Protein separations using polyelectrolyte multilayer coatings with molecular micelles in open tubular capillary electrochromatography

Novel polyelectrolyte multilayer (PEM) coatings for enhanced protein separations in open tubular CEC (OT-CEC) are reported. Use of four cationic polymers (poly-L-lysine, poly-L-ornithine, poly-L-lysine-serine, and poly-L-glutamic acid-lysine), and three anionic molecular micelles, sodium poly(N-undecanoyl-L-leucyl-alaninate) (poly-L-SULA), sodium poly(N-undecanoyl-L-leucyl-valinate) (poly-L-SULV), and sodium poly(undecylenic sulfate) (poly-SUS) were investigated in PEM coatings for protein separations. The simultaneous effects of cationic polymer concentration, number of bilayers, temperature, applied voltage, and pH of the BGE on the separation of four basic proteins (alpha-chymotrypsinogen A, lysozyme, ribonuclease A, and cytochrome c) were analyzed using a Box Behnken experimental design. The influence of NaCl on the run-to-run reproducibility was investigated for PEM coatings containing each cationic polymer. All coatings exhibited excellent reproducibilities with a %RSD of the EOF less than 1% in the presence of NaCl. Optimal conditions were dependent on both the cationic and anionic polymers used in the PEM coatings. Poly-L-glutamic acid-lysine produced the highest resolution and longest migration time. The use of molecular micelles to form PEM coatings resulted in better separations than single cationic coatings. Chiral poly-L-SULA and poly-L-SULV resulted in higher protein resolutions as compared to the achiral, poly-SUS. Furthermore, the use of poly-L-SULV reversed the elution order of lysozyme and cytochrome c when compared to poly-L-SULA and poly-SUS.     

Quaternized celluloses as new dynamic coatings in capillary electrophoresis for basic protein separation

In this work, a series of quaternized celluloses (QCs), homogeneously synthesized in the NaOH/urea aqueous solutions, were studied as dynamic coatings for capillary electrophoresis. Capillaries coated with these cationic cellulose derivatives at the concentration as low as 3 μg/mL were able to generate a stable, reversed electroosmotic flow. The effects of QC molecular parameters, such as the degree of cationic substitution and molecular weight, and the effect of buffer pH on the EOF mobility as well as the separation of basic proteins were investigated in detail. It was shown that the use of QC coatings in CE could drastically reduce the analysis time and improve the separation performance within a broad pH range. Five basic proteins, that is, lysozyme, ribonuclease A, cytochrome C, bovine pancreatic trypsin inhibitor, and chymotrypsinogen were baselinely separated even at pH 8.0. The separation efficiency and analysis reproducibility demonstrated that the QC coatings were efficient in minimizing the adsorption of basic proteins on the fused silica capillary. The successful performance was further demonstrated for biosample analysis.     

The effect of sodium dodecyl sulfate and Pluronic F127 on the electrophoretic separation of protein and polypeptide test mixtures at acid pH

Using a test mixture consisting of standard proteins (cytochrome c, chymotrypsinogen A, hen egg albumin, bovine serum albumin, aldolase, catalase and ferritin) and synthetic polypeptides (polylysine, polyaspartic, polyglutamic acid and polyproline) it was revealed that using sodium dodecyl sulfate (SDS) as background electrolyte modifier at acid pH (2.5) allows selective separation of highly positively charged polypeptides (polylysine) provided that their relative molecular mass is sufficiently low (3300 Da). The altered elution sequence of standard proteins as compared to a separation done without SDS may help their identification. Addition of Pluronic F127 offers clear-cut separations of standard proteins up to a relative molecular mass of 5 x 10(4) Da and allows to reveal protein/polypeptide microheterogeneity where applicable. None of the systems tested is suitable for the separation of acidic polypeptides and polyproline.     

Preparative high-yield electroelution of proteins after separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and its application to analysis of amino acid sequences and to raise antibodies.

A method for the preparative high-yield electroelution of proteins from sodium dodecyl sulphate (SDS) polyacrylamide gel strips was established. The method consisted of SDS-polyacrylamide gel electrophoresis, detection of proteins with sodium acetate and electrophoretic elution at 200 V for 3 h by utilizing a horizontal flat-bed gel electrophoresis apparatus. Standard proteins with molecular masses of 14-66 kilodalton (cytochrome c, aldolase, ovalbumin and bovine serum albumin) were recovered with an average yield of 73.6 +/- 2.3%. A membrane-bound protein, rat skeletal muscle Ca(2+)-ATPase (100 kilodalton) was also well recovered (over 60%). This method was applicable to the purification of proteins required for N-terminal amino acid sequencing and to raise antibodies.     

