarium oxysporum ranged from 36. 2- 65.8% and 7.2 -2 0.1% under different pH and incubation time. pH 7 and 7 days incubation time supported the highest production. β-D-fructofuranosidase production ranged from 16.5 - 44.0% and 27.8 - 68.8% when different organic and inorganic carbon sources were used. Potato peels and fructose supported the highest β-D-fructofuranosidase production. Peptone supported the highest β-D-fructofuranosidase production (37.9%) by Fusarium oxysporum. β-D-fructofuranosidase produced from Fusarium oxysporum was purified by Acetone precipitation method and gel filtration respectively. The crude and purified β-D-fructofuranosidase are 65.79% and 19.57%. pH 7, 35oC and 30oC incubation temperature, 2.0 mg/L of honey and molasses and 1.0 mg/L sucrose supported optimum activity of the crude and purified β-D-fructofuranosidase. The enzyme Kinetic parameters (Km and Vmax) are 0.1285mg/mL and 17.36U/mg. in conclusion, Fusarium oxysporum produced Invertase which can be harness for industrial uses. Keyword: Fusarium oxysporum, Sucrose, Enzyme activity, Purification, Peptone

Characterization of Partially Purified and Immobilized Protease Extracted from Silver Catfish (Pangasius Sutchi) Viscera

Viscera is known as one of the richest sources of proteolytic enzymes. However, the biggest problem regarding enzyme is the rapid deterioration and lose in functional properties due to inefficient treatment and improper storage condition. Therefore, in this study, protease from silver catfish viscera have been extracted, partially purified by acetone precipitation method and immobilized in the calcium alginate beads. Concentrations of 2.99% (w/v) sodium alginate and 0.30 M calcium chloride were used to produce the immobilized protease. Then, the partially purified and immobilized protease were characterized in different range of pH, temperature and storage period in order to establish a greater protease stability during storage and increase in its efficiency. Proteolytic activity of the partially purified and immobilized protease in the alginate beads were measured by using casein as a substrate. The highest proteolytic activity for partially purified protease was recorded at pH 4 (5709.76 CDU/mg) and 40 °C (6061.14 CDU/mg) and for immobilized protease at pH 5 (3987.85 CDU/mg) and 50 °C (4956.54 CDU/mg). For storage stability, the partially purified protease has optimum storage temperature at – 40 °C while the immobilized protease at 4 °C. At optimum storageondition, the partially purified protease and immobilized protease have storage efficiency of 17.44 and 24.50%, respectively until 6th days of storage. Overall, the results showed that immobilization of protease improved enzyme limitation such as proteolytic activity, stability and the purity.

Trichloroacetic Acid/Acetone Precipitation Method to Optimize Canine Synovial Fluid for One and Two-Dimensional Gel Electrophoresis Studies

The challenge associated with the use of synovial fluid for osteoarthritic proteome studies is the optimization step, which involves the depletion of high abundant proteins from the samples. The objective of this study was to develop a cost efficient and effective method to remove albumin from canine synovial fluid for proteome studies. Pooled synovial fluid samples were obtained from clinically healthy dogs (n=5), with no radiographic features of osteoarthritis. The acetone precipitation method and 10% w/v of trichloroacetic acid/acetone were chosen to deplete the albumin from canine synovial fluid and the outcome from the different methods were compared using one dimensional and two-dimensional gel electrophoresis studies. The results showed that the 10% w/v TCA/acetone precipitation method removed highly abundant proteins from synovial fluid for gel electrophoresis studies compared to the acetone precipitation method. The 10% w/v TCA/acetone precipitation method provides an effective method to remove albumin from the synovial fluid using gel electrophoresis, especially two-dimensional gel electrophoresis. The accessibility and cost of TCA and acetone make this method of precipitation a simple and cost-effective technique in preparing a canine synovial fluid for two-dimensional gel electrophoresis analysis.

Evaluation of Protein Purification Techniques and Effects of Storage Duration on LC-MS/MS Analysis of Archived FFPE Human CRC Tissues.

