Effect of buffer pH and peptide composition on the selectivity of peptide separations by capillary zone electrophoresis

A series of 10 synthetic peptides containing varying degrees of charge and hydrophobicity was used to study the effects of peptide composition and buffer pH on the selectivity of separations by capillary zone electrophoresis (CZE). A simple model is used to explain the effect of buffer pH on the separation. It was found that pH is an important parameter affecting the selectivity of CZE separations. Furthermore, it is shown that the selectivity of the separation is such that peptides differing in neutral amino acid composition can be resolved, and that even differences in a peptide's amino acid sequence can be detected. A protease digest of beta-lactoglobulin A is shown as a practical example of a separation of a complex peptide mixture.     

Optimization of capillary coating by hydroxyethyl methacrylate for capillary zone electrophoresis of proteins

This work describes further improvements of coating fused silica capillaries with 2-hydroxyethyl methacrylate (HEMA) by atom transfer radical polymerization (ATRP). First, endcapping with a sterically less bulky silanyl reagent reduces the electrosmotic flow (EOF) by 25% in addition to the 40% EOF reduction caused by HEMA coating compared to a bare fused silica capillary. An additional hydrolysis step was introduced into the preparation of HEMA coated capillaries and leads to better reproducible migration times. The influence of the solvent during ATRP and the resulting polymer coating was investigated by replacement of DMF with water or water-methanol mixtures. The quality of the optimized coating was characterized by protein separations at pH 3. HEMA coated capillaries reveal up to 746000 plates. The polyvinyl alcohol (PVA) coated capillary provides only half of this efficiency. A long-term test at pH 9 shows good stability of the HEMA coated capillaries in basic medium. Also the numbers of plates in this medium was about 30% higher than for separations with the PVA capillary. In addition, the phosphate buffer was replaced by a volatile ammonium acetate buffer for later use with mass spectrometry (MS).     

High-performance separation methods in the analysis of a new peptide family: the galanins

An analytical investigation of a new peptide family, the human galanins and their fragments, was carried out by reversed-phase HPLC, capillary zone electrophoresis (CZE) at different pH values and micellar electrokinetic capillary chromatography (MECC) in phosphate-borate-sodium dodecyl sulphate buffer. None of the methods seems to be superior to the others. The complementary nature of the electrophoretic methods is obvious when the profiles of peptides are compared; impurities not separated by HPLC are separated by CZE or MECC and vice versa. With these three different separation methods, a more complex analytical control of the synthetic work can be achieved.     

Separation of poly(amidoamine) (PAMAM) dendrimer generations by dynamic coating capillary electrophoresis

The separation of compounds possessing amino groups (peptides, proteins, polyamino compounds) by capillary zone electrophoresis suffers from the interaction (sticking) of these solutes with the capillary wall. This sticking can result in the absence or incomplete separation of compounds or even in their retention in the capillary. Polyamidoamine (PAMAM) dendrimers are a class of spherical polymers with primary amino groups at the surface. These compounds can be separated reasonably well at acidic pH but not at neutral pH. A new method based on the dynamic coating of the capillary was developed for the separation of these compounds at pH 7.4. The method comprises separation in a fused-silica capillary (57 cm total length, 50 cm to the detector, ID 75 microm) and a background electrolyte consisting of a Tris-phosphate buffer (50 mmol/L, pH 7.4) and 0.05% (w/v) polyethyleneimine. This system is suitable for the separation of 7 generations of dendrimers (generations 0-6). The dynamic coating agent (polyethyleneimine) also improves the separation at acid pH.     

Enantiomeric separation of chiral ruthenium(II) complexes using capillary electrophoresis

Capillary zone electrophoresis (CZE) and micellar capillary electrophoresis (MCE) were applied for the enantiomeric separation of nine mononuclear tris(diimine)ruthenium(II) complexes as well as the separation of all stereoisomers of a dinuclear tris(diimine)ruthenium(II) complex. Nine cyclodextrin (CD) based chiral selectors were examined as run buffer additives to evaluate their effectiveness in the enantiomeric separation of tris(diimine)ruthenium(II) complexes. Seven showed enantioselectivity. Sulfated gamma-cyclodextrin (SGC), with four baseline and three partial separations, was found to be the most useful chiral selector. In CZE mode, the derivatized gamma-CDs were more effective than beta-CDs while sulfated CDs work better than carboxymethyl CDs. In MCE mode, hydroxypropyl beta-CD separated the greatest number of tris(diimine) ruthenium(II) complexes. The effects of chiral selector concentration, run buffer pH and concentration, the concentration ratio between chiral selector and other factors were investigated.     