A cationic β‐cyclodextrin as a dynamic coating for the separation of proteins in capillary electrophoresis

A cationic cyclodextrin was used as dynamic coating for the capillary electrophoresis of a model mixture of proteins (i.e., ubiquitin, α‐lactoglobulin, cytochrome‐c, and myoglobin) as positively charged species in a fused silica capillary. An interesting feature of the coating is that by simple adjustment of the concentration of cyclodextrin added into the background electrolyte, a neutral or positively charged surface, which was beneficial in preventing protein adsorption at the inner capillary wall surface, was obtained. This is the first demonstration of a dynamic coating that yielded a neutral surface for protein separations in capillary electrophoresis. Based on electro‐osmotic flow measurements, addition of 0.05 to 0.10 mg/mL quaternary β‐cyclodextrin in a low pH electrolyte resulted in a neutral or positive surface (undetectable to very slow anodic electro‐osmotic flow). The coating approach afforded the electrophoretic separation of the mixture of proteins at positive polarity with good repeatability and separation performance.     

Native red electrophoresis--a new method suitable for separation of native proteins

A new type of native electrophoresis was developed to separate and characterize proteins. In this modification of the native blue electrophoresis, the dye Ponceau Red S is used instead of Coomassie Brilliant Blue to impose uniform negative charge on proteins to enable their electrophoretic separation according to their relative molecular masses. As Ponceau Red S binds less tightly to proteins, in comparison with Coomassie Blue, it can be easily removed after the electrophoretic separation and a further investigation of protein properties is made possible (e.g. an enzyme detection or electroblotting). The tested proteins also kept their native properties (enzyme activity or aggregation state).     

N-辛基-2-吡咯烷酮甲磺酸盐离子液体动态涂敷毛细管电泳分离碱性蛋白质

研究了新型吡咯烷酮类离子液体N-辛基-2-吡咯烷酮甲磺酸盐([NOP]~+CH_3SO~-_3)作为毛细管动态涂敷试剂时,对3种常见碱性蛋白质标准物质(溶菌酶、细胞色素c、α-胰凝乳蛋白酶原)的分离情况。系统考察了缓冲溶液类型、缓冲溶液p H值、离子液体添加剂浓度对分离效果的影响。结果表明,该动态涂敷方法简单快速,3种碱性蛋白质在8min内实现了高效分离,理论塔板数分别为2.9×10~5、5.8×10~5、1.8×10~4N/m。     

Protein-protein interactions studied by EPR relaxation measurements: cytochrome c and cytochrome c oxidase

The complex formed between cytochrome c oxidase from Paracoccus denitrificans and its electron-transfer partner cytochrome c has been studied by multi-frequency pulse electron paramagnetic resonance spectroscopy. The dipolar relaxation of a fast-relaxing paramagnetic center induced on a more slowly relaxing center can be used to measure their distance in the range of 1-4 nm. This method has been used here for the first time to study transient protein-protein complex formation, employing soluble fragments for both interacting species. We observed significantly enhanced transversal relaxation of the CuA center in cytochrome c oxidase due to the fast-relaxing iron of cytochrome c upon complex formation. The possibility to measure cytochrome c oxidase in the presence and absence of cytochrome c permitted us to separate the dipolar relaxation from other relaxation contributions. This allowed a quantitative simulation and interpretation of the relaxation data. The specific temperature dependence of the dipolar relaxation together with the high orientational selectivity achieved at high magnetic field values may provide detailed information on distance and relative orientation of the two proteins with respect to each other in the complex. Our experimental results cannot be explained by any single well-defined structure of the complex of cytochrome c oxidase with cytochrome c, but rather suggest that a broad distribution in distances and relative orientations between the two proteins exist within this complex.     

Cationic detergent in agarose for improved electrophoresis of cationic proteins

Inclusion of the cationic detergent N-cetyl-N,N,N-trimethylammonium bromide (CTAB) in normal agarose gels increases the electrophoretic separation and banding pattern of cationic proteins. This is shown for an extract of cationic granular proteins from polymorphonuclear granulocytes.     

