Proteins In Capillary Electrophoresis Research Articles

Dual Contributions of Analyte Adsorption and Electroosmotic Inhomogeneity to Separation Efficiency in Capillary Electrophoresis of Proteins.

Improving separation efficiency in capillary electrophoresis (CE) requires systematic study of the influence of the electric field (or solute linear velocity) on plate height for a better understanding of the critical parameters controlling peak broadening. Even for poly(diallyldimethylammonium chloride) (PDADMAC)/poly(sodium styrenesulfonate) (PSS) successive multiple ionic-polymer layer (SMIL) coatings, which lead to efficient and reproducible separations of proteins, plate height increases with migration velocity, limiting the use of high electric fields in CE. Solute adsorption onto the capillary wall was generally considered as the main source of peak dispersion, explaining this plate height increase. However, experiments done with Taylor dispersion analysis and CE in the same conditions indicate that other phenomena may come into play. Protein adsorption with slow kinetics and few adsorption sites was established as a source of peak broadening for specific proteins. Surface charge inhomogeneity was also identified as a contribution to plate height due to local electroosmotic fluctuations. A model was proposed and applied to partial PDADMAC/poly(ethylene oxide) capillary coatings as well as PDADMAC/PSS SMIL coatings. Atomic force microscopy with topography and recognition imaging enabled the determination of roughness and charge distribution of the PDADMAC/PSS SMIL surface.

Modifying last layer in polyelectrolyte multilayer coatings for capillary electrophoresis of proteins

Protein adsorption on the inner wall of the fused silica capillary wall is an important concern for capillary electrophoresis (CE) analysis since it is mainly responsible for separation efficiency reduction. Successive Multiple Ionic-polymer Layers (SMIL) are used as capillary coatings to limit protein adsorption, but even low residual adsorption strongly impacts the separation efficiency, especially at high separation voltages. In this work, the influence of the chemical nature and the PEGylation of the polyelectrolyte deposited in the last layer of the SMIL coating was investigated on the separation performances of a mixture of four model intact proteins (myoglobin (Myo), trypsin inhibitor (TI), ribonuclease a (RNAse A) and lysozyme (Lyz)). Poly(allylamine hydrochloride) (PAH), polyethyleneimine (PEI), ε-poly(L-lysine) (εPLL) and α-poly(L-lysine) (αPLL) were compared before and after chemical modification with polyethyleneglycol (PEG) of different chain lengths. The experimental results obtained by performing electrophoretic separations at different separation voltages allowed determining the residual retention factor of the proteins onto the capillary wall via the determination of the plate height at different solute velocities and demonstrated a strong impact of the polycationic last layer on the electroosmotic mobility, the separation efficiency and the overall resolution. Properties of SMIL coatings were also characterized by quartz microbalance and atomic force microscopy, demonstrating a glassy structure of the films.

Sodium dodecyl sulfate-capillary gel electrophoresis with native fluorescence detection for analysis of therapeutic proteins

A 280 nm light-emitting diode (LED) was used as the excitation source for native fluorescence detection (NFD) of proteins in capillary electrophoresis. The NFD scheme was evaluated in sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) for monoclonal antibody (mAb) characterization. Utilizing a technique by which we filtered the LED emission through a 280 nm bandpass filter, we were able to increase overall concentration sensitivity of SDS-CGE-NFD ~2.3-fold. Under the optimized conditions, the assay linear dynamic range was > 4 orders of magnitude with a correlation coefficient (r2) of 0.9999, and the limit of detection was 8.3 ng/mL. The SDS-CGE-NFD assay was applied to quantitation and purity analysis of Etanercept, a therapeutic protein. Over the range of 50 – 150% of the target concentration, 200 µg/mL, recoveries were in the range of 97.02 – 101.6%. The SDS-CGE-NFD assay allowed for simultaneous quantitation of high- and low-molecular-weight species in Etanercept.