Capillary zone electrophoresis of collagen type I CNBr peptides in acid buffers

Collagen type-I CNBr peptides were separated under acidic conditions by capillary electrophoresis. Separation conditions were: 100 mM phosphate buffer pH 2.5, 50 cm × 50 μm capillary (placed in a cartridge), 8 kV, running time 30–45 min, detection by UV at 200 nm. The peptides were separated strictly by their molecular mass and the overall pattern was well comparable to RP-HPLC separations of these analytes. It is proposed that the separation mechanism may involve hydrophobic sorptions to the capillary wall.     

Capillary electrochromatographic separation of aromatic amino acids possessing peptides using porphyrin derivatives as the inner wall modifiers

Two different porphyrin derivatives (H2TPP(m-OPh)4 and Rh(III)TPP(m-OPh)4) were investigated with respect to their capability to help resolution of five model aromatic peptides in capillary electrophoresis/open tubular capillary electrochromatography. Though the main separation mechanism was preferentially based on the ionic properties of the separated analytes, involvement of particularly H2TTPP(m-OPh)4-peptide interactions at alkaline pH (8.0) was clearly demonstrated. In combination with Tris-phosphate buffer, a speed up of the separation was observed at pH 2.25 (particularly if Rh(III)TPP(m-OPh)4 was used as capillary coating); in spite of the speed up of the separation the selectivity of the system was sufficient and resulted in a complete separation of the five model peptides. It can be expected that Rh(III)TPP(m-OPh)4 capillary coating in combination with Tris-phosphate buffer can be generally used for a considerable speeding up of lengthy separations of peptides in acidic media with some decrease in the separation power of the system.     

Highly efficient protein separations in high-performance capillary electrophoresis,using hydrophilic coatings on polysiloxane-bonded columns

Three methods for preparing hydrophilic coatings on polysiloxane bonded CElect H-type capillary electrophoresis columns have been shown. The polyalkylsiloxane-bonded phase is the first coating layer on the capillary surface, and nonionic surfactant, hydrophilic polymer, or polymer surfactant, adsorbed onto this first layer through hydrophobic interactions, forms the second coating layer. The resultant capillary surfaces are inert, hydrophilic, and suitable for highly efficient protein separations. The effectiveness and applicability of these capillary surface modification methods were tested for the separations of a variety of proteins over a wide range of buffer pH values under different capillary electrophoretic operation modes.     

Carrier ampholytes as potential buffers in electrophoresis: physico-chemical study

Joule heating is a limiting factor when separating proteins in capillary zone electrophoresis (CZE). Low conductivity buffers, are required for high-speed separations. We investigated the use of carrier ampholytes (CA) as background electrolytes (BGE) in CZE. We prepared 25 "narrow pH cuts" of wide pH range (3-10) CA mixture in order to know if these fractions were suitable to be used as BGE in CZE. Each fraction was characterised by CZE analysis, giving an idea of its heterogeneity (number and relative abundance of molecular ampholytes). Conductivities and buffering capacities of each fraction have been also measured. Our conclusion is that "narrow pH cuts" of CA might be well suited buffers for electrophoretic separations.     

Separation and quantitation of azimilide and its putative metabolites by capillary electrophoresis

Reliable methods based on capillary electrophoresis (CE) have been developed for the separation and quantitation of azimilide, an antiarrhythmic drug under development at Procter & Gamble Pharmaceuticals (P&GP). Both capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) were employed in the separation of azimilide from its impurities, degradants and/or metabolites. Separation of azimilide from NE-11178, F-410, F-1054 and F-1292 was obtained by MECC at pH 9 with 50 mM sodium dodecyl sulfate (SDS). The separation of azimilide and NE-10171, a key metabolite of azimilide, was difficult because their structures differ by only a single methyl group. The best separation was achieved under acidic pH conditions with cetyltriethyl ammonium chloride (CTAC) additive in the buffer. All of the CE separations were completed within a substantially shorter time and with better resolution than the corresponding high-performance liquid chromatography (HPLC) separations. Quantitation was done with azimilide and NE-10171. Calibration curves ranging from 10 to 1000 μg/ml were obtained with R² greater than 0.997 for both azimilide and NE-10171. The back-calculated concentrations of the calibration standards and the recoveries of the quality control (QC) samples were within the acceptance range currently used for HPLC methods. These results demonstrated the viability of CE as an alternative technique for drug metabolism studies in support of pharmaceutical development.     

