Electrophoretic mobility for peptides with post-translational modifications in capillary electrophoresis

Several semiempirical models for peptide electrophoretic mobility have been tested for capillary electrophoresis (CE) separation with a positively charged capillary using on-line CE combined with electrospray ionization-mass spectrometry (ESI-MS). In the current system with pH 2.7, the expression q/M(0.56) provided the best correlation with the electrophoretic mobility in the analysis of a set of 18 standard samples, where q is the calculated net charge and M is the molecular weight. The peptides resulting from various digests of horse heart myoglobin or bovine hemoglobin were used to demonstrate the validity of this correlation. Post-translationally modified peptides from tryptic digest of human myelin basic protein were also investigated and were found to provide excellent correlation with the linear plot when the total charge of the peptide was correctly calculated. If the total charge was not properly calculated then the post-translationally modified peptides fell off the linear plot. Using this method five arginine residues (residues 5, 49, 54, 97 and 130) were found to be partially citrullinated, four glutamine residues (residues 8, 103, 121 and 147) were found to be partially deamidated and both methionines at residues 21 and 167 were found to be partially oxidized. Three peptides were found with phosphorylation; TPPPSQGK (residue 98 to 105), FSWGAEGQR (residue 114 to 122), and SGSPMAR (residue 163 to 169) and Arginine at 107 was found to be partially monomethylated or dimethylated. The method may provide an excellent means of identifying the presence of peptides with post-translational modifications in conjunction with MS.     

Capillary electrophoresis in aqueous-organic media. Ionic strength effects and limitations of the Hubbard-Onsager dielectric friction model

The mobilities of three aromatic sulfonates, ranging in charge from -1 to -3, were investigated by capillary electrophoresis using buffers containing 0 to 75% ethanol or 2-propanol. Absolute mobilities were determined by extrapolation of the effective mobilities to zero ionic strength according to the Pitts' equation. For all buffers studied, ions of higher charge experienced larger ionic strength effects. The resulting ionic strength-induced selectivity alterations were more dramatic when organic solvents were present in the media. Furthermore, for different organic modifier types and contents, the magnitude of the ionic strength effect was governed to a large extent by the 1/(eta epsilon 1/2) dependence in the electrophoretic effect of the Pitts' equation. Addition of ethanol or 2-propanol to the electrophoretic media resulted in changes in the absolute mobilities of the ions. These solvent-induced mobility changes are attributed to dielectric friction. As predicted by the Hubbard-Onsager model, dielectric friction increased with increasing organic content and with increasing analyte charge. As a result, dramatic changes in the relative absolute mobilities were observed, such as a reversal in migration order between sulfonates of -1 and -3 charge in 75% 2-propanol. Within the alcohols, the Hubbard-Onsager model was successful at predicting the relative mobility trends upon changing solvent. However, the relative trends observed between acetonitrile-water and alcohol-water media were not consistent with the model. This may be explained by the continuum nature of the model, whereby the different ion-solvent interactions characteristic to each solvent class are not taken into account.     

Electrophoretic behaviour of quinolones in capillary electrophoresis. Effect of pH and evaluation of ionization constants.

Quinolones are a family of antibacterial agents that are used extensively in both human and veterinary clinics. Their antibacterial activity is pH-dependent, and therefore an examination of protonation equilibria in quinolone solution is essential. In this work, dissociation constants of quinolones in water were obtained using capillary electrophoresis (CE). The method is based on measuring the electrophoretic mobility of the solute as a function of pH. Mobility and pH data are fitted using different models. These developed equations have two advantages. They permit the determination of pKa of analytes with the advantages of CE and also permit the prediction of the effect of pH on the electrophoretic behaviour of substances and then the prediction of the pH optimum for the separation methods, using the minimum of experimental measurements.     