Quantification of Adsorption and Optimization of Separation of Proteins in Capillary Electrophoresis.

The improvement of separation efficiency for protein analysis in capillary electrophoresis (CE) is a challenging topic in which protein adsorption onto the capillary wall plays a crucial role. In this work, a simple method allowing the quantification of the adsorption of proteins onto the coated or untreated inner surface of the fused silica capillary was developed based on the determination of the retention factor by measuring separation efficiency of individual proteins at different separation voltages (i.e., different linear velocities). This approach was applied to the quantification of the residual adsorption of four test proteins on five-layer polyelectrolyte coatings and bare fused silica capillary. It allows to get a fair ranking of the coating performances toward protein adsorption, whatever their apparent electrophoretic mobilities (migration times) are. Due to the existence of (even low) residual adsorption, the electrophoretic operating conditions (electric field, capillary length, and internal diameter) can be optimized to improve the separation performances resulting in experimental separation efficiency up to ∼600 000 plates.m-1 in conditions compatible with MS coupling. This approach represents a crucial step in the course to get antifouling coatings for protein separation in CE. It can be used for the evaluation and ranking of virtually any coating (neutral or charged) in CE.

Factors affecting the separation performance of proteins in capillary electrophoresis

Capillary electrophoresis (CE) is an effective tool for protein separation and analysis. Compared with capillary gel electrophoresis (CGE), non-gel sieving capillary electrophoresis (NGSCE) processes the superiority on operation, repeatability and automaticity. Herein, we investigated the effect of polymer molecular weight and concentration, electric field strength, and the effective length of the capillary on the separation performance of proteins, and find that (1) polymer with high molecular weight and concentration favors the separation of proteins, although concentrated polymer hinders its injection into the channel of the capillary due to its high viscosity. (2) The resolution between the adjacent proteins decreases with the increase of electric field strength. (3) When the effective length of the capillary is long, the separation performance improves at the cost of separation time. (4) 1.4% (w/v) hydroxyethyl cellulose (HEC), 100 V/cm voltage and 12 cm effective length offers the best separation for the proteins with molecular weight from 14,400 Da to 97,400 Da. Finally, we employed the optimal electrophoretic conditions to resolve Lysozyme, Ovalbumin, BSA and their mixtures, and found that they were baseline resolved within 15 min.

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 0.10mg/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.

A fully automated linear polyacrylamide coating and regeneration method for capillary electrophoresis of proteins.

Surface modification of the inner capillary wall in CE of proteins is frequently required to alter EOF and to prevent protein adsorption. Manual protocols for such coating techniques are cumbersome. In this paper, an automated covalent linear polyacrylamide coating and regeneration process is described to support long-term stability of fused-silica capillaries for protein analysis. The stability of the resulting capillary coatings was evaluated by a large number of separations using a three-protein test mixture in pH 6 and 3 buffer systems. The results were compared to that obtained with the use of bare fused-silica capillaries. If necessary, the fully automated capillary coating process was easily applied to regenerate the capillary to extend its useful life-time.

Discrimination of Glycoproteins from Unglycosylated Proteins in Capillary Electrophoresis: Two-Color LIF Detection Coupled with Post-column Derivatization.

Glycosylation is one of the most important posttranslational modifications (PTMs) which lead to the functionalization of proteins. Here, we describe one method for discriminating glycosylated proteins from unglycosylated ones in their mixture sample by capillary electrophoretic separation and two-color laser-induced fluorescence detection coupled with post-column derivatization. Two lasers emitting at 450 and 532 nm permit the detection of amino groups of proteins derivatized by naphthalene-2,3-dicarboxaldehyde and a fluorescently labeled lectin, tetramethylrhodamine-labeled concanavalin A (Rh-Con A), respectively. When a protein mixture react with Rh-Con A, the glycoproteins bound with Rh-Con A exhibit signals at the same migration time in two electropherograms obtained by 450- and 532-nm lasers whereas unbound proteins show a signal only in the electropherogram of the 450-nm laser. So, when one protein is glycosylated it is detected at the same migration time in the electropherograms obtained by two lasers.