Separation and detection of vitellogenin in fish plasma by capillary zone electrophoresis

A method for coupling capillary zone electrophoresis (CZE) with rapid membrane chromatography purification (RMCP) was established for the analysis of vitellogenin (VTG) in male fish plasma induced with 17ss-estrodiol. CZE analyses of purified VTG were performed in a buffer containing 25 mM sodium borate (pH 8.4). A 50 microm i.d. fused-silica capillary was used for separation and the detection was carried out by UV-diode array at 214 nm. Inter- and intra-assay variabilities of the proposed method were less than 10.06 and 1.95%, respectively. The method has good linear relationship over the scope of 15-2250 microg/ml with a correlation coefficient of R2 = 0.9965 and a detection limit of 7.0 microg/ml. The established CZE method was also applied to directly separate and identify VTG from fish plasma. The results indicated this method could minimize interferences from plasma proteins, allowing the detection of at least 62.5 microg/ml of VTG proteins in total proteins. This is a rapid and easy method to determine the quantity and purity of VTG compared to Bradford method and SDS-PAGE.     

Bottom‐up proteome analysis of E. coli using capillary zone electrophoresis‐tandem mass spectrometry with an electrokinetic sheath‐flow electrospray interface

The Escherichia coli proteome was digested with trypsin and fractionated using SPE on a C18 SPE column. Seven fractions were collected and analyzed by CZE‐ESI‐MS/MS. The separation was performed in a 60‐cm‐long linear polyacrylamide‐coated capillary with a 0.1% v/v formic acid separation buffer. An electrokinetic sheath‐flow electrospray interface was used to couple the separation capillary with an Orbitrap‐Velos operating in higher‐energy collisional dissociation mode. Each CZE‐ESI‐MS/MS run lasted 50 min and total MS time was 350 min. A total of 23 706 peptide spectra matches, 4902 peptide IDs, and 871 protein group IDs were generated using MASCOT with false discovery rate less than 1% on the peptide level. The total mass spectrometer analysis time was less than 6 h, the sample identification rate (145 proteins/h) was more than two times higher than previous studies of the E. coli proteome, and the amount of sample consumed (<1 μg) was roughly fourfold less than previous studies. These results demonstrate that CZE is a useful tool for the bottom‐up analysis of prokaryote proteomes.     

Separation of the eleven priority pollutant phenols by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) provides highly efficient separation of the eleven US EPA priority pollutant phenols. All of these phenols are completely resolved in fewer than 15 min in a 100 cm × 75 μm I.D. uncoated fused-silica capillary at 22.5 kV using a pH 9.8 phosphate-borate buffer. Buffer pH is the most critical parameter controlling resolution and separation time. A simple theoretical treatment greatly simplifies the pH optimization procedure. The effects on the separation of buffer concentration, applied voltage, and sample quantity injected were studied. Good calibration data were obtained for phenol concentrations up to 50 mb/l. Limits of detection for all phenols were less than 1 ppm.     

On-line combination of monolithic immobilized pH gradient-based capillary isoelectric focusing and capillary zone electrophoresis via a partially etched porous interface for protein analysis

An integrated platform consisting of monolithic immobilized pH gradient-based capillary isoelectric focusing (M-IPG CIEF) and capillary zone electrophoresis (CZE) coupled by a partially etched porous interface was established. Since carrier ampholytes (CAs) were immobilized on monolith in M-IPG CIEF to form a stable pH gradient, subsequent depletion of CAs at the interface to prevent the interference on CZE separation and detection were avoided. Moreover, a partially etched porous capillary column, which was facile for fabrication and durable for operation, was exploited as the interface to combine M-IPG CIEF and CZE. The RSD values in terms of the migration time for M-IPG CIEF separation, transfer protein from the first dimension to the second dimension, and CZE separation, were 2.4%, 3.9% and 2.3%, respectively. With a 6-protein mixture as the sample, two-dimensional capillary electrophoresis (2D-CE) separation was successfully completed within 116 min, yielding a peak capacity of ～200 even with minute sample amount down to 5.0 μg/mL. The limit of detection was 0.2 μg/mL. In addition, proteins extracted from milk were used to test the performance of such a 2D-CE separation platform. We expect that such a novel 2D-CE system would provide a promising tool for protein separation with high throughput and high peak capacity.     

Selectivity change in the separation of proteins and peptides by capillary electrophoresis using high-molecular-mass polyethyleneimine

A study of the capillary electrophoretic separations of proteins and peptides using high-molecular-mass polyethyleneimine (PEI) is presented. Experiments were performed in the PEI-coated capillaries together with the use of this polymer as a buffer additive under different separation conditions. The effects of pH and the concentration of PEI in the buffer on the electroosmotic flow and the migration orders of biopolymers were investigated. The use of the cationic polymer offers an alternative for the modification of the separation selectivity and resolution of biopolymers.     

Electrokinetic studies of inorganic coated capillaries

The procedures for the preparation of silica capillaries coated with titanium oxide or aluminum oxide are developed. These inorganic coated capillaries are studied for their applicability in capillary electrophoresis. The points of zero charge are measured as pH 5 and pH 7 for titanium oxide- and aluminum oxide-coated capillaries, respectively. Both titanium oxide and aluminum oxide coatings give better protein separations in comparison to the use of fused-silica capillaries. Separation efficiency of lysozyme as model protein is measured in the range of 20 000 theoretical plates/m of inorganic coated capillaries. However, the hydrophobic interaction between proteins and modified capillary wall possibly contributes to the tailing of observed protein peaks.     

