Effective Protein Separation Research Articles

Interfacial modification of hydrogel composite membranes for protein adsorption with cavitands as nano molecular containers

Protein adsorption is of great significance for bioengineering, biomedicine, drug release, etc. Herein, poly-N-isopropylacrylamide (PNIPAm) and polyethylene glycol diacrylate (PEGDA) based hydrogel materials were used to introduce self-synthesized cavitands with pore cage structures and hydrophobic characteristics to construct high-efficiency hydrogel composite membrane (HCM) for the adsorption of bovine serum albumin (BSA). The hydrophobic cavitand cage can improve the interfacial polymerization process by impacting the monomer diffusion and then modifying the interface properties of the hydrogel molecular network, which is validated by the molecular dynamic simulation. The adsorption mechanism can be interpreted as the reorganization of the polymer pores using the cavitands, and hence the surface charge and hydrophobic property consequently changed, which is consistent with the measurement results as well as validated by the molecular dynamic simulation. Thus, the fabricated HCM exhibits good structural stability, thermal stability, and robust BSA adsorption performance. The results indicated that the developed HCM materials can be promising candidates for effective protein separation.

Construction of magnetic Fe3O4@NH2-MIL-100(Fe)-C18 with excellent hydrophobicity for effective protein separation and purification

Due to the appearance of amino and carboxyl groups on the surface of NH2-MIL-100(Fe), a stable Metal-Organic Frameworks (MOFs), it is ready to be modified with other functional ligands to provide versatile applications. In this study, we have successfully employed aliphatic C18 carbon chain to functionalize the magnetite-doped Fe3O4@NH2-MIL-100(Fe). The remarkable hydrophobic surface of Fe3O4@NH2-MIL-100(Fe)-C18 with a water contact angle of 98.93° allowed it to selectively form hydrophobic interaction with a protein in a high concentration of sodium chloride environment. The complex was easily harvested with a magnet, and the bound protein was able to be eluted by reducing sodium chloride concentration. The maximum protein binding capacity reached approximately 28.74 μg of BSA/mg with Fe3O4@NH2-MIL-100(Fe)-C18. Our results have demonstrated that the Dulbecco’s phosphate buffer could provide a better “salt-out” effect and afford a higher resolution to separate proteins in a mixture. In addition, we have successfully employed the magnetic Fe3O4@NH2-MIL-100(Fe)-C18 in the purification of recombinant green fluoresce protein by replacing the stage of using a commercial hydrophobic interaction resin, and achieved almost the same purification efficiency. Fe3O4@NH2-MIL-100(Fe)-C18 also provides us with the higher protein binding capacity than other commercial products, reliable performance for fifteen repeat uses, and more convenient and affordable method for protein purification in laboratory.

Construction of Magnetic Fe3o4@Nh2-Mil-100(Fe)-C18 with Excellent Hydrophobicity for Effective Protein Separation and Purification

Construction of Magnetic Fe3o4@Nh2-Mil-100(Fe)-C18 with Excellent Hydrophobicity for Effective Protein Separation and Purification

High-surface-area interconnected macroporous nanofibrous cellulose microspheres: a versatile platform for large capacity and high-throughput protein separation

With the rapid development of biopharmaceutical industry, conventional chromatographic media cannot meet the increasing demand for high-quality proteins. Here, high-surface-area interconnected macroporous nanofibrous cellulose microspheres (NCMs) are fabricated by combining emulsion technique and template method. The macroporous nanofibrous structure of NCMs contributes to the fast transfer of liquid and improves processing efficiency, and the high specific surface area endows NCMs with numerous active sites for the immobilization of adsorption ligands. Benefiting from the advantages of microstructure, NCMs are highly suitable as a versatile adsorbent platform for highly effective protein separation. As a proof of concept, we prepare three NCM-based protein adsorbents with different adsorption mechanisms including anion ion-exchange adsorbent, Cu2+-chelating affinity adsorbent, and hydrophobic interaction adsorbent, which all exhibit high adsorption capacity and fast adsorption kinetic, outstanding recyclability and structure stability for targeted protein, much superior to commercial and most of previously reported same type of adsorbents. We expect that the NCM would provide a promising candidate for the design and construction of high-performance protein adsorbent.