Tuning capillary surface properties by charged polymeric coatings

Separation of proteins in capillary electrophoresis (CE) is often hindered by their interactions with the silanols groups on the inner surface of the fused silica capillary. In particular, the positive charges of alkaline proteins establish electrostatic interactions with the negative charges of the surface, leading to peak broadening and loss of separation efficiency. Moreover, uncontrolled electroosmotic flow (EOF), severely impacts on analyte mobility causing either loss of resolution or loss of efficiency. Among several strategies, coatings are widely employed to mask capillary surface silanols and so to reduce protein adsorption and EOF.Here we report on the synthesis and characterization of a novel family of adsorbed polymeric coatings, which provide improved performance in terms of prevention of protein adsorption and EOF regulation. In particular, we have added to the polymer backbone, made of N, N-dimethylacrylamide, different ionizable monomers (weak acrylamido acids and bases commercially available with the trade name of Immobilines) to confer a net positive or negative charge to the polymer chain depending on the buffer pH. As a consequence, the separation of alkaline protein is drastically improved in different pH conditions, because the interactions with the inner capillary wall were completely prevented by electrostatic repulsion. The content of these ionogenic monomers can be adjusted, permitting a perfect control of the surface charge density, so EOF is finely and precisely regulated. We also investigated the conformational variation of the polymer on the surface by changing buffer pH using Dual Polarization Interferometry (DPI). The coating procedure is very simple and fast as it consists in the adsorption of a diluted aqueous solution of the polymer on the capillary walls. In addition, the coating is very stable under harsh conditions, can be used for several runs without any re-conditioning or re-coating steps and it is compatible with MS volatile buffers.

Chemiluminescence detection of protein in capillary electrophoresis using aptamer-functionalized gold nanoparticles as biosensing platform

Highly sensitive and selective detection of disease-related proteins play critical roles in clinical practice and diagnostic assays. Herein, we proposed a highly selective and ultrasensitive chemiluminescence (CL) method for protein detection in capillary electrophoresis (CE) using aptamer-functionalized gold nanoparticles (AuNPs) as biosensing platform. In this protocol, AuNPs were synthesized and conjugated with aptamer to form AuNPs-aptamer. Using thrombin and thrombin binding aptamer as an initial proof-of-concept recognization pair, AuNPs-aptamer was linked to thrombin to produce an AuNPs-aptamer–thrombin complex. The resulted complex and unbound AuNPs-aptamer were separated in CE and detected with luminol–H2O2 CL system. The developed strategy produced an ultrasensitive detection of thrombin down to 13.5fmol/L (S/N=3) with a linear range from 0.033 to 66.0pmol/L. The application of the present protocol was demonstrated by analyzing thrombin in human plasma samples with the recoveries of 87.6–116.8%. This novel strategy has many outstanding merits including high specificity of aptamer, excellent catalysis behavior of AuNPs, high sensitivity of CL detection, and high separation efficiency of CE.

A novel, post-column micro-membrane reactor for fluorescent analysis of protein in capillary electrophoresis

Based on the semipermeability of hollow fiber membranes, a post-column membrane reactor was developed for capillary electrophoresis (CE)-laser induced fluorescence (LIF) analysis of proteins by using a hollow fiber membrane to connect the separation and detection capillaries. The membrane length between the separation and detection capillaries was 1 mm. Driven by the chemical potential difference between the separation buffer inside the membrane and the fluorescence derivatization solution outside the membrane, the derivatization reagent can be easily drawn into hollow fiber membrane to react with proteins. Also, the separation buffer can be adjusted by the derivatization solution to match the conditions of derivatization without sample loss. The effect of the separation buffer on the derivatization reaction was investigated and the results showed that even a strong acidic solution and multiple additives can be adopted in the separation buffer without destroying the post-column derivatization of proteins. Under the optimized conditions, the highly sensitive detection of BSA was achieved with a detection limit of 3.3 nmolL(-1) and a linear calibration range from 0.007 to 0.1 mgmL(-1). The proposed CE-LIF system with a post-column membrane reactor was also successfully applied to the separation and detection of proteins in rat liver and loach muscle.

