Biomimetic fabrication of hydroxyapatite-coated zirconia-magnesia composite and its application in the separation of proteins

In this work, biomimetic technique was proposed for the first time to prepare chromatographic packings (2HAp-ZM) for protein separation. By mimicking the mineralization procedures in vivo, this type of matrix with hydroxyapatite (HAp) coating and zirconia-magnesia (ZM) composite core was fabricated. Systematic characterizations, including scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD) and specific surface area analysis, were carried out to investigate the properties of the material. Results showed that the surface of 2HAp-ZM was composed of bead-like, amorphous or nanocrystalline HAp. The specific surface area, total pore volume, and average pore diameter of the resultant material were 25 m²/g, 0.09 cm³/g, and 14 nm, respectively. Furthermore, 2HAp-ZM exhibited good mechanical stability through repeated testing and its application as stationary phases for protein separation was then studied. Five model proteins (bovine serum albumin, trypsin, lysozyme, ribonuclease A, and cytochrome c) were successfully separated on 2HAp-ZM. Finally, the mass recovery of protein and the dynamic loading capacity were studied; the results were calculated to be no less than 93% and 80 μg/mL of blank column volume, respectively.     

Surface-modified polystyrene beads as photografting imprinted polymer matrix for chromatographic separation of proteins

A new and facile fabricating method for lysozyme molecularly imprinted polymer beads (lysozyme-MIP beads) in aqueous media was presented. Mesoporous chloromethylated polystyrene beads (MCP beads) containing dithiocarbamate iniferter (initiator transfer agent terminator) were used as supports for the grafting of lysozyme imprinted copolymers with acrylamide and N,N′-methylenebisacrylamide through surface initiated living-radical polymerization (SIP). After the polymerization, a layer of lysozyme-MIP was formed on the MCP beads. The SIP allowed an efficient control of the grafting process and suppressed solution propagation. Therefore, the obtained lysozyme-MIP beads had a large quantity of well-distributed pores on the surface without any visible gel formation in solution and were more advantageous comparing with traditional MIPs which were prepared by traditionally initiated radical polymerization. The obtained composites were characterized by Fourier transform infrared spectroscopy, elemental analysis, nitrogen sorption analysis and scanning electron microscopy. Chromatographic behaviors of the column packed with lysozyme-MIP beads exhibited ability in separating lysozyme from competitive protein (bovine hemoglobin, bovine serum albumin, ovalbumin or cytochrome c) in aqueous mobile phase.     

Preparation of poly(N-isopropylacrylamide)-grafted polymer monolith for hydrophobic interaction chromatography of proteins

Poly(N-isopropylacrylamide)-grafted polymer monolith has been achieved using a surface-initiated atom transfer radical polymerization grafting polymerization within the pores of poly(chloromethylstyrene-divinylbenzene) macroporous monolith contained in a 100 mm × 4.6 mm I.D. stainless steel column. The grafted-poly(N-isopropylacrylamide) on the surface of the grafted monolith that was used as chromatographic stationary phase showed a response to the variation of temperatures and/or salt concentrations. This study focus on its salt concentration responsive property and it has been revealed that the hydrophobicity of the grafted monolith can be adjusted by changing salt concentrations in the range of 0.05-2.0 mol/L. A variety of salts including sodium sulfate, ammonium sulfate and sodium chloride exhibited different effects on the alteration of hydrophobicity of the grafted monolith, and the effect of the salts was in the order of sodium sulfate > ammonium sulfate > sodium chloride. Based on this response to salt concentrations, the grafted monolith was applied in hydrophobic interaction chromatography of proteins, and the base-line separation of a six proteins mixture consisting of cytochrome c, myoglobin, ribonuclease A, bovine serum albumin, ovalbumin and thyroglobulin bovine was achieved by a salt gradient elution.     