Immunobinding‐induced alteration in the electrophoretic mobility of proteins: An approach to studying the preconcentration of an acidic protein under cationic isotachophoresis

The objective of this study is to explore an approach for analyzing negatively charged proteins using paper‐based cationic ITP. The rationale of electrophoretic focusing the target protein with negative charges under unfavorable cationic ITP condition is to modify the electrophoretic mobility of the target protein through antigen‐antibody immunobinding. Cationic ITP was performed on a paper‐based analytical device that was fabricated using fiberglass paper. The paper matrix was modified with (3‐aminopropyl)trimethoxysilane to minimize sample attraction to the surface for cationic ITP. Negatively charged BSA was used as the model target protein for the cationic ITP experiments. No electrophoretic mobility was observed for BSA‐only samples during cationic ITP experimental condition. However, the presence of a primary antibody to BSA significantly improved the electrokinetic behavior of the target protein. Adding a secondary antibody conjugated with amine‐rich quantum dots to the sample further facilitated the concentrating effect of ITP, reduced experiment time, and elevated the stacking ratio. Under our optimized experimental conditions, the cationic ITP‐based paper device electrophoretically stacked 94% of loaded BSA in less than 7 min. Our results demonstrate that the technique has a broad potential for rapid and cost‐effective isotachphoretic analysis of multiplex protein biomarkers in serum samples at the point of care.     

Application of dodecyldimethyl (2-hydroxy-3-sulfopropyl) ammonium in wall modification for capillary electrophoresis separation of proteins

A zwitterionic surfactant, dodecyldimethyl (2-hydroxy-3-sulfopropyl) ammonium (C12H25N+(CH3)2CH2CHOHCH2SO3-), named dodecyl sulfobetaine (DSB), was used as a novel modifier to coat dynamically capillary walls for capillary electrophoresis separation of basic proteins. The DSB coating suppressed the electroosmotic flow (EOF) in the pH range of 3-12. At high DSB concentration, the EOF was suppressed by more than 8.8 times. The DSB coating also prevented successfully the adsorption of cationic proteins on the capillary wall. Anions, such as Cl-, Br-, I-, SO4(2-), CO3(2-), and ClO4-, could be used as running buffer modifiers to adjust the EOF for better separation of analytes. Using this dynamically coated capillary, a mixture of eight inorganic anions achieved complete separation within 4.2 min with the efficiencies from 24,000 to 1,310,000 plates/m. In the presence of ClO4- as EOF adjustor, the separation of a mixture containing four basic proteins (lysozyme, cytochrome c, alpha-chymotrypsinogen A, and myoglobin) yielded efficiencies of 204,000-896,000 plates/m and recoveries of 88%-98%. Migration time reproducibility of these proteins was less than 0.5% relative standard deviation (RSD) from run to run and less than 3.1% RSD from day to day, showing promising application of this novel modifier in protein separation.     

Separation of basic proteins by capillary zone electrophoresis with coatings of a copolymer of vinylpyrrolidone and vinylimidazole

Capillary zone electrophoretic (CZE) separation of basic proteins has been achieved with capillary columns modified with copolymers of vinylpyrrolidone (VP) and vinylimidazole (VI). The copolymerization reaction is performed inside the capillary column and involves chemical bonding of the polymer to silica. The electroosmotic flow (EOF) is greatly decreased by this surface modification. The presence of positive charges on the coating surface, due to the cationic property of vinylimidazole at pH below 7, reduces the adsorption of basic proteins onto the silanol groups of the capillary surface. Acidic proteins are irreversibly adsorbed, but rapid separation and good performance reproducibility are obtained with basic proteins. In the case of capillaries modified with VP, the acidic and basic proteins are eluted within 10 min. In this work, we studied the effects of pH and buffer concentration on the magnitude of the EOF, as well as the effect of copolymer composition on the separation efficiency.     

Liquid membrane transport of cytochrome c using a calix[6]arene carboxylic acid derivative as a carrier

The present study examined the liquid membrane transport of the cationic protein cytochrome c, using the macrocyclic compound calix[6]arene, which is a carboxylic acid derivative, as a carrier. The transport rate was governed by carrier concentration and the pH gradient between the feed and the receiving phases, as well as the salt concentration in the aqueous phases. Transport of cytochrome c was examined using a series of calix[n]arene carboxylic acid derivatives (n = 4, 6 and 8). Cytochrome c successfully permeated membranes in the presence of the calix[6]arene derivative. Liquid membrane separation of cytochrome c from a mixture of cationic proteins was demonstrated under optimal conditions. Cytochrome c was selectively extracted by the calix[6]arene carboxylic acid derivative and 77% of the extracted cytochrome c was recovered into the receiving phase. In this liquid membrane system, which discriminates between the number of lysine residues on the surface of proteins, cationic proteins with similar molecular weights and pIs were separated with macrocyclic compounds.     