Bridged Hybrid Monolithic Column Coupled to High-Resolution Mass Spectrometry for Top-Down Proteomics.

Top-down mass spectrometry (MS)-based proteomics has become a powerful tool for comprehensive characterization of intact proteins. However, because of the high complexity of the proteome, highly effective separation of intact proteins from complex mixtures prior to MS analysis remains challenging. Monolithic columns have shown great promise for intact protein separation due to their high permeability, low backpressure, and fast mass transfer. Herein, for the first time, we developed bridged hybrid bis(triethoxysilyl)ethylene (BTSEY) monolith with C8 functional groups (C8@BTSEY) for highly effective protein separation and coupled it to high-resolution MS for identification of intact proteins from complex protein mixtures. We have optimized mobile phase conditions of our monolith-based reverse-phase chromatography (RPC) for online liquid chromatography (LC)-MS analysis and evaluated separation reproducibility of the C8@BTSEY column. We further assessed the chromatographic performance of this column by separating a complex protein mixture extracted from swine heart tissue. Using our monolithic column (i.d. 100 μm × 35 cm), we separated over 300 proteoforms (up to 104 kDa) from 360 ng of protein mixture in an 80 min one-dimensional (1D) LC run. The highly effective separation and recovery of intact proteins from this monolithic column allowed unambiguous identification of ∼100 proteoforms including a large protein, αactinin2 (103.77 kDa), by online 1D LC-MS/MS analysis for the first time. As demonstrated, this C8@BTSEY column is reproducible and effective in separation of intact proteins, which shows high promise for top-down proteomics.

Post-translational modifications and their applications in eye research (Review).

Gene expression is the process by which genetic information is used for the synthesis of a functional gene product, and ultimately regulates cell function. The increase of biological complexity from genome to proteome is vast, and the post-translational modification (PTM) of proteins contribute to this complexity. The study of protein expression and PTMs has attracted attention in the post‑genomic era. Due to the limited capability of conventional biochemical techniques in the past, large‑scale PTM studies were technically challenging. The introduction of effective protein separation methods, specific PTM purification strategies and advanced mass spectrometers has enabled the global profiling of PTMs and the identification of a targeted PTM within the proteome. The present review provides an overview of current proteomic technologies being applied in eye research, with a particular focus on studies of PTMs in ocular tissues and ocular diseases.

Gel-Filtration Chromatography.

Gel-filtration chromatography is a versatile method that permits the effective separation of biological molecules in high yield. This article describes the basis of the method, the selection of suitable operating conditions, and contrasts typical matrix types. Applications of the technique are described, with references to the scientific literature.

Simultaneous separation of acidic and basic proteins using gemini pyrrolidinium surfactants and hexafluoroisopropanol as dynamic coating additives in capillary electrophoresis

The separation of acidic and basic proteins using CE has been limited in part due to the adsorption of proteins onto the capillary wall. In this work, the efficient control of EOF and the simultaneous separation of acidic and basic proteins are achieved by use of C18-4-C18PB as a dynamic coating additive, which is a representative surfactant for 1,1′-(butane-1,s-alkyl)bis(1-alkylpyrrolidinium) bromide (Cn-4-CnPB, n=10, 12, 14, 16 and 18). C18-4-C18PB exhibits a powerful capability in the reversal of EOF, and a low concentration even less than 0.001mM is sufficient to reverse EOF at the tested pH values (3.0–9.0). Baseline separation of eight proteins with sharp peaks and high efficiencies (54,000–297,000 plates/m) is obtained with 30mM NaH2PO4 buffer (pH 5.0) containing 4mM C18-4-C18PB. At the same buffer condition, the Cn-4-CnPB with shorter alkyl chain (n=10, 12, 14, 16) cannot achieve the same effective protein separation as C18-4-C18PB. However, the combined use of small amounts (≤0.5%, v/v) of hexafluoroisopropanol (HFIP) and Cn-4-CnPB (n=10, 12, 14, 16) as additives can completely separate all eight proteins with high efficiencies of 81,000–318,000 plates/m. The RSDs of migration time are less than 0.80% and 5.84% for run-to-run and day-to-day assays (n=5), respectively, and the protein recoveries are larger than 90.15%. To the best of our knowledge, this is the first report on the simultaneous separation of acidic and basic proteins using Cn-4-CnPB surfactants or Cn-4-CnPB surfactants combined with HFIP as dynamic coating additives.

