Comparison of Plasma Exosomes by Differential Ultracentrifugation and Solvent Precipitation Methods.

BACKGROUND: Emerging evidence has identified that exosomes play a pivotal role in intercellular signal transmission. However, the standardized purification techniques to isolate high quality exosomes are still deficient at present. This study was to evaluate reproducibility and efficiency of differential ultracentrifugation and solvent precipitation-based kits by isolating plasma-derived exosomes from oral lichen planus patients. METHODS: Morphology, exosomal biomarkers, particle size distribution, proteomic components, and protein yield of isolated exosomes were evaluated by transmission electron microscope, western blot, laser diffraction instrument, Coomassie staining, and BCA protein assay kit, respectively. RESULTS: TEM displayed representative cup-shaped morphology of exosomes and western blot identified exosomal biomarkers CD9 and CD63. The size distribution showed that particles by differential ultracentrifugation were mainly from 26.15 nm to 166.5 nm, while some of the particles obtained by solvent precipitation kits were larger than 1,000 nm. In addition, exosomes isolated by solvent precipitation kits showed a significantly higher amount of protein yield due to plasma albumin contamination. CONCLUSIONS: Both differential ultracentrifugation and precipitation based kits could successfully isolate plasma exosomes, and exosomes by differential ultracentrifugation were purer and more appropriate for further proteomic analysis.

Sample preparation for large-scale bioanalytical studies based on liquid chromatographic techniques.

Quality of the analytical data obtained for large-scale and long term bioanalytical studies based on liquid chromatography depends on a number of experimental factors including the choice of sample preparation method. This review discusses this tedious part of bioanalytical studies, applied to large-scale samples and using liquid chromatography coupled with different detector types as core analytical technique. The main sample preparation methods included in this paper are protein precipitation, liquid-liquid extraction, solid-phase extraction, derivatization and their versions. They are discussed by analytical performances, fields of applications, advantages and disadvantages. The cited literature covers mainly the analytical achievements during the last decade, although several previous papers became more valuable in time and they are included in this review.

Partial purification and characterization of a novel histidine decarboxylase from Enterobacter aerogenes DL-1.

Histidine decarboxylase (HDC) from Enterobacter aerogenes DL-1 was purified in a three-step procedure involving ammonium sulfate precipitation, Sephadex G-100, and DEAE-Sepharose column chromatography. The partially purified enzyme showed a single protein band of 52.4 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH for HDC activity was 6.5, and the enzyme was stable between pH 4 and 8. Enterobacter aerogenes HDC had optimal activity at 40°C and retained most of its activity between 4 and 50°C. HDC activity was reduced in the presence of numerous tested compounds. Particularly with SDS, it significantly (p < 0.01) inhibited enzyme activity. Conversely, Ca(2+) and Mn(2+) showed prominent activation effects (p < 0.01) with activity increasing to 117.20% and 123.42%, respectively. The Lineweaver-Burk plot showed that K m and V max values of the enzyme for L-histidine were 0.21 mM and 71.39 µmol/min, respectively. In comparison with most HDCs from other microorganisms and animals, HDC from E. aerogenes DL-1 displayed higher affinity and greater reaction velocity toward L-histidine.

Preparation and optimization of resveratrol nanosuspensions by antisolvent precipitation using Box-Behnken design.

Resveratrol, a natural polyphenolic component, has inspired considerable interest for its extensive physiological activities. However, the poor solubility of resveratrol circumscribes its therapeutic applications. The purpose of this study was to optimize and prepare resveratrol nanosuspensions using the antisolvent precipitation method. The effects of crucial formulation and process variables (drug concentration, stabilizer, and surfactant contents) on particle size were investigated by utilizing a three-factor three-level Box-Behnken design (BBD) to perform this experiment. Different mathematical polynomial models were used to identify the impact of selected parameters and to evaluate their interrelationship for predictive formulation purposes. The optimal formulation consisted of drug 29.2 (mg/ml), polyvinylpyrrolidone (PVP) K17 0.38%, and F188 3.63%, respectively. The morphology of nanosuspensions was found to be near-spherical shaped by scanning electron microscopy (SEM) observation. The X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) analysis confirmed that the nanoparticles were in the amorphous state. Furthermore, in comparison to raw material, resveratrol nanosuspensions showed significantly enhanced saturation solubility and accelerated dissolution rate resulting from the decrease in particle size and the amorphous status of nanoparticles. Meanwhile, resveratrol nanosuspensions exhibited the similar antioxidant potency to that of raw resveratrol. The in vivo pharmacokinetic study revealed that the C max and AUC0→∞ values of nanosuspension were approximately 3.35- and 1.27-fold greater than those of reference preparation, respectively. Taken together, these results suggest that this study provides a beneficial approach to address the poor solubility issue of the resveratrol and affords a rational strategy to widen the application range of this interesting substance.