A very thin coating for capillary zone electrophoresis of proteins based on a tri(ethylene glycol)-terminated alkyltrichlorosilane

We describe the use of a tri(ethylene glycol)-terminated alkyltrichlorosilane to create a very thin, protein-resistant "self-assembled monolayer" coating on the inner surface of a fused-silica capillary. The same compound has been demonstrated previously on flat silica substrates to resist adsorption of many proteins. As a covalently bound capillary coating, it displays good resistance to the adsorption of cationic proteins, providing clean separations of a mixture of lysozyme, cytochrome c, ribonuclease A, and myoglobin for more than 200 consecutive runs. Electroosmotic flow (EOF) was measured as a function of pH; the coated capillary retains significant cathodal EOF, with roughly 50% of the EOF of an uncoated capillary at neutral pH, making this coating promising for applications requiring some EOF. The EOF was reasonably stable, with a 2.9% relative standard deviation during a 24 h period consisting of 72 consecutive separations of cationic proteins. Efficiencies for cationic protein separations were moderate, in the range of 190,000-290,000 theoretical plates per meter. The coating procedure was simple, requiring only a standard cleaning procedure followed by a rinse with the silane reagent at room temperature. No buffer additives are required to maintain the stability of the coating, making it flexible for a range of applications, potentially including capillary electrophoresis-mass spectrometry (CE-MS).     

CEC separation of peptides using a poly(hexyl acrylate-co-1,4-butanediol diacrylate-co-[2-(acryloyloxy)ethyl]trimethyl ammonium chloride) monolithic column

A polyacrylate-based monolithic column bearing cationic functionalities and designed for capillary electrochromatography (CEC) has been prepared via photopolymerization of a mixture of hexyl acrylate, butanediol diacrylate, 2-(acryloyloxy) ethyltrimethyl ammonium chloride (monomers), azobisisobutyronitrile (photoinitiator), acetonitrile, phosphate buffer, and ethanol (porogens). The polymerization process was initiated with UV light at 360 nm. The column performance was evaluated via the separations of alkylbenzenes, substituted anilines, basic drugs, peptides, and a protein digest. The separation of complex peptide mixtures was then studied since such separations constitute a promising application of capillary electrochromatography. In particular, the effects of mobile phase composition, including ionic strength of the buffer solution and the percentage of acetonitrile on the retention factor, the column efficiency, and the resolution were determined. The separations were affected by both interaction of the peptides with the stationary phase and their own electrophoretic mobility. Excellent separations with column efficiencies of up to 160 000 plates/m were achieved for both a mixture of ten well-defined peptides and a tryptic digest of cytochrome c. The fractions of eluent containing peptides of the digest separated in the monolithic column were collected and characterized using matrix-assisted laser desorption ionization mass spectrometry.     

Polyamidoamine‐grafted silica nanoparticles as pseudostationary phases for capillary electrochromatographic separation of proteins

Polyamidoamine‐grafted silica nanoparticles were synthesized, characterized and investigated for the feasibility as pseudostationary phases in alkaline buffer for separation of cationic and anionic proteins, viz., lysozyme, cytochrome C, gamma globulin, and myoglobin. Neither bare silica nanoparticles nor polyamidoamines nor their mixtures as pseudostationary phases could lead to simultaneous separation of the four proteins. However, polyamidoamine‐grafted silica nanoparticles not only suppressed the irreversible wall adsorption of the cationic lysozyme and cytochrome C, but also provided selectivity toward all the proteins. We found that polyamidoamine generation two‐modified silica nanoparticles were the optimum pseudostationary phases with respect to detection sensitivity and separation efficiency; presence of the nanoparticles at 0.01% in the running buffer of 12.5 mM tetraborate/phosphate at pH 9.1 resulted in baseline resolution of the four proteins.     

