High-throughput screening of holoprotein conformational stability by dynamic ligand exchange-affinity capillary electrophoresis.

Dynamic ligand exchange-affinity capillary electrophoresis (DLE-ACE) is introduced as a convenient platform for assessing the conformational stability and relative affinity of a holoprotein to different ligands without off-line sample pretreatment, since ligand exchange and protein unfolding processes are integrated in-capillary during electromigration.

Strategies for comprehensive analysis of amino acid biomarkers of oxidative stress.

Despite the wide interest in using modified amino acids as putative biomarkers of oxidative stress, many issues remain as to their overall reliability for early detection and diagnosis of diseases. In contrast to conventional single biomarker studies, comprehensive analysis of biomarkers offers an unbiased strategy for global assessment of modified amino acid metabolism due to reactive oxygen and nitrogen species. This review examines recent analytical techniques amenable for analysis of modified amino acids in biological samples reported during 2003-2007. Particular attention is devoted to the need for validated methods applicable to high-throughput analysis of multiple amino acid biomarkers, as well as consideration of sample pretreatment protocols on artifact formation for improved clinical relevance.

Velocity-difference induced focusing of nucleotides in capillary electrophoresis with a dynamic pH junction.

Velocity-difference induced focusing (V-DIF) of nucleotides was achieved by using a dynamic pH junction in capillary electrophoresis (CE) with UV detection. The influence of specific analyte properties, such as nucleotide base structure, sugar structure, and degree of phosphorylation, is examined. The pKa values and borate complexation with vicinal diols are important factors that caused the focusing. Therefore, the pH and borate content in the sample and background electrolyte can be adjusted to optimize the focusing effect. This method allows the injection of large volumes of sample (approximately 300 nL), resulting in at least 50-fold improvement in concentration sensitivity. The detection limit of 4.0 x 10(-8) M for nucleotides can be achieved in favorable conditions. V-DIF can be also applied to nucleotide pool analysis from cell extracts to improve the concentration sensitivity of CE and to reduce the time-consuming steps of desalting and off-line preconcentration that are often required for assays of nucleotides from biological samples.

Selective focusing of catecholamines and weakly acidic compounds by capillary electrophoresis using a dynamic pH junction.

A systematic study of selective analyte focusing in a multisection electrolyte system by capillary electrophoresis (CE) is presented. It was found that a dynamic pH junction between sample and background electrolyte zones can be used to focus zwitterionic catecholamines and weakly acidic compounds without the use of special ampholytes. Differences in pH and concentration of complexing agents, such as borate, in the sample and background electrolyte zones were determined to cause focusing through changes in the local velocity of the analyte in two different segments of the capillary. Velocity-difference induced focusing (V-DIF) of analytes using a dynamic pH junction allowed the injection of large sample volumes and significantly improved the concentration sensitivity of CE. Under optimized conditions, the limit of detection for epinephrine was determined to be about 4 x 10(-8) M (the original sample) with conventional UV absorbance detection. Moreover, separation efficiencies greater than a million theoretical plates can be achieved by focusing such large sample volumes into narrow zones. Multisection electrolyte systems, which lead to the formation of a dynamic pH junction, can be tuned toward improving the concentration sensitivity of specific analytes if their chemical properties are known.

Analysis of epinephrine from fifteen different dental anesthetic formulations by capillary electrophoresis.

A robust method for the quantification of epinephrine from 15 different commercial dental anesthetic formulations is developed using CE. This work presents an extension to a method reported earlier. The solvability of several anesthetic compounds was improved through appropriate dilutions and the addition of sodium dodecyl sulfate to the separation background electrolyte. By controlling the mobility of the analyte at different pH values, a dilute solution of epinephrine is focused into a sharp zone with the injection of about 150 nl of anesthetic solution into the capillary. This on-column concentration technique extended the concentration detection limit of epinephrine to about 5.0 x 10(-7) M using a commercially available UV detector. A correlation plot between the measured and listed epinephrine concentration for the 15 dental anesthetic solutions demonstrated excellent accuracy of this method.

Analysis of gamma-carboxyglutamic acid content of protein, urine, and plasma by capillary electrophoresis and laser-induced fluorescence.

When the properties of an analyte are known, the separation system can be designed to make the analyte of interest migrate at either a much faster or a much slower velocity compared to other molecules in the sample matrix. A simple and sensitive method to analyze the gamma-carboxyglutamic acid (Gla) content of protein, urine, and plasma was developed using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). The separation method is designed according to the specific properties of three amino acids of interest. The number of Gla residues from three vitamin K-dependent proteins were estimated by quantifying the amount of fluorescein thiocarbamyl derivative of Gla after alkaline hydrolysis and fluorescein isothiocyanate labeling. Human prothrombin, blood coagulation factor X, and bovine osteocalcin were calculated to have 10.0 +/- 0.7, 11.0 +/- 0.6, and 2.1 +/- 0.1 Gla residues per mole of protein, respectively, which agreed well with amino acid sequencing data. The analysis of free Gla content in urine and plasma was also demonstrated by this method. It was demonstrated that submicrograms of protein can be characterized by CE-LIF.

