Micellar electrokinetic chromatography separations of dynorphin peptide analogs.

Prodynorphin is a precursor that has multiple cleavage sites to release various dynorphin opioid peptides. The dynorphin analogs used in this study have 18 amino acid residues. A series of dynorphin-like peptides, differing by a single residue (alanine substitution) were assembled by Fmoc solid-phase procedures and purified by preparative high performance liquid chromatography (HPLC). Separation of the Ala-scan dynorphin analogs was investigated by micellar electrokinetic chromatography (MEKC) employing anionic, cationic and zwitterionic surfactants. The role of electrostatic and hydrophobic forces in analyte-surfactant interactions is discussed with respect to the observed elution patterns. Separation of all dynorphin analogs by MEKC using a zwitterionic surfactant shows this technique to be powerful for separating closely related peptide species. It also demonstrates the potential for using MEKC for the prescreening of peptide libraries to determine their biological activity toward specific receptors. Results from the separation of dynorphin analogs by free solution and ion-pairing capillary electrophoresis are also presented.

On-line system for peptide mapping by capillary electrophoresis at sub-micromolar concentrations.

Peptide mapping has been widely used for the identification of modified proteins involved in certain diseases. Despite the fact that capillary electrophoresis (CE) has been shown to be a powerful tool for the separation and detection of tryptic peptide fragments after protein digestion, this technique lacks sensitivity for mapping proteins isolated in very small quantities from biological samples. Consequently, it has been necessary to preconcentrate the protein before adding the proteolytic enzyme for digestion in solution. These experimental steps are quite long, labor intensive and require a lot of sample handling. In this paper, we describe an on-line system allowing digestion of the protein, followed by preconcentration, separation and detection of the tryptic fragments in 4 h. Up to an 800-fold preconcentration factor was achieved for cytochrome c, despite a loss of separation efficiency induced by the multiple-valve design of the system and dispersion of the 60-nl desorption plug. Moreover, our system showed good migration time reproducibility between peptide maps and could be reused for several samples.

Estimation of the pH-independent binding constants of alanylphenylalanine and leucylphenylalanine stereoisomers with beta-cyclodextrin in the presence of urea.

The separation of stereoisomers, particularly enantiomers, is important when their physiological activity differs. We have resolved the four stereoisomers each of alanylphenylalanine (Ala-Phe) and of leucylphenylalanine (Leu-Phe) by capillary electrophoresis using beta-cyclodextrin as a buffer additive and urea to enhance its solubility. A study of the influence of pH and beta-cyclodextrin concentration on the separations showed that weak inclusion complexes were formed between the dipeptides and chiral selector. It was found that pH could alter the migration order of enantiomers L-Ala-L-Phe and D-Ala-D-Phe, as well as L-Leu-L-Phe and D-Leu-D-Phe; however, there was no change in order for the other pairs of optical isomers. Electrophoretic mobility data were used to estimate the acid dissociation constants of the dipeptide isomers at pH < 7 with no chiral selector present. By varying the concentration of beta-cyclodextrin, the chiral selector, the binding constants of Ala-Phe and Leu-Phe optical isomers in their fully protonated and zwitterionic forms were estimated. For the four Ala-Phe stereoisomers, K = 42-66 M(-1) and 4-41 M(-1) for the cationic and zwitterionic forms, respectively. For the four Leu-Phe stereoisomers, K = 43-94 M(-1) and 1-28 M(-1) for the cationic and zwitterionic forms, respectively.

On-line solid-phase preconcentration for sensitivity enhancement in capillary electrophoresis.

Capillary electrophoresis is attractive for the analysis of biological samples because only a few nanoliters of sample need to be injected. Indeed, optimal resolution is achieved when the injected volume is 1% or less of the total capillary volume. Unfortunately, this advantage leads to severe detection limitations compounded by the fact that many analytes in a biological sample are present at very low concentrations. To overcome the detection sensitivity limitations of CE, nonspecific, on-line preconcentration has been employed in a variety of applications. This technique is based on inserting a small quantity of reversed-phase material (e.g., C18 particles or membrane) near the inlet of the CE capillary for sample enrichment by solid-phase extraction. A detachable cartridge containing the solid phase is relatively simple to construct and permits the injection of large sample volumes (1-100 microL) into the capillary. Elution of adsorbed analyte in less than 100 nL of solvent permits a 100-1000 fold improvement in the effective concentration limit of detection, depending on the hydrophobicity of the compound. Detection of analytes present in complex mixtures at concentrations as low as 20 amol/microL has been reported when using an on-line preconcentration device in conjunction with CE-mass spectrometry. In this review, the potential merits of the technique are described and some examples of direct analysis of biological samples without rigorous off-line pretreatment are given.