Capillary electrophoresis-mass spectrometry of intact basic proteins using Polybrene-dextran sulfate-Polybrene-coated capillaries: System optimization and performance

A capillary electrophoresis-mass spectrometry (CE-MS) method using sheath liquid electrospray ionization interfacing was studied and optimized for the analysis of intact basic proteins. To prevent protein adsorption, capillaries with a noncovalent positively charged coating were utilized. Capillaries were coated by subsequent rinsing with solutions of Polybrene, dextran sulfate and Polybrene. The coating proved to be fully compatible with MS detection, causing no background signals and ionization suppression. The composition of the sheath liquid and BGE was optimized using the model proteins α-chymotrypsinogen A, ribonuclease A, lysozyme and cytochrome c. A sheath liquid of isopropanol-water-acetic acid (75:25:0.1, v/v/v) at 2 μL min⁻¹ resulted in optimal signal intensities for most proteins, but caused dissociation of the heme group of cytochrome c. Optimum protein responses were obtained with a BGE of 50 mM acetic acid (pH 3.0), which allowed a baseline separation of the test protein mixture. Several minor impurities present in the mixture could be detected and provisionally identified using accurate mass and a protein modification database. The selectivity of the CE-MS system was investigated by the analysis of acetylated lysozyme. Eight highly related species, identified as non-acetylated lysozyme and lysozyme acetylated in various degrees, could be distinguished. The CE-MS system showed good reproducibility yielding interday (three weeks period) RSDs for migration time and peak area within 2% and 10%, respectively. With the CE-MS system, determination coefficients (R²) for protein concentration and peak area were higher than 0.996, whereas detection limits were between 11 and 19 nM.     

A facile and efficient strategy for one-step in situ preparation of hydrophobic organic monolithic stationary phases by click chemistry and its application on protein separation

A simple one-step in situ “click” modification strategy was developed for the preparation of hydrophobic organic monolithic columns for the first time. The column morphology and surface chemistry of the fabricated monolithic columns were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, respectively. The chromatographic performances of the C8/C18 “click” monoliths were evaluated through the separation of a mixture of five proteins such as ribonuclease A, soybean trypsin inhibitor, cytochrome c, bovine haemoglobin and bovine serum albumin. Compared with the blank column, the higher hydrophobicity stationary phases obtained from the “clicked” modification have longer retention times and higher resolution for the five proteins. The separation of five proteins mixture on click C18 monolith with gradient elution at different flow rates was also investigated, the baseline separation of five proteins could be achieved at three different flow rates.     

Hydrophobic AEROSIL®R972 Fumed Silica Nanoparticles Incorporated Monolithic Nano-Columns for Small Molecule and Protein Separation by Nano-Liquid Chromatography

A new feature of hydrophobic fumed silica nanoparticles (HFSNPs) when they apply to the preparation of monolithic nano-columns using narrow monolithic fused silica capillary columns (e.g., 50-µm inner diameter) was presented. The monolithic nano-columns were synthesized by an in-situ polymerization using butyl methacrylate (BMA) and ethylene dimethacrylate (EDMA) at various concentrations of AEROSIL^®R972, called HFSNPs. Dimethyl formamide (DMF) and water were used as the porogenic solvents. These columns (referred to as HFSNP monoliths) were successfully characterized by using scanning electron microscopy (SEM) and reversed-phase nano-LC using alkylbenzenes and polyaromatic hydrocarbons as solute probes. The reproducibility values based on run-to-run, column-to-column and batch-to-batch were found as 2.3%, 2.48% and 2.99% (n = 3), respectively. The optimized column also indicated promising hydrophobic interactions under reversed-phase conditions, while the feasibility of the column allowed high efficiency and high throughput nano-LC separations. The potential of the final HFSNP monolith in relation to intact protein separation was successfully demonstrated using six intact proteins, including ribonuclease A, cytochrome C, carbonic anhydrase isozyme II, lysozyme, myoglobin, and α-chymotrypsinogen A in nano-LC. The results showed that HFSNP-based monolithic nanocolumns are promising materials and are powerful tools for sensitive separations.     