Resolubilization of precipitated intact membrane proteins with cold formic acid for analysis by mass spectrometry.

Protein precipitation in organic solvent is an effective strategy to deplete sodium dodecyl sulfate (SDS) ahead of MS analysis. Here we evaluate the recovery of membrane and water-soluble proteins through precipitation with chloroform/methanol/water or with acetone (80%). With each solvent system, membrane protein recovery was greater than 90%, which was generally higher than that of cytosolic proteins. With few exceptions, residual supernatant proteins detected by MS were also detected in the precipitation pellet, having higher MS signal intensity in the pellet fraction. Following precipitation, we present a novel strategy for the quantitative resolubilization of proteins in an MS-compatible solvent system. The pellet is incubated at -20 °C in 80% formic acid/water and then diluted 10-fold with water. Membrane protein recovery matches that of sonication of the pellet in 1% SDS. The resolubilized proteins are stable at room temperature, with no observed formylation as is typical of proteins suspended in formic acid at room temperature. The protocol is applied to the molecular weight determination of membrane proteins from a GELFrEE-fractionated sample of Escherichia coli proteins.

Efficient isolation and elution of cellular proteins using aptamer-mediated protein precipitation assay.

Protein precipitation is one of the most widely used methods for antigen detection and purification in biological research. We developed a reproducible aptamer-mediated magnetic protein precipitation method that is able to efficiently capture, purify and isolate the target proteins. We discovered DNA aptamers having individually high affinity and specificity against human epidermal growth factor receptor (EGFR) and human insulin receptor (INSR). Using aptamers and magnetic beads, we showed it is highly efficient technique to enrich endogenous proteins complex and is applicable to identify physiologically relevant protein-protein interactions with minimized nonspecific binding of proteins. The results presented here indicate that aptamers would be applicable as a useful and cost-effective tool to identify the presence of the particular target protein with their specific protein partners.

APols-aided protein precipitation: a rapid method for concentrating proteins for proteomic analysis.

Amphipols (APols) are a newly designed and milder class of detergent. They have been used primarily in protein structure analysis for membrane protein trapping and stabilization. We have recently demonstrated that APols can be used as an alternative detergent for proteome extraction and digestion, to achieve a "One-stop" single-tube workflow for proteomics. In this workflow, APols are removed by precipitation after protein digestion without depleting the digested peptides. Here, we took further advantage of this precipitation characteristic of APols to concentrate proteins from diluted samples. In contrast with tryptic peptides, a decrease in pH leads to the unbiased co-precipitation of APols with proteins, including globular hydrophilic proteins. We demonstrated that this precipitation is a combined effect of acid precipitation and the APols' protein interactions. Also, we have been able to demonstrate that APols-aided protein precipitation works well on diluted samples, such as secretome sample, and provides a rapid method for protein concentration.

Isolation of Escherichia coli mannitol permease, EIImtl, trapped in amphipol A8-35 and fluorescein-labeled A8-35.

Amphipols (APols) are short amphipathic polymers that keep integral membrane proteins water-soluble while stabilizing them as compared to detergent solutions. In the present work, we have carried out functional and structural studies of a membrane transporter that had not been characterized in APol-trapped form yet, namely EII(mtl), a dimeric mannitol permease from the inner membrane of Escherichia coli. A tryptophan-less and dozens of single-tryptophan (Trp) mutants of this transporter are available, making it possible to study the environment of specific locations in the protein. With few exceptions, the single-Trp mutants show a high mannitol-phosphorylation activity when in membranes, but, as variance with wild-type EII(mtl), some of them lose most of their activity upon solubilization by neutral (PEG- or maltoside-based) detergents. Here, we present a protocol to isolate these detergent-sensitive mutants in active form using APol A8-35. Trapping with A8-35 keeps EII(mtl) soluble and functional in the absence of detergent. The specific phosphorylation activity of an APol-trapped Trp-less EII(mtl) mutant was found to be ~3× higher than the activity of the same protein in dodecylmaltoside. The preparations are suitable both for functional and for fluorescence spectroscopy studies. A fluorescein-labeled version of A8-35 has been synthesized and characterized. Exploratory studies were conducted to examine the environment of specific Trp locations in the transmembrane domain of EII(mtl) using Trp fluorescence quenching by water-soluble quenchers and by the fluorescein-labeled APol. This approach has the potential to provide information on the transmembrane topology of MPs.