To elucidate cancer pathogenesis and its mechanisms at the molecular level, the collecting and characterization of large individual patient tissue cohorts are required. Since most pathology institutes routinely preserve biopsy tissues by standardized methods of formalin fixation and paraffin embedment, these archived FFPE tissues are important collections of pathology material that include patient metadata, such as medical history and treatments. FFPE blocks can be stored under ambient conditions for decades, while retaining cellular morphology, due to modifications induced by formalin. However, the effect of long-term storage, at resource-limited institutions in developing countries, on extractable protein quantity/quality has not yet been investigated. In addition, the optimal sample preparation techniques required for accurate and reproducible results from label-free LC-MS/MS analysis across block ages remains unclear. This study investigated protein extraction efficiency of 1, 5, and 10-year old human colorectal carcinoma resection tissue and assessed three different gel-free protein purification methods for label-free LC-MS/MS analysis. A sample size of n = 17 patients per experimental group (with experiment power = 0.7 and α = 0.05, resulting in 70% confidence level) was selected. Data were evaluated in terms of protein concentration extracted, peptide/protein identifications, method reproducibility and efficiency, sample proteome integrity (due to storage time), as well as protein/peptide distribution according to biological processes, cellular components, and physicochemical properties. Data are available via ProteomeXchange with identifier PXD017198. The results indicate that the amount of protein extracted is significantly dependent on block age (p < 0.0001), with older blocks yielding less protein than newer blocks. Detergent removal plates were the most efficient and overall reproducible protein purification method with regard to number of peptide and protein identifications, followed by the MagReSyn® SP3/HILIC method (with on-bead enzymatic digestion), and lastly the acetone precipitation and formic acid resolubilization method. Overall, the results indicate that long-term storage of FFPE tissues (as measured by methionine oxidation) does not considerably interfere with retrospective proteomic analysis (p > 0.1). Block age mainly affects initial protein extraction yields and does not extensively impact on subsequent label-free LC-MS/MS analysis results.

Optimization of Immobilization Condition of Protease Extracted from Silver Catfish (Pangasius Sutchi) Viscera in Calcium Alginate by using Response Surface Methodology (RSM)

The term of immobilized enzymes refers to enzymes that physically entrapped within pores of synthetic or natural polymeric network with no alteration in the enzyme catalytic activities. In this study, protease from silver catfish viscera have been extracted, partially purified by acetone precipitation method and immobilised in the calcium alginate beads. Various range of sodium alginate (1–5% w/v) and calcium chloride (0.1–0.5 M) concentrations were used for the optimization purpose. Proteolytic activity of the protease in the alginate beads was measured as a response to the independent variables by using casein as a substrate. The highest actual and predicted proteolytic activities were at run 12 with 674.77 CDU/mg and 639.26 CDU/mg, respectively, under predetermined factors, in which the sodium alginate and calcium chloride solution concentrations were at 3.00% (w/v) and 0.30 M, respectively. The lowest actual and predicted proteolytic activities were at run 2 with the values of 77.35 CDU/mg and 71.53 CDU/mg, respectively, whereby factors include were a sodium alginate of 4.00% (w/v) and 0.20 M of calcium chloride solution. For the experimental feasibilities, the optimum conditions that was feasible to be carried out was with a sodium alginate of 2.99% (w/v) and 0.30 M calcium chloride solution. Verification for the optimum condition was performed and there was no significant difference (p &gt; 0.05) between the predicted (638.19 CDU/mg) and verified (699.82 CDU/mg) values. Thus, indicating that the model was significant and can be used to produce the immobilize protease under the optimum condition.

Proteomic analysis of drought stress response mechanism in soybean (Glycine max L.) leaves.