Novel poly(vinyl alcohol)-based column coating for capillary electrophoresis of proteins

A novel and simple method for the preparation of chemically bonded poly(vinyl alcohol) (PVA) coating to silica capillary inner wall was developed, and the PVA-coated capillary columns were employed for capillary electrophoresis (CE). The coating procedure included pretreatment of the capillary inner wall, silanization, aldehyde group functionalization and PVA immobilization. Electroosmotic flow of the coated capillary was almost suppressed over a wide pH range (pH 3–10). High-efficiency separations of cationic proteins (including cytochrome c, lysozyme, α-chymotrypsinogen A) at pH 3.0–5.0 and of anionic proteins (including myoglobin and trypsin inhibitor) at pH 10.0 were achieved with the PVA-coated capillary. Moreover, a “dual-opposite-injection” approach was adopted for simultaneous separations of both cationic and anionic proteins at neutral pH with the prepared column. In this CE mode, positively charged proteins migrated from one end of the column to the detector while negatively charged proteins from the other end to the detection window. Good run-to-run repeatability was obtained in all of the protein CE separations performed in this work. The PVA-coated column can also afford long-term stable uses for protein separations, as demonstrated in 100 repeated uses using a single capillary with the relative standard deviation values of the retention times less than 0.9%. Moreover, good column-to-column reproducibility was demonstrated by protein CE separation with 10 different columns. The results indicate that the present method for PVA-coated capillary preparation is promising for protein CE applications.

Synthesis of hydroxyethylcellulose‐graft‐poly(N, N‐dimethylacrylamide) copolymer by ATRP and as dynamic coating in capillary electrophoresis

AbstractHydroxyethylcellulose‐graft‐poly (N, N‐dimethylacrylamide) was synthesized by successive atom transfer radical polymerization (ATRP) of N,N‐dimethylacrylamide (DMA) monomer using HEC‐Br as initiator, CuBr and 5,5,7,12,12,14‐hexamethyl‐1,4,8,11‐tetraazamacrocyclotetradecane (Me6[14]aneN4) as catalyst and ligand, with molar ratio DMA: HEC‐Br (CBr): CuBr: Me6[14]aneN4 = 100 : 1 : 1 : 3. HEC–Br macroinitiator was synthesized by esterification of HEC with 2‐bromoisobutyryl bromide. GPC and 1H NMR studies show that the molecular weight of the resulting PDMA increased linearly with the conversion. Within 6 h, the polymerization can reach almost 60% of conversion. The copolymer is applied for the separation of basic proteins in capillary electrophoresis. The results show that this medium has a powerful capability in resisting basic proteins adsorption because the polymer forms noncovalent coating in silica capillaries. With a broad range of pH 2–7, proteins were separated with sufficient efficiencies above 200,000 plates/m. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010

Lamp-based native fluorescence detection of proteins in capillary electrophoresis