Peptide mapping by capillary electrophoresis with Pluronic F127

Separation of peptides and proteins by capillary zone electrophoresis suffers from the interaction of these solutes with the capillary wall which results in the formation of broad peaks and low resolution. To minimize the protein/peptide-capillary wall interaction we tried to use Pluronic F127, a triblock copolymer of the general formula (polyethylene oxide)(x)(polypropylene oxide)(y)(polyethylene oxide)(z) when x=106, y=70 and z=106 which can be considered a surfactant capable of self-association both into isotropic and anisotropic gels. The analytes studied were enzymatic digests (obtained by trypsin or pepsin treatment) of insoluble matrix proteins from avian eggshell. The best separations were obtained by a system exploiting 10% Pluronic F127 in 20 mmol/l phosphate buffer, pH 2.5. Electrophoretic peptide profiles obtained were very complex owing to the complicated nature of the samples (the exact composition of the proteinous insoluble part of the eggshell is still unknown). The separation in phosphate buffer only offered complex maps of incompletely resolved peaks. The use of Pluronic F127 distinctly improved the separation with a considerably better resolution regarding both the number of peaks obtained and the quality of the separation.     

Sodium dodecyl sulfate solution is an effective between-run rinse for capillary electrophoresis of samples in biological matrices

It is common practice in capillary electrophoresis to perform some sort of capillary washing step(s) between separations. In many analyses little consideration is given to optimization of the wash, and typically a rather standard washing procedure is used involving a few minutes wash with 0.1M NaOH followed by a few minutes reconditioning with the run buffer. As an alternative to this procedure, we have investigated the use of wash solutions containing sodium dodecyl sulfate (SDS). This type of wash has been used in the analyses of both small molecules and proteins, with encouraging results. After the SDS wash, the electroosmotic flow has been shown to be restored to values close to normal in a capillary which had previously been coated with plasma proteins. Separation efficiency for a test compound (dextromethorphan) is improved if an SDS rather than a HCl---NaOH wash is used after injection of plasma. In a direct-injection analysis of plasma proteins using a pH 10 borate buffer, an SDS-based washing procedure (total time, 1 min) gave better migration-time reproducibility than an NaOH-based wash, which took 5 min in total.     

Monitoring equilibria and kinetics of protein folding/unfolding reactions by capillary zone electrophoresis

A method is described here for studying conformational transitions of proteins due to denaturing agents: capillary zone electrophoresis (CZE) in acidic, isoelectric buffers. The sample is run in 50 mM isoelectric glutamic acid (pH = pI = 3.2) added with 1 mM oligoamine (tetraethylene pentamine) for quenching protein interaction to the capillary wall (final pH = 3.3). Muscle acylphosphatase (AcP), in this buffer, exhibited a free solution mobility of 2.63 x 10(-4) cm(2) V(-1) s(-1). By studying the unfolding kinetics, as a function of time of incubation in 7 M urea, it was possible to measure the rate constant of the unfolding reaction, estimated to be 0.00030+/-0.00006 s(-1). The same measurements, when repeated via spectroscopic monitoring of intrinsic fluorescence, gave a value of 0.00034+/-0.00002 s(-1), thus in excellent agreement with CZE data. By equilibrium unfolding CZE studies, it was possible to construct the typical sigmoidal transition of unfolding vs urea molarity: the midpoint of this transition, at which the folded and unfolded states should be equally populated, was estimated to be at 4.56 M urea. Similar experiments by fluorometric analysis gave a value of 4.60 M urea as midpoint of the unfolding curve.     

Determination of poorly separated monoclonal serum proteins by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) technique was developed for the determination of poorly separated monoclonal serum proteins by agarose gel electrophoresis (AGE). A P/ACE 5500 capillary instrument (Beckman) was used under the following conditions: 57 cm x 50 microm I.D. fused-silica capillary, pH 9.6 borate buffer, and 214 nm on-line detection. Sixty patients (61 +/- 13 years) with a well isolated (n=24, group A) or poorly separated monoclonal band(s) by AGE (n=36, group B) were included in this study. Within- and between-run precision for CZE was below 4% for albumin and 7% for gamma-globulin. A 100% (group A) or 61% agreement (group B, more bands detected by CZE in 10 cases) was obtained between CZE and AGE for the number of monoclonal bands. In group B, quantification was possible in 92% of samples by CZE vs. 64% by AGE (P<0.05, chi-square). The proposed CZE method appears as an additional helpful technique for the determination of poorly separated monoclonal serum proteins by AGE.