Knowledge of the physiological and molecular mechanisms of drought responses is fundamental for developing genetically drought tolerant and high yielding crops. To understand molecular mechanism of drought tolerance of soybean (Glycine max L.), we compared leaf proteome patterns of in two genotypes GN‐3074 (drought tolerant) and GN‐2032 (drought‐sensitive) under drought stress during vegetative stage. Proteins were extracted from leaves of well‐watered and drought‐treated plants by using the trichloroacetic acid (TCA)–acetone precipitation method and analyzed by two‐dimensional polyacrylamide gel electrophoresis. Out 488 reproducibly detected and analyzed on two‐dimensional electrophoresis gels, 26 proteins showed significant changes in at least one genotype. The identification of 20 differentially expressed proteins using mass spectrometry revealed a coordinated expression of proteins involved in cellular metabolisms including photosynthesis, oxidative stress defense, respiration, metabolism process, signal transduction, phosphorus transduction, and methyl transduction which enable plant to cope with drought conditions. The most identified proteins involved in photosynthesis and oxidative stress defense system. The up‐regulation of several photosynthetic proteins and also high abundance of oxidative stress defense proteins in GN‐3074 genotypes as compare to GN‐2032 genotypes might reflect the fact that drought tolerance of GN‐3074 is due to effective photosynthetic machinery and more defense against oxidative stress. Our results suggest that soybean plant might response to drought stress by applying efficiently stay‐green mechanism through coordinated gene expression during vegetative stage.

Psp140: an immunodominant antigen in the supernatant of Streptococcus pneumoniae culture.

Background and Objectives:Streptococcus pneumoniae causes many lethal infections. Due to its reduced sensitivity to commonly used antibiotics, development of new strategies against pneumococcal infections seems to be necessary. We aimed to investigate immunodominant antigens in S. pneumoniae culture supernatant in order to develop novel targets for pneumococcal vaccines.Materials and Methods:In this study S. pneumoniae ATCC49619 was sub-cultured into BHI broth from overnight culture at 37°C for 4 h. The supernatant proteins were precipitated using acetone precipitation method. A rabbit was intramuscularly immunized with alum adjuvant and 100 μg pneumococcal supernatant proteins, 6 times at 14 days’ intervals to produce hyperimmune serum. ELISA assay was performed to determine the antibody level response to pneumococcal secretory proteins. Then dot blot applied for rapid evaluation of hyperimmune serum reactivity to pneumococcus supernatant proteins. The western blot was also used to determine the interaction of supernatant proteins with immunogenic rabbit’s hyperimmune-serum.Results:According to the western blot analysis, the immunodominant protein had 140KDa molecular weight and designated as pneumococcal secretory protein140 (Psp140).Conclusion:The Psp140 protein in the supernatant of S. pneumoniae culture is an immunodominant protein and it is likely related to pneumococcal secretory protein or surface exposed protein which released into culture supernatant during bacterial growth.

STAT3 Inhibition by Centipede Scolopendra Extract in Liver Cancer HepG2 Cells and Orthotopic Mouse Models of Hepatocellular Carcinoma