Imprinting unique motifs formed from protein-protein associations

Lysozyme and cytochrome c were imprinted in aqueous media, both as individual proteins and in combination, together with the functional monomer 3-aminophenylboronic acid (APBA) using ammonium persulphate as the initiator. The polymers were formed as films on the gold surfaces of quartz crystal microbalance (QCM) electrodes. It was shown that the lysozyme imprinted polymer was capable of selective template recognition. Micro-calorimetry measurements were used to determine the ratio of lysozyme and cytochrome c giving rise to the maximum enthalpy change when combined in the presence of the functional monomer. Using this procedure a maximum enthalpic change was found when the two proteins were present in an equimolar ratio. A polymer, formed by jointly imprinting the proteins in this ratio, exhibited minimal recognition for the individual template proteins, but was however able to recognise them in combination, suggesting that the proteins when imprinted together interact to form a ‘new’ imprintable motif.The introduction of a series of protein solutions, comprising the imprint proteins in various ratios, to the lysozyme/cytochrome c imprinted films, showed that the films exhibit maximum affinity towards the proteins when they are presented in approximately the same mole ratio (57% cytochrome c and 43% lysozyme) as was used to form the original imprint (equimolar ratio).Frequency response profiles of the QCM electrodes carrying the films, as a function of time, showed the establishment of a new stable baseline (−4.3 Hz) after the electrode was challenged with template protein (1.39 × 10⁻⁹ mol) in less than 3 min.     

Phenylalanine functionalized zwitterionic monolith for hydrophobic interaction electrochromatography

A novel phenylalanine (Phe) functionalized zwitterionic monolith for hydrophobic electrochromatography was prepared by a two‐step procedure involving the synthesis of glycidyl methacrylate based polymer monolith and subsequent on‐column chemical modification with Phe via ring‐opening reaction of epoxides. Benefitting from the hydrophobicity of both methacrylate‐based matrix and aromatic group of Phe, this monolith could exhibit good hydrophobic interaction for the separation. Typical RP chromatographic behavior was observed toward various solutes. The well‐controlled cathodic or anodic EOF of the prepared column could be facilely switched by altering the pH values of running buffers. The separation mechanism of this Phe functionalized zwitterionic monolith is discussed in detail. Two mixed‐mode mechanisms of RP/cation exchange and RP/anion exchange could be further realized on the same monolith in different pH condition of the mobile phase. Versatile separation capabilities of neutral, basic, and acidic analytes have been successfully achieved in this zwitterionic monolith by CEC method.     

Epoxy-based monoliths for capillary liquid chromatography of small and large molecules

A versatile epoxy-based monolith was synthesised by polycondensation polymerisation of glycidyl ether 100 with ethylenediamine using a porogenic system consisting of polyethylene glycol, M _w?=?1000, and 1-decanol. Polymerisation was performed at 80 °C for 22 h. A simple acid hydrolysis of residual epoxides resulted in a mixed diol-amino chemistry. The modified column was used successfully for hydrophilic interaction liquid chromatography (HILIC) of small molecule probes such as nucleic acid bases and nucleosides, benzoic acid derivatives, as well as for peptides released from a tryptic digest of cytochrome c. The mixed-mode chemistry allowed both hydrophilic partitioning and ion-exchange (IEX) interactions to contribute to the separation, providing flexibility in selectivity control. Residual epoxide groups were also exploited for incorporating a mixed IEX chemistry. Alternatively, the surface chemistry of the monolith pore surface rendered hydrophobic via grafting of a co-polymerised hydrophobic hydrogel. The inherent hydrophilicity of the monolith scaffold also enabled high performance separation of proteins under IEX and hydrophobic interaction modes and in the absence of non-specific interactions.     

Synthesis of novel racemic 3,4-dihydroferroceno[c]pyridines via the Ritter reaction

A new approach for the synthesis of functionalized racemic 3,4-dihydroferroceno[c]pyridines via the Ritter reaction of 2-methyl-1-ferrocenylpropan-1-ol with nitriles in the presence of methansulfonic acid was developed. The scope and limitations of the reaction were evaluated. Selected racemic 3,4-dihydroferroceno[c]pyridines were successfully separated by preparative HPLC on a Chiralcel OD-H column. The absolute configuration of the enantiomers was determined by X-ray crystal structure analysis.     