Undercarboxylation of recombinant prothrombin revealed by analysis of gamma-carboxyglutamic acid using capillary electrophoresis and laser-induced fluorescence.

The gamma-carboxyglutamic acid (Gla) content of several variants of human prothrombin has been measured by using capillary electrophoresis and laser-induced fluorescence (CE-LIF). Both plasma-derived prothrombin and recombinant prothrombin contain ten residues of Gla per molecule of protein. In contrast, a variant of human prothrombin (containing the second kringle domain of bovine prothrombin) was separated into two populations that differed in their Gla content. Direct measurement of the Gla content showed an association with the presence or absence of the calcium-dependent conformational change that is required for prothombinase function. Thus, the CE-LIF assay is useful in determining the carboxylation status of recombinant proteins.

Quantitative assay for epinephrine in dental anesthetic solutions by capillary electrophoresis.

A simple and robust method for the separation and quantification of epinephrine in dental anesthetic solutions was developed. The method allows the direct injection of high salt solutions without sample pre-treatment. Large sample plugs (5.7% of the total capillary length) are used for epinephrine determination by selective analyte focusing in capillary electrophoresis. The concentration detection limit for epinephrine is about 5.0 x 10(-7) M (90 ng ml-1) with a commercial UV detector. The separation protocol was validated in terms of its precision, linearity, accuracy and specificity.

The effects of a mixture of charged and neutral additives on analyte migration behavior in capillary electrophoresis.

Multicomponent additives, such as derivatized cyclodextrins with various degrees of substitution, can be considered single-component additives as long as the fraction of each component remains constant. In this paper, equations are derived describing the effect of such additives on the migration behavior of analytes. These equations are used in the study of capillary electrophoresis (CE) systems with differentially charged cyclodextrins as additives. For weakly acidic analytes, the binding with highly negatively charged sulfobutyl ether beta-cyclodextrin (SBE-beta-CD) increases their negative electrophoretic mobility, while the binding with neutral hydroxypropyl-beta-cyclodextrin (HP-beta-CD) decreases their negative mobility. By obtaining the equilibrium constants and mobilities for each additive with each analyte (in this case, phenol, 2-naphthol and 1-naphthol), the migration behavior of these analytes in CE systems is quantitatively predicted at various concentrations of mixtures of the two additives. The properties of the contour lines in the binding isotherm surfaces of such CE systems are discussed.

A water-soluble tetraethylsulfonate derivative of 2-methylresorcinarene as an additive for capillary electrophoresis.

A water-soluble tetraethylsulfonate derivative of 2-methylresorcinarene (TESMR), an aromatic-based, bowl-shaped macrocycle, was used as an additive in capillary electrophoresis. Several phenol derivatives are used as analytes to demonstrate the effect of this highly charged additive. TESMR is observed to interact differently with a mixture of positional isomers and other types of phenol derivatives. A comparative study of separations with two charged additives, TESMR and sulfobutyl ether-ß-cyclodextrin (SBE-ß-CD), provided insight into the selectivity exhibited by these additives. The influence of buffer pH, ionic strength, and organic modifier content on separation and peak shape is investigated. Peak asymmetry caused by the use of highly charged additives at lower pH is minimized by the addition of small amounts of a polar aprotic organic solvent to the run buffer. The effects of mixing a charged additive with a neutral additive are also discussed.

Quantitative description of analyte migration behavior based on dynamic complexation in capillary electrophoresis with one or more additives.

A comprehensive theory is proposed to describe the migration behavior of analytes in capillary electrophoresis (CE) when one or more additives are present in the buffer solution. This theory amalgamates and extends the previous work done by others. The capacity factor (k') in this theory is defined as the product of the equilibrium constant and the additive concentration, thus, k' changes linearly with additive concentration. The net electrophoretic mobility of an analyte is a function of k', therefore, it can be changed by varying the additive concentration. Three parameters are needed to predict the mobility of an analyte in a one-additive CE system: the mobility of the free analyte, the mobility of the complex, and the equilibrium constant for the analyte-additive interaction (which determines the fraction of the free analyte at different additive concentrations). When additives are used, the change in viscosity obscures this relationship, therefore, a viscosity correction factor is required to convert all mobilities to an ideal state where the viscosity remains constant. The migration behavior of an analyte in a solution with multiple additives can be predicted and controlled, once the equilibrium constants of the interactions between the analyte and each of the additives are obtained separately. beta-Cyclodextrin and hydroxypropyl-beta-cyclodextrin are used as additives and the migration behavior of phenol, p-nitrophenol, and benzoic acid are studied as a model system to verify this theory. When the necessary viscosity correction factor is included, the net electrophoretic mobilities of the analytes obtained from experimental results agree with the values predicted by the theory based on dynamic complexation. Although only experiments with one and two additives were carried out to verify the theory, the equations apply to situations when more than two additives are used. The relationship between the theories of electrophoresis and chromatography is clarified.