ObjectiveTo observe the effects of Centipede Scolopendra extraction (CSE) on human liver cancer HepG2 cells and the nude mouse tumor model of liver orthotopic transplantation, and to explore the anti-liver cancer mechanism of the extract. MethodsHepG2 cells were respectively treated with CSE250 (250 μg/mL), CSE500 (500 μg/mL) and 5-FU, and control group was established. An enzymatic hydrolysis and acetone precipitation method was used to separate and purify CSE, which was then used to treat HepG2 cells. The CCK8 assay was used to detect the inhibition of cell proliferation and the half maximal inhibitory concentration (IC50) was calculated. Flow cytometry was used to analyze the cell cycle, and western blot was used to detect the expression of signal transduction and activator of transcription 3 (STAT3) pathway-related proteins in HepG2 cells treated with CSE. A nude mouse model with an orthotopic liver tumor was prepared. The mice were randomly divided into four groups, each containing 12 animals: the model group, the 5-FU group, the CSE10 group [10 mg/(kg·d)] and the CSE50 group [50 mg/(kg·d)]. The volume and mass changes in the nude mice with orthotopic transplanted tumors were observed. Western blot method was used to test the protein expression levels of p-STAT3 and p38 mitogen-activated protein kinase (p38MAPK). Tissues from the liver of mice in the model group and the CSE50 group were analyzed by using a protein tyrosine kinase (PTK) chip, and the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) function enrichment analysis of the differentially expressed proteins was performed. ResultsThis study showed that CSE significantly inhibited the proliferation of HepG2 cells (P < 0.05). After 48 h of CSE treatment, the cell cycle of HepG2 cells manifested as S phase and G2/M phase; p-STAT3 protein levels in the CSE groups were significantly lower than that in the control group (P < 0.05). Analysis of the tumor inhibition in the mice showed that the tumor masses and volume in CSE groups were lower (P < 0.05). The protein levels of p-STAT3 and p38MAPK in CSE50 group and 5-FU group decreased significantly (P < 0.05). PTK antibody chip screening results showed that CSE groups had a bidirectional regulation trend, and there were 23 up-regulated PTKs and six down-regulated PTKs. The GO and KEGG analyses showed that CSE exerted its anticancer effects through regulation of biological processes, including mitogen-activated protein kinase (MAPK) cascade, chemotaxis, cell invasion, cell adhesion, angiogenesis and other biological processes, and through signaling pathways, including the MAPK, phosphatidylinositol-3-kinase/serine threonine protein kinase (PI3K/AKT), and RAS signaling pathways. ConclusionsCSE can effectively inhibite the proliferation of HepG2 cells and effectively inhibite the growth of liver cancer orthotopic transplantation tumor. Its mechanism may be closely related to the regulation of STAT3, MAPK and PI3K/AKT signaling pathways.

Novel Cytoplasmic Bacteriocin Compounds Derived from Staphylococcus epidermidis Selectively Kill Staphylococcus aureus, Including Methicillin-Resistant Staphylococcus aureus (MRSA).

Staphylococcus aureus (S. aureus) is one of the well-known agents causing atopic dermatitis (AD) in susceptible individuals, and Staphylococcus epidermidis (S. epidermidis) produces class I thermostable bacteriocins that can selectively kill S. aureus, suggesting protective roles against AD. There is a large need for developing precise therapies only to target S. aureus and not to harm the beneficial microbiome. On the agar well diffusion assay, live planktonic S. epidermidis showed clear zones of inhibition of S. aureus growth, but heat-killed cells and cell-free supernatants did not show this. These results would lead us to hypothesize that cytoplasmic bacteriocin from S. epidermidis will be a promising agent to inhibit S. aureus growth. Therefore, we have extracted a novel thermolabile cytoplasmic bacteriocin from S. epidermidis using trichloroactic acid (TCA)/acetone precipitation method after cell lysis with a SDS-containing buffer. These bacteriocin selectively exhibited antimicrobial activity against S. aureus and methicillin-resistance Staphylococcus aureus (MRSA), presenting no active actions against S. epidermidis, E. coli, and Salmonella Typhimurium. The extracted cytoplasmic bacteriocin compounds revealed several diffuse bands of approximately 40–70 kDa by SDS-PAGE. These findings suggest that these cytoplasmic bacteriocin compounds would be a great potential means for S. aureus growth inhibition and topical AD treatment.

Improved Method for the Determination of Polychlorinated Dibenzo-p-dioxins and Dibenzofurans (PCDD/Fs) in Sanitary Napkins

An improved method was developed for determining highly toxic 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins and polychlorinated dibenzo-p-furans (PCDD/Fs) in sanitary napkins. Samples were extracted by Soxhlet (50% dichloromethane/hexane) and sonication (hexane), and a multi-layer silica column was used for clean-up after acetone precipitation to separate the superabsorbent polymers. Improved separation efficiency of PCDD/Fs from sanitary napkins with a high level of superabsorbent polymers was achieved using the acetone precipitation method. Hexane was then applied as an alternative solvent to remove residual interfering substances including sticky components contained on sanitary napkins via alumina column chromatography in the second clean-up step. Recoveries approached 100% with an average relative standard deviation of less than 15%, and recoveries of internal standards were from 70.5 to 114.8% and 0.6 to 13.6% for the two extraction methods. The mean method detection limit was 0.075–0.968 pg/g for Soxhlet extraction and 0.0032–0.091 pg/g for sonication extraction. The established method meets the quality criteria for the screening of dioxins stated in EU Regulations 589/2014 and 709/2014, and therefore provides a suitable alternative for the determination of dioxins in sanitary napkins containing superabsorbent polymers.