Comparison of enantioseparation of amino acid derivatives in aqueous and mixed aqueous-organic media by capillary zone electrophoresis

The chiral separation of dansyl-amino acids has been performed by capillary zone electrophoresis using ?β-cyclodextrin as a chiral selector, urea as an additive and 2-propanol and methanol as organic modifiers. The enantiomeric separations of dansyl-amino acids were investigated in aqueous medium and compared with the separation in mixed aqueous-organic medium as background electrolytes. The separation conditions, (concentration of buffer, β-cyclodextrin, methanol, urea and the pH value of buffer) were optimized. In the absence of organic modifier, only five pairs of 8 separated dansyl-amino acids were resolved when run separately. A mixture of up to eight chiral amino acids can be baseline resolved in less than 19 min by β-cyclodextrin-modified capillary zone electrophoresis with a buffer of 60 mmol L^–1 H₃BO₃-KCl/40 mmol L^–1 NaOH (pH 9.0), 4 mol L^–1 urea, 100 mmol L^–1β-cyclodextrin and 10% (v/v) methanol. Received: 15 March 1999 / Revised: 10 May 1999 / Accepted: 12 May 1999     

Comparison of enantioseparation of amino acid derivatives in aqueous and mixed aqueous-organic media by capillary zone electrophoresis

The chiral separation of dansyl-amino acids has been performed by capillary zone electrophoresis using ¶β-cyclodextrin as a chiral selector, urea as an additive and 2-propanol and methanol as organic modifiers. The enantiomeric separations of dansyl-amino acids were investigated in aqueous medium and compared with the separation in mixed aqueous-organic medium as background electrolytes. The separation conditions, (concentration of buffer, β-cyclodextrin, methanol, urea and the pH value of buffer) were optimized. In the absence of organic modifier, only five pairs of 8 separated dansyl-amino acids were resolved when run separately. A mixture of up to eight chiral amino acids can be baseline resolved in less than 19 min by β-cyclodextrin-modified capillary zone electrophoresis with a buffer of 60 mmol L^–1 H₃BO₃-KCl/40 mmol L^–1 NaOH (pH 9.0), 4 mol L^–1 urea, 100 mmol L^–1β-cyclodextrin and 10% (v/v) methanol.     

Dynamic pH junction technique for on-line preconcentration of peptides in capillary electrophoresis

A method based on the presence of a dynamic pH junction within the capillary to induce band narrowing for enhanced detection sensitivity for some peptides is presented. This technique is predicated on a sharp reduction in an analyte's migration velocity following a reversal of its electrophoretic direction from the acidic sample zone to the basic BGS zone. Larger-than-usual injection volumes of samples in relatively high-conductivity matrices were enabled, without degrading peak shape, resolution and efficiency. The size of the original sample plug was reduced by as much as 38-fold, and improvement in detector response in terms of peak height by as much as 124-fold was obtained. The effects of pH and concentration of the sample matrix, and the length of sample injection on the efficiency of the technique are discussed.     

Determination of effective charges and ionic mobilities of polycationic antimicrobial peptides by capillary isotachophoresis and capillary zone electrophoresis

Capillary ITP (CITP) and CZE were applied to the determination of effective charges and ionic mobilities of polycationic antimicrobial peptides (AMPs). Twelve AMPs (deca‐ to hexadecapeptides) containing three to seven basic amino acid residues (His, Lys, Arg) at variable positions of peptide chain were investigated. Effective charges of the AMPs were determined from the lengths of their ITP zones, ionic mobilities, and molar concentrations, and from the same parameters of the reference compounds. Lengths of the ITP zones of AMPs and reference compounds were obtained from their CITP analyses in cationic mode using leading electrolyte (LE) composed of 10 mM NH₄OH, 40 mM AcOH (acetic acid), pH 4.1, and terminating electrolyte (TE) containing 40 mM AcOH, pH 3.2. Ionic mobilities of AMPs and singly charged reference compounds (ammediol or arginine) were determined by their CZE analyses in the BGE of the same composition as the LE. The effective charges numbers of AMPs were found to be in the range 1.65–5.04, i.e. significantly reduced as compared to the theoretical charge numbers (2.86–6.99) calculated from the acidity constants of the analyzed AMPs. This reduction of effective charge due to tightly bound acetate counterions (counterion condensation) was in the range 17–47% depending on the number and type of the basic amino acid residues in the AMPs molecules. Ionic mobilities of AMPs achieved values (26.5‐38.6) × 10⁻⁹ m²V⁻¹s⁻¹ and in most cases were in a good agreement with the ratio of their effective charges and relative molecular masses.     