Triamine‐bonded stationary phase for open tubular capillary electrochromatography

A multi‐functional separation column modified with 3‐[2‐(2‐aminoethylamino)ethylamino] propyl‐trimethoxysilane was developed for open tubular capillary electrochromatography. This functional hydrophilic triamine‐bonded open tubular column could generate both anodic and cathodic EOF. When the pH of the running buffer was below 5.3 (30% 3‐[2‐(2‐aminoethylamino)ethylamino] propyl‐trimethoxysilane, v/v), the anodic EOF was exhibited, which greatly prevented the undesired adsorptions of basic proteins on the capillary inner wall. Favorable separation of four basic proteins (viz. trypsin, ribonuclease A, lysozyme and cytochrome c) was successfully achieved at pH 3.5 of 10 mmol/L phosphate buffer. The column efficiencies of proteins were in the range from 87 000 to 110 000 plates/m, and the RSD values for migration time of four proteins were less than 1.2% (run‐to‐run, n=5). The ionic analytes were also separated efficiently in the co‐electroosmotic mode. The average efficiencies ranged from 81 000 to 190 000 plates/m for seven aromatic acids and 186 000–245 000 plates/m for four nucleoside monophosphates, respectively, and good capillary column repeatability was gained with RSD of the migration time not more than 3.0%. The triamine‐bonded open tubular capillary column is favorable to be an alternative functional medium for the further analysis of basic proteins and anionic analytes.     

Preparation of a boronate-functionalized affinity hybrid monolith for specific capture of glycoproteins

A novel strategy for preparation of a boronate affinity hybrid monolith was developed using a Cu(I)-catalyzed 1,3-dipolar azide–alkyne cycloaddition (CuAAC) reaction of an alkyne–boronate ligand with an azide-functionalized monolithic intermediate. An azide-functionalized hybrid monolith was first synthesized via a single-step procedure to provide reactive sites for click chemistry; then the alkyne–boronate ligands were covalently immobilized on the azide-functionalized hybrid monolith via an in-column CuAAC reaction to form a boronate affinity hybrid monolith under mild conditions. The boronate affinity monolith was characterized and evaluated by means of elemental analysis, Fourier transform infrared spectroscopy, and scanning electron microscopy. The boronate affinity hybrid monolith exhibited excellent specificity toward nucleosides and glycoproteins, which were chosen as test cis-diol-containing compounds under neutral conditions. The binding capacity of the monolith for the glycoprotein ovalbumin was 2.36 mg?·?g^-1 at pH 7.0. The practicability of the boronate affinity hybrid monolithic material was demonstrated by specific capture of the glycoproteins ovalbumin and ovotransferrin from an egg sample.

Rapid Separation of Proteins by Capillary HPLC on a Short Polymethacrylate-based Strong Cation-exchange Monolithic Column

A polymethacrylate‐based strong cation‐exchange capillary monolithic column was prepared by in‐situ copolymerization for the fast separation of proteins. Glycidyl methacrylate (GMA) was used as monomer, ethylenedimethacrylate (EDMA) as cross link agent and the mixture of 1‐propanol, 1,4‐butanediol and water as porogen solvent. The monolith was sulfonated using 1 mol/L Na₂SO₃ based on a ring opening of epoxides. The influences of the contents of the porogen solvent and GMA and the various concentration ratios of 1‐propanol to 1,4‐butanediol in the polymerization mixture on the morphology, porosity, globule size, stability and column efficiency were investigated. The morphology and pore size distribution of the monolith were characterized by SEM and mercury intrusion porosimetry, respectively. Using only 1.5 cm length of this monolithic capillary column, four kinds of proteins, trypsin, cytochrome C, lysozyme (egg white) and egg albumin, were successfully separated from each other in 5 min at a high flow rate of 110 mm/s.     