Secretory Laccase from Pestalotiopsis Species CDBT-F-G1 Fungal Strain Isolated from High Altitude: Optimization of Its Production and Characterization

Microorganisms producing laccases may be used for the pretreatment of lignocellulosic biomass to recover fermentable sugar. Very few fungi and other microbes growing in high altitudes have been tested for this purpose. As part of this study, we have collected soil samples from different parts of the Kathmandu Valley and the Rautah at district of Nepal (1600 to 2303 m above sea level) and successfully cultured 53 different isolates of microorganisms. Among the 53 isolates obtained 30 were Actinomycetes, 20 were Streptomycetes, and three were fungi). These isolates were tested for laccase expression using guaiacol, tannic acid, and 1-naphthol as substrates. Twelve of the 53 isolates tested positive for the expression of laccase. Among the laccase- positive isolates, a fungal species designated as CDBT-F-G1was found to produce high levels of laccase. This isolate was identified as Pestalotiopsis species based on 18S rRNA sequencing. Pestalotiopsis spp. CDBT-F-G1 isolate grows efficiently in PDB media containing 1% Kraft lignin at pH 5 and 30 °C and secretes 20 ± 2 U/mL laccase in culture medium. Further optimization of growth conditions reveled that addition of (i) metal salts, e.g., 1 mM magnesium sulfate (51 ± 25 U/mL); (ii) agitation of cultures at 200 rpm (51 ± 9U/mL); (iii) surfactants, e.g., 0.75 mM Tween 80 (54 ± 14 U/mL); (iv) 40% dissolved O2 (57 ± 2 U/mL) and inducers, e.g., 1 mM gallic acid (69 ± 11 U/mL), further promote laccase production by Pestalotiopsis spp. CDBT-F-G1 isolate. On the other hand, 0.1 mM cysteine inhibited laccase production. The secretory laccase obtained from fermentation broth of CDBT-F-G1 was partially purified by ammonium sulfate (13-fold purification with specific activity 26,200 U/mg) and acetone (14-fold purification with specific activity 31,700 U/mg) precipitation methods. The enzyme has an approximate molecular mass of 43 kDa, pH and temperature optima werepH6 and 60 °C, respectively. Vmax and Km were 100 μmol/min and 0.10 mM, respectively, with ABTS as the substrate. Given the above characteristics, we believe Pestalotiopsis spp. CDBT-F-G1 strain native to high altitudes of Nepal could be used to pretreat lignocellulosic biomass to efficiently recover fermentable sugars.

Development of a Rapid and Simple Method for Preparing Tea-Leaf Saponins and Investigation on Their Surface Tension Differences Compared with Tea-Seed Saponins.

The relative overcapacity in China's tea-leaf production and the potential application of tea-leaf saponins in soil remediation encouraged in-depth developments and comprehensive utilizations of tea-leaf resources. Through variables optimizations using Box–Behnken designs for ultrasonic power, temperature as well as ultrasonic treatment time in ultrasonic-assisted water extraction and single-variable experiments for acetone-extraction solution ratio in acetone precipitation, a rapid and simple method was developed for preparing tea-leaf saponins. Tea-leaf saponins with the concentration of 3.832 ± 0.055 mg/mL and the purity of 76.5% ± 1.13% were acquired under the optimal values of 78 w, 60 °C, 20 min and 0.1 ratio of acetone-extraction solution. Both Fourier transform-infrared (FT-IR) spectra and ultraviolet (UV) spectra revealed slight composition differences between tea-leaf saponins and tea-seed saponins, while these differences were not reflected in the critical micelle concentration (CMC) and the surface tension of tea-leaf saponins and tea-seed saponins, indicating there was no need to distinguish them at the CMC. Further research attention on where tea-leaf saponins were in low concentrations is deserved to discover whether they had differences in comparison with tea-seed saponins, which was beneficial to apply them in the phytoremediation of contaminated soils.