Electrophoretic behavior study and determination of some active components in Chinese medicinal preparations by capillary electrophoresis

The determination of icariin (IC), rhein (RH), chrysophanol (CH), physcion (PHY), glycyrrhetic acid (GE), and glycyrrhizic acid (GI), in traditional Chinese preparations, Anshen Bunao oral liquid and Maren pill, has been investigated by micellar electrokinetic capillary electrophoresis. With borate buffer (10 mM), SDS (20 mM) and acetonitrile (10%) as background electrolyte (pH 9.55), 20 kV applied voltage and 254 nm UV detection, the six active compounds were completely separated within 10 min. The effects of buffer pH, concentration of borate, SDS and modifier on electrophoretic behavior and separation are discussed. Regression equations revealed linear relationships (correlation coefficients: 0.9960-0.9999) between the peak-area of each component and the content. In addition, the levels of the six active compounds in two kinds of traditional Chinese medicinal preparations were easily determined with recoveries of from 94.7% to 106.4%.     

Electrophoretic behavior and pKa determination of quinolones with a piperazinyl substituent by capillary zone electrophoresis

Electrophoretic behavior and pKa determination of six quinolones with a piperazinyl substituent, together with two quinolones without a piperazinyl substituent and 1-phenylpiperazine, were investigated by capillary zone electrophoresis. The results indicate that quinolones with a piperazinyl substituent involve three protonation/deprotonation equilibria. The results also suggest that the contribution of the zwitterionic species of these quinolones to the effective mobility may not be neglected. This is probably due to a slightly incomplete protonation of the piperazinyl moiety in the pH range of 6.0-8.0, compared with the complete dissociation of the carboxylic group. Consequently, the zwitterionic species of ciprofloxacin, in particular, is slightly negatively charged. With the aid of computer simulation, three pKa values were determined for quinolones with a piperazinyl substituent, thus allowing us to rationalize precisely the influence of pH on the electrophoretic behavior of these compounds.     

Estimation of acidity constants,ionic mobilities and charges of antimicrobial peptides by capillary electrophoresis

Capillary electrophoresis (CE) was employed for the determination of thermodynamic acidity constants (pK_a) and actual ionic mobilities of polycationic antimicrobial peptides (AMPs). The effective electrophoretic mobilities of AMPs were measured by CE in a series of the background electrolytes within a wide pH range (2.00–12.25), at constant ionic strength (25 mM) and ambient temperature, using polybrene coated fused silica capillaries to suppress sorption of cationic AMPs to the capillary wall. Eventually, Haarhoff–Van der Linde peak fitting function was used for the determination of correct migration times of some AMPs peaks that were distorted by electromigration dispersion. The measured effective mobilities were corrected to 25°C. Mixed acidity constants, , and actual ionic mobilities, m_i, of AMPs were determined by the nonlinear regression analysis of pH dependence of their effective mobilities. The values were recalculated to thermodynamic pK_as using the Debye–Hückel theory. Thermodynamic pK_a of imidazolium group of histidine residues was found to be in the range 3.72–4.98, pK_a of α‐NH₃⁺ group was in the range 6.14–6.93, and pK_a of ε‐NH₃⁺ group of lysine spanned the interval 7.26–9.84, depending on the particular amino acid sequence of the AMPs. Actual ionic mobilities of AMPs with positive charges from one to six elementary units achieved values (9.8 – 36.5) × 10^?9 m²V^?1s^?1.     

Chromatographic and physical studies of tropomyosin in aqueous-organic media at low pH

Non-cross-linked and disulfide-cross-linked two-chain molecules comprising the alpha and/or beta chains of rabbit skeletal tropomyosin were studied by electrophoretic, chromatographic and physical methods. Elution order on C4 reversed-phase high-performance liquid chromatography depends markedly on the number and position of the cross-links. In the C4 reversed-phase elution medium, cross-linked and non-cross-linked species are greater than 85% helical by circular dichroism, but the non-cross-linked elute later from high-performance size-exclusion chromatography (G4000) and have molecular mass of 31,000-41,000 dalton by equilibrium ultracentrifugation. The data suggest that in the C4 reversed-phase high-performance liquid chromatography elution medium non-cross-linked tropomyosin exists as amphipathic single-chain alpha-helices.     

pKa values of peptides in aqueous and aqueous-organic media. Prediction of chromatographic and electrophoretic behaviour