High binding capacity surface grafted monolithic columns for cation exchange chromatography of proteins and peptides

Poly(glycidyl methacrylate-co-ethylene methacrylate) monoliths have been prepared in 100 μm i.d. capillaries and their epoxy groups hydrolyzed to obtain poly(2,3-dihydroxypropyl methacrylate-co-ethylene methacrylate) matrix. These polymers were then photografted in a single step with 2-acrylamido-2-methyl-1-propanesulfonic acid and acrylic acid to afford stationary phases for a strong and a weak cation exchange chromatography, respectively. Alternatively, poly(ethylene glycol) methacrylate was used for grafting in the first step in order to enhance hydrophilicity of the support followed by photografting with 2-acrylamido-2-methyl-1-propanesulfonic acid or acrylic acid in the second step. These new columns were used for the separation of proteins and peptides. A mixture of ovalbumin, α-chymotrypsinogen, cytochrome c, ribonuclease A and lysozyme was used to assess the chromatographic performance for large molecules while a cytochrome c digest served as a model mixture of peptides. All tested columns featured excellent mass transfer as demonstrated with very steep breakthrough curves. The highest binding capacities were found for columns prepared using the two step functionalization. Columns with sulfonic acid functionalities adsorbed up to 21.5 mg/mL lysozyme while the capacity of the weak cation exchange column functionalized with acrylic acid was 29.2 mg/mL.     

Antifungal evaluation of cholic acid and its derivatives on Candida albicans by microcalorimetry and chemometrics

Multi-hydroxyl amines including tris(hydroxymethyl)aminomethane (Tris), serinol and ethanolamine were selected as weak affinity ligands using a rapid screening by quartz crystal microbalance (QCM) biosensor. Based on the specific recognition between the ligands and two proteins, lysozyme (LZM) and cytochrome c (Cyt c), a weak affinity chromatography method was developed for specific separation of the two proteins. The frontal analysis results showed that the apparent dissociation constants (K_D) of ligand–protein complexes were all in the order of weak affinity (10⁻⁴ M). By weak affinity columns modified with the three multi-hydroxyl amines individually, LZM and Cyt c were baseline separated as retarded peaks from non-specific protein and each other in a single cycle of loading and eluting. Moreover, the Tris-modified column typically showed the satisfactory repeatability and stability as a new type of weak affinity columns. The present strategy composed of QCM selecting and affinity chromatography separating was promising to extend the variety of weak affinity ligands and develop inexpensive specific affinity methods for separation and purification of multiple proteins on one single column.     

High-efficiency liquid chromatography–mass spectrometry separations with 50 mm, 250 mm,and 1 m long polymer-based monolithic capillary columns for the characterization of complex proteolytic digests

In this study, high-efficiency LC–MS/MS separations of complex proteolytic digests are demonstrated using 50 mm, 250 mm, and 1 m long poly(styrene-co-divinylbenzene) monolithic capillary columns. The chromatographic performance of the 50 and 250 mm monoliths was compared at the same gradient steepness for gradient durations between 5 and 150 min. The maximum peak capacity of 400 obtained with a 50 mm column, increased to 485 when using the 250 mm long column and scaling the gradient duration according column length. With a 5-fold increase in column length only a 20% increase in peak capacity was observed, which could be explained by the larger macropore size of the 250 mm long monolith. When taking into account the total analysis time, including the dwell time, gradient time and column equilibration time, the 50 mm long monolith yielded better peptide separations than the 250 mm long monolithic column for gradient times below 80 min (n_c = 370). For more demanding separation the 250 mm long monolith provided the highest peak production rate and consequently higher sequence coverage. For the analysis of a proteolytic digest of Escherichia coli proteins a monolithic capillary column of 1 m in length was used, yielding a peak capacity of 1038 when applying a 600 min gradient.     

Coupled affinity-hydrophobic monolithic column for on-line removal of immunoglobulin G, preconcentration of low abundance proteins and separation by capillary zone electrophoresis