The potential of a recently developed lamp-based fluorescence detector for the analysis of underivatised proteins by capillary electrophoresis (CE) was investigated. Fluorescence detection (Flu) was achieved using optical light guides to deliver excitation light from a Xenon–Mercury lamp to the capillary detection window and to collect fluorescence emission and lead it to a photomultiplier. The performance of the detector was evaluated by monitoring the native fluorescence of the amino acid tryptophan and the proteins α-chymotrypsinogen A, carbonic anhydrase II, lysozyme and trypsinogen upon excitation at 280 nm. The test compounds were analysed using background electrolytes (BGEs) of sodium phosphate at pH 3.0 and 11.3. The results were compared to experiments of CE with UV absorbance detection. For tryptophan, a linear fluorescence response was obtained with a dynamic range of over 4 orders of magnitude, and a limit of detection (LOD) of 6.7 nM. This LOD was a factor of 200 more favourable than UV detection at 280 nm, and a factor of 20 better than detection at low-UV wavelengths. All tested proteins showed linear fluorescence responses up to 250 μg/mL. LODs were typically in the 10–20 nM range. These LODs were a factor of 25 lower than for UV detection at 280 nm, and comparable to UV detection at low-UV wavelengths. Overall, Flu yields much more stable baselines, especially with a BGE of high pH. The applicability of CE–Flu is demonstrated by the analysis of a degraded protein mixture, and of an expired formulation of the protein drug human growth hormone, indicating that protein degradation products can be selectively detected.

Effect of somatic cell counts on ewes' milk protein profile and cheese-making properties in different sheep breeds reared in Spain

Bulk tank ewe's milks from Assaf, Castellana and Churra breeds categorized within three different Somatic Cell Count (SCC) groups (LSCC: <500,000; MSCC: 1,000,000 to 1,500,000; and HSCC: 2,500,000 to 3,000,000 cells ml-1) were used to investigate changes in capillary electrophoresis protein profiles and cheese-making properties. The results do not reveal a significant effect of SCC on total casein contents, because the sum of beta-caseins decreased as SCC increased; no statistically significant differences were observed for the sum of alpha-caseins, and the values of kappa-casein were higher in the HSCC milk. However, the soluble proteins other than alpha-lactalbumin and beta-lactoglobulin increased with SCC. Regarding the effect of breed, the Assaf breed had the lowest contents of kappa-CN, alpha s1-I-CN, alpha s1-II-CN 1-CN, alpha s1-III-CN, beta1-CN and beta2-CN. The protein profile was significantly correlated with curd textural properties. alpha s1-I-CN was the most influential variant because it was positively correlated with a large number of textural parameters. Cheese yield was positively correlated with all casein variants except alpha s1-III-CN, showing that the milk from local breeds were more suitable for cheese-making due to their higher contents of all the casein variants. Regarding curd texture properties LSCC milk curds showed more cohesiveness, associated with its lower content of alpha s1-III-CN and Castellana milk curds showed the highest values for firmness owing to their higher content of alpha s1-I-CN.

Long‐chained gemini surfactants for semipermanent wall coatings in capillary electrophoresis of proteins

In this paper, we presented the first example of using gemini surfactants as semipermanent coatings in CE for protein separation. These coatings are based on the self-assembly of a series of cationic gemini surfactants, alkanediyl-alpha,omega-bis(dimethylalkylammonium bromide) (m-s-m), on the capillary wall. The coatings can keep stable for a long time without surfactant in the buffer, e.g., after the surfactants were removed from the buffer, the reversed EOF only decreased by 3.6 and 3.9% for 18-2-18 and 16-2-16 coatings over 60 min under continuous electrophoretic conditions. The coating stability increased with the alkyl chain length m. The double long chains of geminis (m > or = 14) yielded a good coating stability; meanwhile, the spacer group acted as an EOF modifier. Thus, this bifunctional surfactant coating provided a new buffer-independent method for EOF control. For 18-s-18 series, the best coating stability and largest EOF were obtained at s = 10. Ranging s from 3 to 10 yielded a linear fine-tuning of EOF and thereby allowed the adjustment of the protein apparent mobility. Highly efficient separation (>500 000 plates/m) was achieved with all the 18-s-18 coatings. Excellent run-to-run and day-to-day reproducibility (RSD of migration time </=0.7 and 4.3% for run-to-run and day-to-day assays, respectively) and fine recoveries (97.5-100.1%) of the protein samples indicated that the gemini semipermanent coatings were quite effective for the wall adsorption suppression of proteins.