Collection and preservation of urinary proteins, using a fluff pulp diaper.

Change is the most fundamental property of a biomarker. In contrast to the blood, which is under homeostatic controls, urine reflects changes in the body earlier and is more sensitive, thus making it a better biomarker source. Moreover, drawing blood from infants and toddlers is difficult and not tolerated well. For patients limited by language, communicating their chief complaint is difficult. Thus, monitoring biomarkers in urine can provide valuable clues for the diagnosis of diseases, especially pediatric diseases. Collecting urine from young children and some adult patients is more challenging than collecting it from healthy adults. Here, we propose a method that uses a fluff pulp diaper to collect urine. Urinary proteins are then eluted and adsorbed onto a piece of nitrocellulose membrane, which can be dried and stored in a vacuum bag. SDS-PAGE and LC-MS/MS analysis indicated that this method is reproducible, and similar proteins were identified as those obtained by an acetone precipitation method. With this simple and economical method, it is possible to collect and preserve urine samples from infants, toddlers, and patients with special needs, even for large-scale biomarker studies.

Expression analysis of apolipoproteins AI & AIV in hepatocellular carcinoma: A protein-based hepatocellular carcinoma-associated study.

Background & objectives:Hepatocellular carcinoma (HCC) is one of the leading causes of cancer mortality. The objective of this study was to find out the differential expression of apolipoproteins (ApoAI and ApoAIV) in HCC and cases of liver cirrhosis and chronic hepatitis (controls) without HCC and to compare ApoAI and ApoAIV expression with alpha-foetoprotein (AFP), the conventional marker in HCC.Methods:Fifty patients with HCC and 50 controls comprising patients with liver cirrhosis (n=25) and chronic hepatitis (n=25) without HCC were included in this study. Total proteins were precipitated using acetone precipitation method followed by albumin and IgG depletion of precipitated protein using depletion kit. Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The expression changes of ApoAI and ApoAIV were confirmed by western blotting using specific primary and secondary polyclonal antibodies followed by densitometric protein semi-quantitative estimation. ApoAI, ApoAIV and AFP were measured in the plasma samples by ELISA method.Results:Semi-quantitative densitometric image analysis of the western blot images and the comparison between HCC patients with those without HCC (control) revealed differential expression of ApoAI and ApoAIV. Levels of ApoAI were significantly higher in patients with HCC compared to controls without HCC (0.279±0.216 vs 0.171±0.091 and 0.199±0.014; P <0.001). Levels of ApoAIV were significantly lower in patients of HCC compared to controls without HCC (0.119±0.061 vs 0.208±0.07 and 0.171±0.16; P <0.01). ELISA assays of apolipoproteins (ApoAI and ApoAIV) revealed similar results of expression of ApoAI and ApoAIV as detected in western blotting densitometric image analysis.Interpretation & conclusions:Increased expression of ApoAI and decreased expression of ApoAIV in HCC patients compared to controls without HCC revealed the abnormalities in HCC. These molecules need to be studied further for their use as potential biomarkers in the future diagnostic tools along with other conventional biomarkers for screening of HCC cases. It needs further analysis in higher number of patient population.

Electrokinetic Removal of Dodecyl Sulfate Micelles from Digested Protein Samples Prior to Electrospray-Ionization Mass Spectrometry.