Novel thermo-responsive fucose binding ligands for glycoprotein purification by affinity precipitation.

Novel thermo-responsive affinity sugar binders were developed by fusing a bacterial fucose lectin with a thermo-responsive polypeptide. These designer affinity ligand fusions were produced using an Escherichia coli system capable of extracellular secretion of recombinant proteins and were isolated with a high recovery yield (95%) directly from growth medium by Inverse Temperature Cycling (ITC). With horse radish peroxidase (HRP) as a model protein, we demonstrate here that the designer thermo-responsive ligands are capable of interacting with glycans on a glycoprotein, a property that was used to develop a novel affinity precipitation method for glycoprotein purification. The method, requiring only simple process steps, affords full recovery of a target glycoprotein, and is effective at a target glycoprotein concentration as low as 1.4 pM in the presence of large amounts of contaminants. By developing other sugar binders in the similar fashion, the method should be highly useful for glycoprotein purification and detection.

Optimization of the fractional precipitation of paclitaxel from a Taxus chinensis cell culture using response surface methodology and its isolation by consecutive high-speed countercurrent chromatography.

A consecutive preparation method for the isolation and purification of paclitaxel from the Taxus Chinensis cell culture was developed in this study. The process involved alkaline Al2O3 chromatography, fractional precipitation, and high-speed countercurrent chromatography. The original cell culture materials were first extracted with methanol using ultrasound-assisted extraction, and then the extract (the content of paclitaxel is 1.5%) was separated by alkaline Al2O3 column chromatography. Subsequently, fractional precipitation was used to obtain paclitaxel. In particular, response surface methodology was used to optimize the factors of fractional precipitation (methanol concentration, material-to-solvent ratio, and precipitating time were optimized as 48.14%, 8.85 mg/mL, and 48.71 h, respectively) and the yield of fractional precipitation product was 30.64 ± 0.60 mg (the content of paclitaxel is 89.3%, 27.37 ± 0.54 mg) from a 100 mg fraction by Al2O3 column separation (the content of paclitaxel is 32.4%). Then, the product was used for further isolation by high-speed countercurrent chromatography. About 1.00 g paclitaxel (200 ± 2 mg in each loading) with a purity up to 99.61% was isolated from 1.25 g of fractional precipitation product with a solvent system of n-hexane/ethyl acetate/methanol/water (1.2:1.8:1.5:1.5, v/v/v/v) in one run of five consecutive sample loadings without exchanging a new solvent system.

Highly visible-light-responsive photocatalytic AgCl/BiOCl hetero-nanostructures synthesized by a chemical coprecipitation method.

AgCl/BiOCl heteronanostructures were synthesized by a room-temperature chemical coprecipitation method. The as-obtained products were characterized by energy-dispersive X-ray spectroscopy (EDS), transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and UV-Vis diffuse spectra, which show the structures, morphologies, and optical properties. The results revealed that the absorption edge of AgCl/BiOCl shifted towards visible light regions. Meanwhile, the AgCl/BiOCl heteronanostructures showed better photocatalytic properties than the pure BiOCl to degrade rhodamine B and the 5% AgCl/BiOCl showed the best photocatalytic ability, which completely decomposed the target molecules in 17 minites with the visible-light illumination. The formation of heteronanostructures might improve the separation of photogenerated electrons and holes derived from the coupling effect of BiOCl and AgCl heteroarchitectures, which was regarded as the main reason for the high photocatalytic activity.

Properties and in vitro characterization of polyhydroxybutyrate-chitosan scaffolds prepared by modified precipitation method.

Porous polyhydroxybutyrate (PHB)-chitosan biopolymer scaffolds were prepared by co-precipitation from biopolymer solutions with propylene carbonate and acetic acid as solvents. A change of the fibrous character of chitosan precipitates to globular shaped forms with a polyhydroxybutyrate addition was found in suspensions. Scaffolds differ by porosity and morphology of polymers in microstructures, while chitosan represented more compact plate-like fibers and PHB characterized mainly fine fibrous globular agglomerates. Two structurally dissimilar phase regions were verified in blended scaffolds. A rise in the number of smaller pores, and fine structured polymer forms with PHB content were observed in the scaffolds. A significant reduction in the average molecular weight of biopolymers was found in pure chitosan scaffold, this after precipitation of the chitosan in the presence of propylene carbonate and in blends after mutual biopolymer mixing. Interactions between shortened chitosan chains, PHB and chitosan biopolymers in the blends were observed. An excellent fibroblast proliferation was found in scaffolds prepared from biopolymer blends.