Optimization of lactoferrin and bovine serum albumin separation using ion-exchange membrane chromatography

Lactoferrin (LF), which is a high value minor whey protein, has recently received extensive attention from research scientists and industry due to its multifunction and potential therapeutic applications. In this study, the separation of two similar-sized proteins: bovine serum albumin (BSA) and LF was investigated using strong cation and anion exchange membrane chromatography (MC). Single protein and BSA–LF mixture adsorption were performed on Sartobind Q75 and S75 at pH between the LF and BSA isoelectric points. Identical breakthrough curves were obtained for both single protein and binary protein mixture, which suggests that there is no protein adsorption competition at the binding sites. The process optimization was further studied to yield optimum buffer and operating conditions. The highest BSA flux per membrane area (728.00gm−2h−1) was obtained using 100mM phosphate buffer solution at pH 6.0 on the cation exchange membrane, whereas LF was bound to the membrane with the dynamic binding capacity at 10% breakthrough (DBC10%) of about 60mg. On the anion exchange membrane, LF was collected in the effluent at the flux of 287.46gm−2h−1 using 5mM phosphate buffer at pH 6.0, while BSA was retained on the membrane with DBC10% equal to 60.96mg. The combination effect between pH change and hydrophobic interaction improved the eluted protein mass for both anion and cation exchangers. Furthermore, the completed separation cycle was operated with the Sartobind S75 device with a short process time of 34.19min and optimal LF productivity over 2628.84mgmL−1h−1. This study confirms the advantage of MC for the separation of biomolecules with similar molecular weight and different isoelectric points, such as BSA–LF mixture separation. This fast and effective protein separation method could be applied at an industry scale.

Three dimensional liquid chromatography coupling ion exchange chromatography/hydrophobic interaction chromatography/reverse phase chromatography for effective protein separation in top-down proteomics.

To address the complexity of the proteome in mass spectrometry (MS)-based top-down proteomics, multidimensional liquid chromatography (MDLC) strategies that can effectively separate proteins with high resolution and automation are highly desirable. Although various MDLC methods that can effectively separate peptides from protein digests exist, very few MDLC strategies, primarily consisting of 2DLC, are available for intact protein separation, which is insufficient to address the complexity of the proteome. We recently demonstrated that hydrophobic interaction chromatography (HIC) utilizing a MS-compatible salt can provide high resolution separation of intact proteins for top-down proteomics. Herein, we have developed a novel 3DLC strategy by coupling HIC with ion exchange chromatography (IEC) and reverse phase chromatography (RPC) for intact protein separation. We demonstrated that a 3D (IEC-HIC-RPC) approach greatly outperformed the conventional 2D IEC-RPC approach. For the same IEC fraction (out of 35 fractions) from a crude HEK 293 cell lysate, a total of 640 proteins were identified in the 3D approach (corresponding to 201 nonredundant proteins) as compared to 47 in the 2D approach, whereas simply prolonging the gradients in RPC in the 2D approach only led to minimal improvement in protein separation and identifications. Therefore, this novel 3DLC method has great potential for effective separation of intact proteins to achieve deep proteome coverage in top-down proteomics.

Proteomic analysis of human bladder epithelial cells by 2D blue native SDS-PAGE reveals TCDD-induced alterations of calcium and iron homeostasis possibly mediated by nitric oxide.