In the present work, models describing the effect of the pH on the chromatographic and electrophoretic behaviour for polyprotic peptides were compared. The proposed models can be simultaneously used for determination of dissociation constants and selection of the optimum pH for the separation of peptides, in water and acetonitrile-water mixtures widely used in liquid chromatography and in capillary electrophoresis. The models use the pH value measured in the acetonitrile-water mixture instead of the pH value in water and take into account the effect of the activity coefficients. They permit the determination of the acidity constants in the aqueous and hydro-organic mobile phase from chromatographic retention and electrophoretic migration measurements, respectively. The values obtained by both proposed techniques agree with the potentiometric values previously determined. The suitability of the proposed models for predicting chromatographic and electrophoretic behaviour of compounds studied from a limited number of experimental data was also compared. The separation between solutes by both techniques in a complex mixture can be easily predicted, making simple and rapid pH selection to achieve optimum separation.     

Electrophoretic behavior of individual nuclear species as determined by capillary electrophoresis with laser-induced fluorescence detection

We determined the feasibility of using capillary electrophoresis with postcolumn laser-induced fluorescence (CE-LIF) detection to characterize electrophoretic properties of isolated cell nuclei and impurities present in nuclear fractions. These fractions were isolated from NS-1 mouse hybridoma cells, stained with hexidium iodide, a DNA intercalating dye, and analyzed by CE-LIF detection. The corresponding electropherograms display two features: (i) broad peaks (6-90 s wide) caused by the cell culturing media and by free-DNA intercalated with hexidium iodide, and (ii) a large number of narrow peaks (180 ms wide), resulting from DNA associated with individual intact or disrupted nuclei. We confirmed that the narrow peaks were not caused by contaminating mitochondria. The overall electrophoretic mobility range of disrupted nuclei is 0 to -5 x 10(-4)cm(2)/Vs, while intact nuclei seem to have mobilities in the -1.5 to -3.5 x 10(-4)cm(2)/Vs range. Furthermore, the highly sensitive CE-LIF method reveals a high abundance of disrupted nuclei that cannot be directly observed by confocal microscopy.     

Flavonoid separation by capillary electrophoresis. Effect of temperature and pH

Summary The use of capillary electrophoresis for the analysis of selected flavonols present in fruit juices and wines (kaempferol-3-rutinoside, rutin, avicularin, quercitrin, isoquercitrin, isorhamnetin, kaempferol and quercetin) was explored, and the effect of pH and temperature on the separation studied. The method had good reproducibility and analyses were carried out in less than 10 minutes.     

Flavonoid separation by capillary electrophoresis. Effect of temperature and pH

Summary The use of capillary electrophoresis for the analysis of selected flavonols present in fruit juices and wines (kaempferol-3- rutinoside, rutin, avicularin, quercitrin, isoquercitrin, isorhamnetin, kaempferol and quercetin) was explored, and the effect of pH and temperature on the separation studied. The method had good reproducibility and analyses were carried out in less than 10 minutes.     

The influence of ionic strength, nutrients and pH on bacterial adhesion to metals

Bacteria-metal interactions in aqueous solutions are important in biofilm formation, biofouling and biocorrosion problems in the natural environment and engineered systems. In this study, the adhesion forces of two anaerobes (Desulfovibrio desulfuricans and Desulfovibrio singaporenus) and an aerobe (Pseudomonas sp.) to stainless steel 316 in various aqueous systems were quantified using atomic force microscopy (AFM) with a cell probe. Results show that the nutrient and ionic strength of the solutions influence the bacteria-metal interactions. The bacteria-metal adhesion force was reduced in the presence of the nutrients in the solution, because a trace organic film was formed and thus decreased the metal surface wettability. Stronger ionic strength in the solution results in a larger bacteria-metal adhesion force, which is due to the stronger electrostatic attraction force between the positively charged metal surface and negatively charged bacterial surface. Solution pH also influences the interaction between the bacterial cells and the metal surface; the bacteria-metal adhesion force reached its highest value when the pH of the solution was near the isoelectric point of the bacteria, i.e. at the zero point charge. The adhesion forces at pH 9 were higher than at pH 7 due to the increase in the attraction between Fe ions and negative carboxylate groups.