A butyl methacrylate-co-ethylene dimethacrylate (BuMA-co-EDMA) monolith was synthesized by UV initiated polymerization at the inlet end of a 75 microm I.D. fused silica capillary that had been previously coated with a protein compatible polymer, poly(vinyl)alcohol. The monolith was used for on-line preconcentration of proteins followed by capillary electrophoresis (CE) separation. For the analysis of standard proteins (cytochrome c, lysozyme and trypsinogen A) this system proved reproducible. The run-to-run %RSD values for migration time and corrected peak area were less than 5%, which is typical of CE. As measured by frontal analysis using lysozyme as solute, saturation of a 1cm monolith was reached after loading 48 ng of protein. Finally, the BuMA-co-EDMA monolithic preconcentrator was coupled to a protein G monolithic column via a zero dead volume union. The coupled system was used for on-line removal of IgG, preconcentration of standard proteins and CE separation. This system could be a valuable sample preparation tool for the analysis of low abundance proteins in complex samples such as human serum, in which high abundance proteins, e.g., human serum albumin (HSA) and immunoglobulin G (IgG), hinder identification and quantification of low abundance proteins.     

Novel surface modified molecularly imprinted polymer using acryloyl-β-cyclodextrin and acrylamide as monomers for selective recognition of lysozyme in aqueous solution

A novel protein imprinted polymer for selective recognition of lysozyme was obtained. Acryloyl-β-cyclodextrin, which offered hydrophilic exterior and hydrophobic cavity that were allowed to self-assemble with the template protein through hydrogen interaction and hydrophobic interaction, was synthesized and used as the functional monomer. Polymerization was carried out in the presence of acrylamide as an assistant monomer, which resulted in a new type of protein imprinted polymer. Langmuir adsorption model was employed to describe the isotherms, and maximum adsorption capacity was evaluated. The performance of such imprinted polymer was further demonstrated by high-performance liquid chromatography, and the results showed that the column packed with the lysozyme imprinted silica beads could effectively separate lysozyme from the mixture of lysozyme–cytochrome c, lysozyme–bovine serum albumin, lysozyme–avidin or lysozyme–methylated bovine serum albumin, which showed its high selectivity.     

The effect of feed composition on the behavior of chemically selective displacement systems

In this paper we examine whether adding a more retained protein to the feed will mitigate displacer–protein interactions in the column, thus affecting the displacement modality that occurs (chemically selective vs. traditional displacement chromatography). STD-NMR experiments were carried out to probe displacer–protein interactions for the chemically selective displacer chloroquine diphosphate and the results indicated that this displacer only had measurable interactions with the protein α-chymotrypsinogen A. For a two component feed mixture containing ribonuclease A and α-chymotrypsinogen A, the separation resulted in the displacement of ribonuclease A, with the more hydrophobic α-chymotrypsinogen A remaining on the column. On the other hand, when the experiment was repeated with cytochrome c added to the feed, all three feed proteins were displaced. Column simulations indicated that the combination of sample self-displacement occurring during the introduction of the feed, along with the dynamics of the initial displacement process at the column inlet was responsible for this behavior. These results indicate that for this class of hydrophobic-based selective displacers, in order for the protein to be selectively retained, the protein should be the most strongly retained feed component.     

Synthesis of boronate-silica hybrid affinity monolith via a one-pot process for specific capture of glycoproteins at neutral conditions

In this study, a boronate-silica hybrid affinity monolith was prepared for specific capture of glycoproteins at neutral pH condition. The monolith was synthesized via a facile one-pot procedure in a stainless steel column by concurrently mixing hydrolyzed alkoxysilanes tetramethoxysilane and vinyltrimethoxysilane, organic monomer 3-acrylamidophenylboronic acid and initiator 2,2′-azobisisobutyronitrile together. The polycondensation of alkoxysilanes and copolymerization of organic monomer and vinyl-silica monolith were carried out successively by reacting at different temperatures. After optimizing the preparation conditions, the resulting hybrid affinity monolith was systematically characterized and exhibited excellent affinity to both cis-diol-containing small molecules and glycoproteins at neutral and physiological pH, including adenosine, horseradish peroxidase, transferrin and ovalbumin. The binding capacity of ovalbumin on monolith was measured to be 2.5 mg g^?1 at pH 7.0. Furthermore, the hybrid affinity monolith was applied to the separation of transferrin from bovine serum sample at a physiological condition. Good repeatability was obtained and the relative standard deviations of retention time were 1.15 and 4.77 % (n?=?5) for run-to-run and column-to-column, respectively.