A simple and rapid method to isolate purer M13 phage by isoelectric precipitation.

M13 virus (phage) has been extensively used in phage display technology and nanomaterial templating. Our research aimed to use M13 phage to template sulfur nanoparticles for making lithium ion batteries. Traditional methods for harvesting M13 phage from Escherichia coli employ polyethylene glycol (PEG)-based precipitation, and the yield is usually measured by plaque counting. With this method, PEG residue is present in the M13 phage pellet and is difficult to eliminate. To resolve this issue, a method based on isoelectric precipitation was introduced and tested. The isoelectric method resulted in the production of purer phage with a higher yield, compared to the traditional PEG-based method. There is no significant variation in infectivity of the phage prepared using isoelectric precipitation, and the dynamic light scattering data indirectly prove that the phage structure is not damaged by pH adjustment. To maximize phage production, a dry-weight yield curve of M13 phage for various culture times was produced. The yield curve is proportional to the growth curve of E. coli. On a 200-mL culture scale, 0.2 g L(-1) M13 phage (dry-weight) was produced by the isoelectric precipitation method.

Optimization of parameters for preparation of docetaxel-loaded PLGA nanoparticles by nanoprecipitation method.

The purpose of this study was to develop docetaxel-poly (lactide-co-glycolide) (PLGA) loaded nanoparticles by using nanoprecipitation method and optimize the relative parameters to obtain nanoparticles with higher encapsulation efficiency and smaller size. The physicochemical characteristics of nanoparticles were studied. The optimized parameters were as follows: the oil phase was mixture of acetone and ethanol, concentration of tocopheryl polyethylene glycol succinate (TPGS) was 0.2%, the ratio of oil phase to water phase was 1:5, and the theoretical drug concentration was 5%. The optimized nanoparticles were spherical with size between 130 and 150 nm. The encapsulation efficiency was (40.83±2.1)%. The in vitro release exhibited biphasic pattern. The results indicate that docetaxel-PLGA nanoparticles were successfully fabricated and may be used as the novel vehicles for docetaxel, which would replace Taxotere® and play great roles in future.

Phosphorus recovery potential from a waste stream with high organic and nutrient contents via struvite precipitation.

Recovery of NH4(+)-N and PO(3-)-P via struvite precipitation (SP) was evaluated from liquor of thermally pretreated waste activated sludge, containing high levels of nutrients (1500 mg NH4(+)-N/L and 650 mg PO(3-)-P/L), organics (45.5 g COD/L) and suspended solids (3.5 g TSS/L), with reference to anaerobically digested sludge centrate. In a series of jar tests, the order of pH adjustment and chemical addition were first tested for the digested sludge centrate. The effects of MgCl2 and MgO, as Mg2+ sources, on SP were evaluated in both waste streams. Up to 80% of the dissolved PO4(3-)-P was recovered using MgO (pH = 9.2) from the pretreated sludge liquor and more than 86% of NH4(+)-N from the digested sludge centrate (pH = 8.0-8.5) regardless of the Mg2+ source used. NH4(+)-N recovery from digested sludge centrate required the addition of alkali, Mg2+ source and PO4(3-)-P, making the process less viable. The precipitates contained mostly struvite and some levels of Ca2+, Fe2+ and other Mg2+ phosphates. The levels of solids, inorganics and organics in the waste streams influenced SP, specifically struvite crystal formation and settleability in the pretreated sludge liquor, which suggests that the applicability of SP for nutrient recovery from complex waste streams requires case-by-case testing, and process optimization.

Simple sodium dodecyl sulfate-assisted sample preparation method for LC-MS-based proteomics applications.

Sodium dodecyl sulfate (SDS) is one of the most popular laboratory reagents used for biological sample extraction; however, the presence of this reagent in samples challenges LC-MS-based proteomics analyses because it can interfere with reversed-phase LC separations and electrospray ionization. This study reports a simple SDS-assisted proteomics sample preparation method facilitated by a novel peptide-level SDS removal step. In an initial demonstration, SDS was effectively (>99.9%) removed from peptide samples through ion substitution-mediated DS(-) precipitation using potassium chloride (KCl), and excellent peptide recovery (>95%) was observed for <20 µg of peptides. Further experiments demonstrated the compatibility of this protocol with LC-MS/MS analyses. The resulting proteome coverage obtained for both mammalian tissues and bacterial samples was comparable to or better than that obtained for the same sample types prepared using standard proteomics preparation methods and analyzed using LC-MS/MS. These results suggest the SDS-assisted protocol is a practical, simple, and broadly applicable proteomics sample processing method, which can be particularly useful when dealing with samples difficult to solubilize by other methods.