Extraction and separation of a lysine-rich protein by formation of supramolecule between crown ether and protein in aqueous two-phase system

The macrocyclic calixarenes and crown ethers have recently been found to form hydrophobic complexes with the cationic protein cytochrome c (Cyt-c), by recognizing lysine residues on the protein surface. In the present study, it was found that the distribution of cytochrome c in Li₂SO₄/PEG aqueous two-phase system (ATPS) can be controlled by complexation with the crown ether dicyclohexano-18-crown-6 (DCH18C6). The protein was quantitatively extracted into the PEG-rich phase in the presence of DCH18C6 and perchlorate ion. Of various crown ethers and their analogues that were investigated, only DCH18C6 was able to extract cytochrome c into the PEG-rich phase. Extraction of cytochrome c in the ATPS using DCH18C6 is complete within 5 min. Cytochrome c complexed with DCH18C6 in the PEG-rich phase was quantitatively recovered into a salt-rich phase using K₂SO₄ by ion exchange of potassium ion and cationic protein in the cationic protein complex with DCH18C6. Selective extraction of cationic proteins was demonstrated in the ATPS. Under optimum conditions, the lysine-rich protein cytochrome c was selectively extracted over other cationic proteins using DCH18C6.     

Simultaneous separation of anionic and cationic proteins by capillary electrophoresis using high concentration of poly(diallyldimethylammonium chloride) as an additive

The simultaneous separation of anionic and cationic proteins has been achieved by addition of high concentration of poly(diallyldimethylammonium chloride) (PDDAC) in capillary electrophoresis. A capillary was filled with PDDAC so that it would act as ion-pair reagents in the separation of anionic proteins. On the other hand, the PDDAC can also be used as coating additives for the analysis of cationic proteins. Increasing the concentration of PDDAC in the separation buffer had the ability to improve the separation efficiency, change the electrophoretic mobility, and alter the separation selectivity; however, this was not true in the case of analyzing proteins by using the PDDAC larger than 1.6%. By both using a buffer containing 1.6% PDDAC and applying pH-stepwise techniques, 13 proteins with a wide range of pI (4.7-11.1) and molecular masses (6.5-198.0 kDa) could be separated within 30 min in a single run. In addition to this separation, we observed not only more peaks from alpha-chymotrypsinogen A and aprotinin but also the bovine serum albumin (BSA) dimer and trimer. With the 50 nL protein injection sample, the limits of detections at signal-to-noise of 3 for proteins are in the range of 0.07-0.79 microM. Except for BSA, the relative standard derivation values of migration time and peak height for all proteins were <1.3 and <6.9%, respectively. We suggested that this proposed method is a promising approach for clinical diagnosis and proteomics applications.     

Weak interactions and molecular recognition in systems involving electron transfer proteins

Electrostatic interactions and other weak interactions between amino acid side chains on protein surfaces play important roles in molecular recognition, and the mechanism of their intermolecular interactions has gained much interest. We established that charged peptides are useful for investigating the molecular recognition character of proteins and their molecular interaction induced structural changes. Positively charged lysine peptides competitively inhibited electron transfer from reduced cytochrome f (cyt f or cytochrome c (cyt c) to oxidized plastocyanin (PC), due to neutralization of the negatively charged site of PC by formation of PC-lysine peptide complexes. Lysine peptides also inhibited electron transfer from cyt c to cytochrome c peroxidase. Likewise, negatively charged aspartic acid peptides interacted with the positively charged sites of cytfand cyt c, and competitively inhibited electron transfer from reduced cytfor cyt c to oxidized PC and from [Fe(CN)6]4- to oxidized cyt c. Changes in the geometry and a shift to a higher redox potential of the active site Cu of PC on oligolysine binding were detected by spectroscopic and electrochemical measurements, owing to the absence of absorption in the visible region for lysine peptides. Structural and redox potential changes were also observed for cyt f and cyt c by interaction with aspartic acid peptides.     

Admittance measurements on protein layers adsorbed at the Pt/solution interface: effect of d.c. potential and a.c. field

The effect of changes in the electric field on the structure of protein layers adsorbed at the Pt electrode/solution interface has been investigated by means of admittance measurements. The measurements have been performed over a wide range of d.c. potentials, at different frequencies and amplitudes of the a.c. electric field, and at various pH values. The proteins used, cytochrome C and serum albumin, differ considerably with respect to their molecular masses, points of zero charge and structure stabilities. In contrast to serum albumin, cytochrome C has a relatively strong electric dipole moment. Nevertheless, the results for the two proteins are very similar. Both proteins stay adsorbed at the interface over the d.c. potential range studied and at every pH, irrespective of any electrostatic repulsion. Apparently, for both proteins factors other than electrostatic interactions are dominant in their final binding to the Pt/solution interface. In line with this, no indications were obtained that the orientation of adsorbed cytochrome C molecules is modulated by low-frequency (200–1000 Hz) reversal of the electric field of the interface. A strong a.c. field leads to irreversible structural changes in the adsorption layer for both proteins.