In proteomics, dodecyl sulfate (DS-) as sodium salt is commonly used in protein solubilization prior to tryptic digestion, but the presence of the DS- hampers the electrospray ionization mass spectrometric (ESI-MS) analysis. The development of DS- depletion techniques is therefore important especially when dealing with small samples where there could be poor sensitivity due to sample loss or dilution during sample preparation. Here, we present a simple and fast electrokinetic removal method of DS- from small volumes of peptide and digested protein samples prior to ESI-MS. The selective removal was accomplished using an acidic extraction solution (ES) containing acetonitrile (ACN) inside a fused-silica capillary that was dipped into the sample. The use of acidic ES suppressed the electroosmotic flow; allowing the electrokinetic movement of DS- monomers and micelles into the capillary. The high amount of ACN present at the tip of the capillary served to collapse the micelles migrating into the capillary, thereby releasing the peptides that were bound to these micelles, facilitating peptide retention in the sample and efficient DS- removal. Increased % MS signal intensity (SI) restoration of the peptide was observed, while DS- removal was unaffected when the amount of ACN in the ES was increased. This is because of the micelle to solvent stacking mechanism (effective electrophoretic mobility reversal) working at high concentration of ACN for the improved recovery of the peptides. % MS SI restoration for the Z-Gly-Gly-Val and bradykinin peptides were 75-83% while % MS SI reduction of DS- was up to 99% under optimal conditions, that is, 40% ACN in the ES. Higher % peptide recoveries from digested protein samples were obtained using the proposed method compared to the conventional cold acetone precipitation method.

Proteomic prospects for tolerance of sunflower (Helianthus annuus) to drought stress during the flowering stage

In order to understanding proteomic basis of drought tolerance in sunflower (Helianthus annuus L.), two contrasting inbred lines were subjected to drought stress during the flowering stage for two years. Proteins were extracted from leaves of well-watered and drought-treated plants by using the TCA–acetone precipitation method and analysed by two-dimensional polyacrylamide gel electrophoresis followed by nanoscale liquid chromatography coupled to tandem mass spectrometry for identification of affected proteins. When comparing proteomic patterns, 18 proteins were changed by drought stress in sensitive lines and 24 proteins in tolerant lines. Concurrent down-expressions of oxygen-evolving enhancer and ferredoxin-NADP reductase were considered as primary drought sensors that mediate downstream pathways to cope with drought conditions. Differential and line-specific proteomic changes were attributed as the source for contrasting response to drought stress. According to the results, scavenging of reactive oxygen species, conservation of energy and water, and cell-structure integrity constituted the major aspects of drought tolerance in sunflower.

Extraction and Solubilization Methods Suitable for Proteomic Analysis of Common Bean Seed of Ivory Coast

The extraction and solubilization of proteins from seeds of Phaseolus vulgarisms for two-dimension polyacrylamide gel electrophoresis (2D-PAGE) and analysis by mass spectrometry are very sensitive procedures. In this study, we used two methods of extraction and solubilization of proteins, the urea/thiourea method and the trichloroacetic acid (TCA)/acetone precipitation method, in order to determine their effectiveness in separating proteins from bean seeds by 2D-PAGE. In both methods, proteins were well separated by 2D PAGE with minor variations in the protein pattern. These two extraction methods showed that it was possible to separate hundreds of very resolvent proteins by 2D electrophoresis. A protein spot was selected on the 2D-PAGE gel, digested with trypsin and analyzed by mass spectrometry (LCMS/ MS). The results suggest that thiourea/urea and TCA methods were effective and reliable for the extraction and solubilization for 2D analysis of proteins from seeds of Phaseolus vulgaris .

A simple procedure for preparing chitin oligomers through acetone precipitation after hydrolysis in concentrated hydrochloric acid

Chitin oligomers are of interest because of their numerous biologically relevant properties. To prepare chitin oligomers containing 4–6 GlcNAc units [(GlcNAc)4–6], α- and β-chitin were hydrolyzed with concentrated hydrochloric acid at 40°C. The reactant was mixed with acetone to recover the acetone-insoluble material, and (GlcNAc)4–6 was efficiently recovered after subsequent water extraction. Composition analysis using gel permeation chromatography and MALDI-TOF mass spectrometry indicated that (GlcNAc)4–6 could be isolated from the acetone-insoluble material with recoveries of approximately 17% and 21% from the starting α-chitin and β-chitin, respectively. The acetone precipitation method is highly useful for recovering chitin oligomers from the acid hydrolysate of chitin. The changes in the molecular size and higher-order structure of chitin during the course of hydrolysis were also analyzed, and a model that explains the process of oligomer accumulation is proposed.