Characterization of a solid-phase extraction device for discontinuous on-line preconcentration in capillary electrophoresis-based peptide mapping.

Peptide mapping by capillary electrophoresis (CE) with UV detection is problematic for the characterization of proteins that can only be obtained at low micromolar concentrations. Dilution of peptide fragments during digestion of the protein can further reduce the detection sensitivity in peptide mapping to the point where analysis at sub-micromolar concentrations is not possible. A remedy to this problem is preconcentration (sample enrichment) of the proteolytic digest by solid-phase extraction (SPE). To minimize non-specific adsorptive losses during sample handling, on-line SPE-CE is preferred. However, packed-inlet SPE-CE is not always feasible due to either instrument or sample limitations. We describe here a simple method of preconcentration by discontinuous on-line SPE-CE, specifically applied to peptide mapping in low-pH separation buffer after protein digestion in a solid-phase enzyme microreactor. The SPE-CE system does not require application of a low pressure during electrophoretic separation to overcome reversed electroosmotic flow because the preconcentrator device is disconnected from the separation capillary before the electric field is applied. Up to a 500-fold preconcentration factor can be achieved with this device, which can be reused for many samples. Parameters such as the volume of desorption solution, the adsorption/desorption (chromatographic) process, reproducibility of packing the SPE preconcentrator and effects of sample concentration on the peptide map are investigated.

Capillary electrophoresis with laser-induced native fluorescence detection for profiling body fluids.

Laser-induced native fluorescence detection with a KrF excimer laser (lambda=248 nm) was used to investigate the capillary electrophoretic (CE) profiles of human urine, saliva and serum without the need for sample derivatization. All separations were carried out in sodium phosphate and/or sodium tetraborate buffers at alkaline pH in a 50-microm I.D. capillary. Sodium dodecyl sulfate was added to the buffer for micellar electrokinetic chromatography (MEKC) analysis of human urine. Although inherently a pulsed source, the KrF excimer laser was operated at a high pulse repetition rate of 553, 1001 or 2009 Hz to simulate a continuous wave excitation source. Detection limits were found to vary with pulse rate, as expected, in proportion to average excitation power. The following detection limits (3sigma) were determined in free solution CE: tryptophan, 4 nM; conalbumin, 10 nM; alpha-lactalbumin, 30 nM. Detection limits for indole-based compounds and catecholamine urinary metabolites under MEKC separation conditions were in the range 7-170 nM.

Micellar electrokinetic chromatographic study of the interaction between enkephalin peptide analogs and charged micelles.

The relative hydrophobicity/hydrophilicity of the pentapeptides leucine enkephalin (LE), methionine enkephalin (ME) and five analogs, differing in their uncharged side chain and/or chirality, was investigated by micellar electrokinetic capillary chromatography (MEKC) employing anionic and cationic surfactants. The effect of sodium dodecyl sulfate (SDS) concentration on peptide mobility was studied at pH 8.8, a value that is well above the peptide isoelectric point, to minimize electrostatic interaction with the anionic micelles. Similarly, the effect of cetyltrimethylammonium bromide (CTAB) cationic micelles on peptide migration was studied at pH 4.1. The migration order from MEKC experiments was compared to the peptide hydrophobicity calculated from reversed-phase HPLC-derived hydrophobicity coefficients. Although relative peptide hydrophobicity was, in general, positively correlated with effective electrophoretic mobility, a tryptophan-containing analog showed only weak interaction with micelles compared to the less hydrophobic peptides. The enkephalins studied were zwitterionic in character from pH 3 to 8, and their migration as a function of pH under MEKC conditions demonstrated that electrostatic forces were at least as important as hydrophobic interactions in pentapeptide-micelle complexation.

Miniaturized protein microsequencer with PTH amino acid identification by capillary electrophoresis I. An argon pressurized delivery system for adsorptive and covalent sequencing.