A proteomic analysis of the interaction among multiprotein complexes involved in 2,3,7,8-dibenzo-p-dioxin (TCDD)-mediated toxicity in urinary bladder epithelial RT4 cells was performed using two-dimensional blue native SDS-PAGE (2D BN/SDS-PAGE). To enrich the protein complexes, unexposed and TCDD-exposed cells were fractionated. BN/SDS-PAGE of the resulting fractions led to an effective separation of proteins and protein complexes of various origins, including cell membrane, mitochondria, and other intracellular compartments. Major differences between the proteome of control and exposed cells involved the alteration of many calcium-regulated proteins (calmodulin, protein S100-A2, annexin A5, annexin A10, gelsolin isoform b) and iron-regulated proteins (ferritin, heme-binding protein 2, transferrin). On the basis of these findings, the intracellular calcium concentration was determined, revealing a significant increase after 24 h of exposure to TCDD. Moreover, the concentration of the labile iron pool (LIP) was also significantly elevated in TCDD-exposed cells. This increase was strongly inhibited by the calmodulin (CaM) antagonist W-7, which pointed toward a possible interaction between iron and calcium signaling. Because nitric oxide (NO) production was significantly enhanced in TCDD-exposed cells and was also inhibited by W-7, we hypothesize that alterations in calcium and iron homeostasis upon exposure to TCDD may be linked through NO generated by CaM-activated nitric oxide synthase. In our model, we propose that NO produced upon TCDD exposure interacts with the iron centers of iron-regulatory proteins (IRPs) that modulate the alteration of ferritin and transferrin, resulting in an augmented cellular LIP and, hence, increased toxicity.

Magnetic separation of proteins by a self-assembled supramolecular ternary complex.

The easy and effective separation of proteins from a mixture is crucial in proteomics. A supramolecular method is described to selectively capture and precipitate one protein from a protein mixture upon application of a magnetic field. A multivalent complex self-assembles in a dilute aqueous solution of three components: magnetic nanoparticles capped with cyclodextrin, non-covalent cross-linkers with an adamantane and a carbohydrate moiety, and lectins. The self-assembled ternary complex is precipitated in a magnetic field and readily redispersed with the aid of a non-ionic surfactant and competitive binding agents. This strategy to purify proteins by supramolecular magnetic precipitation is highly selective and efficient.

Effective protein separation by coupling hydrophobic interaction and reverse phase chromatography for top-down proteomics.

One of the challenges in proteomics is the proteome’s complexity, which necessitates the fractionation of proteins prior to the mass spectrometry (MS) analysis. Despite recent advances in top-down proteomics, separation of intact proteins remains challenging. Hydrophobic interaction chromatography (HIC) appears to be a promising method that provides high-resolution separation of intact proteins, but unfortunately the salts conventionally used for HIC are incompatible with MS. In this study, we have identified ammonium tartrate as a MS-compatible salt for HIC with comparable separation performance as the conventionally used ammonium sulfate. Furthermore, we found that the selectivity obtained with ammonium tartrate in the HIC mobile phases is orthogonal to that of reverse phase chromatography (RPC). By coupling HIC and RPC as a novel two-dimensional chromatographic method, we have achieved effective high-resolution intact protein separation as demonstrated with standard protein mixtures and a complex cell lysate. Subsequently, the separated intact proteins were identified by high-resolution top-down MS. For the first time, these results have shown the high potential of HIC as a high-resolution protein separation method for top-down proteomics.