Capillary electrophoresis of proteins 2005–2007

This review article with 239 references describes recent developments in capillary electrophoresis of proteins, and covers the two years since the previous review (V. Dolník, Electrophoresis 2006, 27, 126-141) through spring 2007. It includes topics related to CE of proteins, such as sample pretreatment, wall coatings, improving separation, various forms of detection, and special electrophoretic techniques including ACE, CIEF, capillary ITP, and CEC. The paper describes applications of CE to analysis of proteins in real-world samples including human body fluids, food and agricultural samples, protein pharmaceuticals and recombinant protein preparations.

Simple luminescence detectors using a light-emitting diode or a Xe lamp, optical fiber and charge-coupled device, or photomultiplier for determining proteins in capillary electrophoresis: A critical comparison

The performance of two homemade fluorescence-induced capillary electrophoresis detectors, one based on light-emitting diode (LED) as the excitation source and a charge-coupled device (CCD) photodetector and the other based on a commercial luminescence spectrometer (Xe lamp) as the excitation source and a photomultiplier tube as a detector, were compared for the determination of fluorescent proteins R-phycoerythrin and B-phycoerythrin. Both devices use commercially available, reasonably priced optical components that can be used by nonexperts. After fine optimization of several optical and separation parameters in both devices, a zone capillary electrophoresis methodology was achieved with 50 mM borate buffer (pH 8.4) and 10 mM phytic acid for the determination of two phycobiliproteins. Detection limits of 0.50 and 0.64 μg/ml for R-phycoerythrin and B-phycoerythrin, respectively, were achieved by using the LED-induced fluorescence capillary electrophoresis (LED-IF-CE) system, and corresponding detection limits of 2.73 and 2.16 μg/ml were achieved by using the Xe lamp-IF-CE system. Analytical performance and other parameters, such as cost and potential to miniaturization, are compared for both devices.

On‐column capture of a specific protein in capillary electrophoresis using magnetic beads

A method for capturing specific molecules separated by CE has been explored. To demonstrate on-column capture of migrating analyte molecules, two detection windows were fabricated on a capillary. Magnetic beads containing immobilized molecules that react with the specific molecules under study were placed between the detection windows in the capillary using magnets. Molecules in a sample solution injected into the capillary were separated and detected at the first detection window. After passing through the first detection window, the separated molecules encountered the magnetic beads, where the specific analyte was captured. As a result, the peak area for those analyte molecules decreased or disappeared completely at the second detection window. Rabbit IgG and carbonic anhydrase were employed to demonstrate on-column capture of a specific molecule. For rabbit IgG, magnetic beads containing the immobilized antibody (anti-rabbit IgG) were used. Rabbit IgG molecules were captured on the magnetic beads during CE migration. Furthermore, the capture of carbonic anhydrase was demonstrated by the reaction between magnetic beads (containing immobilized anti-rabbit IgG) and anti-carbonic anhydrase (rabbit IgG), before the beads were packed in the capillary. After packing the magnetic beads in the capillary, a mixture of two proteins was injected into the capillary. Two proteins were detected at the first detection window, while the peak corresponding to carbonic anhydrase disappeared at the second detection window. The results show that using an appropriate antibody, the present technique would be applicable to any proteins.

On-line preconcentration of protein in capillary electrophoresis with an end-column cellulose acetate-based porous membrane

A simple on-line preconcentration method of protein for capillary electrophoresis (CE) using a cellulose acetate (CA)-coated porous membrane was proposed. CA membrane is fabricated at one of the ends of the column that allows the passage of buffer ions but excludes larger protein molecules. Protein sample is continuously electrokinetically loaded and trapped by the membrane. When injection is completed, the direction of the electric field is switched and the trapped proteins are then separated by conventional CE procedure. The results achieved showed that the preconcentration mechanism of this method was based on size-exclusion effect. Bovine serum albumin (BSA) was used for model protein sample, and signal enhancement of 550-fold with 15 min injection time was achieved.