The design of a simple, highly miniaturized instrument for manual microsequence analysis of proteins and peptides is described. The reaction chamber is made of fused silica capillary tubing with all reagents and solvents necessary for coupling and cleavage delivered via two valves and a syringe-based dispenser. Only two pressure regulators are required. A section of the flow-through reaction chamber is heated by thermoelectric modules to control the sequencing reaction temperature. Conversion of the extracted amino acid product to the more stable phenylthiohydantoin (PTH) form is performed off-line so that it may be dissolved in 1 mu1 buffer for identification. Approximately 0.1% of this PTH product is analyzed by micellar electrokinetic capillary chromatography (MECC) with thermo-optical absorbance detection (TOAD), providing femtomole detection of the phenylthiohydantoin amino acids. Preliminary results at the 50 pmol level for both adsorptive and covalent sequencing methods are presented.

Miniaturized protein microsequencer with PTH amino acid identification by capillary electrophoresis II. A syringe-pump-based system for covalent sequencing.

A miniaturized protein and peptide microsequencer consisting of a fused silica capillary reaction chamber is described. Extremely small volumes of reagents, 2 mul or less, were delivered directly to the reaction chamber through individual fused silica capillary lines using low pressure syringe pumps. Other than an argon gas controller, no values were used in the delivery system. The short flow path and very low dead volume were achieved by directly connecting the narrow-bore capillaries to the reaction chamber. This configuration minimized side reactions. The elimination of valves, as well as the use of capillaries for the reaction chamber and delivery lines, greatly simplified the construction of the sequencer. The performance of the sequencer was evaluated by sequencing 8-33 picomoles of myoglobin and insulin chain B that were covalently attached to Sequelon-DITC and Sequelon-AA membranes.

Investigation of a pulsed-laser thermo-optical absorbance detector for the determination of food preservatives separated by capillary electrophoresis.

Thermo-optical absorbance (TOA) detection using a KrF excimer waveguide laser for detection of benzoic acid, dehydroacetic acid and sorbic acid separated by capillary electrophoresis (CE) was studied. Detection limits were, on average, ten times better than those for on-column UV absorbance methods with CE, and two or more times better than those for UV absorbance with HPLC. The influence of increased laser power on TOA detection sensitivity was found to be strong for benzoic and dehydroacetic acids but quite weak for sorbic acid. It was discovered that photoisomerization of sorbic acid (2,4-hexadienoic acid) occurred readily in the detection volume at moderate laser powers (P(ave) = 3 mW) and increased with slow electroosmotic flows (< 6 cm/min). The TOA method described here shows improved detection sensitivity for CE analyses of compounds having only weak absorptivities (< 5% of maximum) at lambda = 248 nm, and thus demonstrates its utility for determination of a variety of analytes in a single separation.

Micellar capillary electrophoresis separation and thermo-optical absorbance detection of products from manual peptide sequencing.

Micellar capillary electrophoresis is optimized for separation of phenylthiohydantoin (PTH) amino acids produced in manual Edman degradation reaction for protein sequencing. There are also two major side-products produced by the Edman degradation reaction: diphenylthiourea and dimethylphenylthiourea. We report the complete separation of 19 PTH amino acids plus the two major side-reaction products in 10 min. Capillary electrophoresis is used to identify the five residues generated by manual Edman degradation sequencing of a pentapeptide.

Attachment of a single fluorescent label to peptides for determination by capillary zone electrophoresis.

Complicated electropherograms are produced in the separation of fluorescently labeled peptides. Incomplete labeling of epsilon-amino groups on lysine residues results in the production of 2n-1 reaction products, where n is the number of alpha and epsilon amino groups in the peptide. A single label is attached to the peptide by first taking the peptide through one cycle of the Edman degradation reaction. All epsilon-amino groups are converted to the phenyl thiocarbamyl and the cleavage step exposes one alpha-amino group at the N-terminus of the peptide; the fluorescent label is attached to the N-terminus.

Capillary zone electrophoresis separation and laser-based detection of both fluorescein thiohydantoin and dimethylaminoazobenzene thiohydantoin derivatives of amino acids.

Capillary zone electrophoresis is employed for the separation and analysis of both fluorescein thiohydantoin and dimethylaminoazobenzene thiohydantoin derivatives of amino acids. Detection of minute amounts of these amino acid derivatives is an important milestone in the development of a high sensitivity protein sequencer. Current detection limits for the fluorescein derivative is on the order of 10(-21) moles whereas detection limits for the dimethylaminoazobenzene derivative is on the order of 10(-16) moles.