Selective precipitation and purification of monovalent proteins using oligovalent ligands and ammonium sulfate.

This paper describes a method for the selective precipitation and purification of a monovalent protein (carbonic anhydrase is used as a demonstration) from cellular lysate using ammonium sulfate and oligovalent ligands. The oligovalent ligands induce the formation of protein-ligand aggregates, and at an appropriate concentration of dissolved ammonium sulfate, these complexes precipitate. The purification involves three steps: (i) the removal of high-molecular-weight impurities through the addition of ammonium sulfate to the crude cell lysate; (ii) the introduction of an oligovalent ligand and the selective precipitation of the target protein-ligand aggregates from solution; and (iii) the removal of the oligovalent ligand from the precipitate by dialysis to release the target protein. The increase of mass and volume of the proteins upon aggregate formation reduces their solubility, and results in the selective precipitation of these aggregates. We recovered human carbonic anhydrase, from crude cellular lysate, in 82% yield and 95% purity with a trivalent benzene sulfonamide ligand. This method provides a chromatography-free strategy of purifying monovalent proteins--for which appropriate oligovalent ligands can be synthesized--and combines the selectivity of affinity-based purification with the convenience of salt-induced precipitation.

Highly efficient precipitation of phosphoproteins using trivalent europium, terbium, and erbium ions.

This study describes a highly efficient method for the selective precipitation of phosphoproteins by trivalent europium, terbium, and erbium metal ions. These metal cations belong to the group of lanthanides and are known to be hard acceptors with an overwhelming preference for oxygen-containing anions such as phosphates to which they form very tight ionic bonds. The method could be successfully applied to specifically precipitate phosphoproteins from complex samples including milk and egg white by forming solid metal-protein complexes. Owing to the low solubility product of the investigated lanthanide salts, the produced metal-protein complexes showed high stability. The protein pellets were extensively washed to remove nonphosphorylated proteins and contaminants. For the analysis of proteins the pellets were first dissolved in 30 % formic acid and subjected to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. For peptide mass-fingerprint analysis the precipitated phosphoproteins were enzymatically digested using microwave-assisted digestion. The method was found to be highly specific for the isolation and purification of phosphoproteins. Protein quantification was performed by colorimetric detection of total precipitated phosphoproteins and revealed more than 95 % protein recovery for each lanthanide salt.

Proteomic characterization of specific minor proteins in the human milk casein fraction.

Human milk contains many bioactive proteins that are likely to support the early development of the newborn. The aim of this study was to identify whether there are specific minor proteins associated with the human milk casein micelle prepared by the acid precipitation method. Protein identification was performed by liquid chromatography tandem mass spectrometry analysis. Eighty-two proteins were identified in the casein micelle, 18 of which are not present in their whey compartment. Thirty-two of these proteins specifically associated with the casein micelle have not previously been identified in human milk or colostrum. Proteins involved in immune function comprised the major part (28%) of total proteins, and another significant part is involved in metabolism/energy production (22%). Most of the proteins were of extracellular or cytoplasmic origin (accounting for 50 and 29%, respectively). This study indicates that various soluble proteins should be considered as part of the casein compartment, prepared by the acid precipitation method. The data provide new insight not only into the proteomic profile of the human milk casein micelle and its physiological significance, but also into the proper proportion of casein and casein-associated proteins to use in infant formula.

Phosphoproteome analysis of Streptomyces development reveals extensive protein phosphorylation accompanying bacterial differentiation.

Streptomycetes are bacterial species that undergo a complex developmental cycle that includes programmed cell death (PCD) events and sporulation. They are widely used in biotechnology because they produce most clinically relevant secondary metabolites. Although Streptomyces coelicolor is one of the bacteria encoding the largest number of eukaryotic type kinases, the biological role of protein phosphorylation in this bacterium has not been extensively studied before. In this issue, the variations of the phosphoproteome of S. coelicolor were characterized. Most distinct Ser/Thr/Tyr phosphorylation events were detected during the presporulation and sporulation stages (80%). Most of these phosphorylations were not reported before in Streptomyces, and included sporulation factors, transcriptional regulators, protein kinases and other regulatory proteins. Several of the identified phosphorylated proteins, FtsZ, DivIVA, and FtsH2, were previously demonstrated to be involved in the sporulation process. We thus established for the first time the widespread occurrence and dynamic features of Ser/Thr/Tyr protein phosphorylation in a bacteria species and also revealed a previously unrecognized phosphorylation motif "x(pT)xEx".