Analysis of Soybean Seed Proteins Using Proteomics

In order to determine the variation of seed proteins that may occur in the crop as a result of genetic modification, accurate and replicable methodology for protein extraction, isolation, and characterization is essential. Modern proteomic tools are being used to study the expression of proteins to examine alterations in protein profiles caused by genetic mutations and environmental stress [2-10]. Proteome analysis is performed using a variety of methods including structural proteomics such as high throughput (HT) X-ray crystallography and HT nuclear magnetic resonance (NMR) spectroscopy; expressional or analytical proteomics such as gel based electrophoresis (IDE, 2DE, 2DIGE), gel-free (LC-MS/MS or multidimensional protein identification technology (Mud PIT), protein chips, DNA chips, mass spectrometry (MS), micro sequencing; and functional or interaction proteomics such as HT functional assays, ligand chips, yeast 2-hybrid, deletion analysis, and motif analysis [11-14]. In this mini review, we discuss some of the expression analysis methodology that we’ve applied to study soybean seed proteins. Because the extraction of soybean seed proteins for accurate 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is challenging, we initially optimized protein extraction techniques by comparing different methods. Extraction of protein suitable for 2D-PAGE is sample-dependent and is achieved by optimizing the concentration of chaotropic agents, detergents, reducing agents, buffers, enzymes and ampholytes. Also helpful in this regard are advances in immobile pH gradient (IPG) technology and the development of electrophoretic instruments have improved the reproducibility of protein separation. The availability of commercial IPG strips in linear and nonlinear gradients with multiple narrow pH ranges [15], allows effective protein separation for various downstream analyses. We compared four different solubilization methods (urea/thiourea, urea, modified trichloroacetic acid (TCA)/ acetone and phenol) for extraction of proteins from soybean seeds for subsequent analysis by 2D-PAGE [16]. Our study demonstrated that the modified TCA/acetone method with urea/thiourea solubilization resolved more protein spots than the urea or phenol extractions. Using the phenol/urea method, resolution of proteins was generally poor and spots diffuse in the high molecular weight region of the gel, particularly when separating the proteins at pH 4.0 to 7.0. Moreover, while overall protein separation was similar in the TCA/acetone and urea/thiourea methods, low molecular weight proteins were more consistently resolved with the TCA/acetone method. In addition, characterization of low abundant proteins is a challenge because they are often masked by highly abundant proteins. Recently, Boschetti and Righetti [17] published a review of plant proteomic methods to isolate

Fe3O4 nanoparticles: protein-mediated crystalline magnetic superstructures

The synthesis of magnetic, monodisperse nanoparticles has attracted great interest in nanoelectronics and nanomedicine. Here we report the fabrication of pure magnetite nanoparticles, less than ten nanometers in size, using the cage-shaped protein apoferritin (Fe3O4–ferritin). Crystallizable proteins were obtained through careful successive separation methods, including a magnetic chromatography that enabled the effective separation of proteins, including a Fe3O4 nanoparticle (7.9 ± 0.8 nm), from empty ones. Macroscopic protein crystals allowed the fabrication of three-dimensional arrays of Fe3O4 nanoparticles with interparticle gaps controlled by dehydration, decreasing their magnetic susceptibilities and increasing their blocking temperatures through enhanced dipole–dipole interactions.

Amino acid side chain-like surface modification on magnetic nanoparticles for highly efficient separation of mixed proteins

This work reports on the realization of specific functionalized silica-coated magnetic nanoparticles (Si-MNPs) for effective protein separation through surface modification with various amino acid side chain-like functional groups such as thiol (SH), disulfide (SS), carbon chain (Cn), carboxyl (COOH), amine (NH2), and aldehyde (CHO). This study also suggests an improved and convenient method for the synthesis of functionalized Si-MNPs by hydrolysis condensation with silan-coupling agents. The protein adsorption effects in a coexistent mixed state are explored using various proteins, which have different isoelectric point (pI) values and molecular weights, in order to elucidate the binding performance of different proteins one solution. The adsorption efficiency of bovine serum albumin (BSA; 66kDa; pI=4.65) and lysozyme (LYZ; 14.3kDa; pI=11) is 70–100% with various amino acid side chain-like functional groups. However, the adsorption efficiency of a mixed protein solution of BSA and LYZ was different. Although the relatively bulky BSA molecule displayed 50% and 20% adsorption corresponding to pH 4.65, and pH 11, respectively, the smaller LYZ provided almost 100% adsorption at both pH 4.65 and pH 11.

Hemoglobin Extraction Using Cosurfactant-Free Nonionic Microemulsions

ABSTRACT Oil-continuous microemulsions provide an effective medium as a liquid membrane for extraction of proteins due to high solubilization and low miscibility in aqueous feed. Unlike the use of reverse micelles, this new method employs a preformulated cosurfactant-free nonionic microemulsion of high water content. The use of nonionic microemulsions for hemoglobin extraction demonstrates the potential for effective separation of proteins, amino acids, and other biochemicals. The extraction can be controlled by adjusting pH and microemulsion composition. It is enhanced by the addition of an appropriate liquid ion exchanger. The extraction results cannot be explained only in terms of ionic interactions.