On-line Capillary Zone Electrophoresis Research Articles

Assaying human neutrophil elastase activity by capillary zone electrophoresis combined with laser-induced fluorescence

Skin aging is a progressive process determining the ultimate skin appearance. Human neutrophil elastase (HNE) has been shown to play an important role in the degradation of the extracellular matrix. In order to assay HNE kinetics, a novel online capillary zone electrophoresis (CZE) assay has been developed in this study for the determination of the maximum velocity (Vmax) and of the Michaelis–Menten constant (Km) of HNE regarding several potential substrates. These assays are based on short-end injection to shorten analysis time, on transverse diffusion of laminar flow profiles (TDLFP) for in-capillary reactant mixing, and on UV or laser-induced fluorescence (LIF) detection. Kinetic constants for a referenced peptidic substrate were determined using not only online assays but also offline (pre-capillary) mode. The results obtained were cross compared and compared to the literature in order to validate the developed assays. The hydrolysis of three new potential fluorogenic substrates by HNE was also monitored. Two new peptidic substrates for HNE were identified through this study. Km values of these novel substrates were successfully determined using online CZE assay (Km ∼0.07mM). This value was in the same order of magnitude of that of the referenced substrate despite the presence of the labeling group 5-carboxyfluorescein (5-FAM). HNE activity has never been assessed using online CZE-based assay, neither with UV nor with LIF detection. The developed assay conducted with the new labeled substrates is particularly sensitive (LOQ of few nM), does not require the presence of micelles in the BGE (which is the case for the reference substrate) and only necessitates few nanoliters of reactants making it particularly adapted for screening studies.

Vinyl functionalized silica hybrid monolith-based trypsin microreactor for on line digestion and separation via thiol-ene “click” strategy

A novel thiol-ene “click” strategy for the preparation of monolithic trypsin microreactor was proposed. The hybrid organic–inorganic monolithic capillary column with ene-functionality was fabricated by sol–gel process using tetramethoxysilane (TMOS) and γ-methacryloxypropyltrimethoxysilane (γ-MAPS) as precursors. The disulfide bonds of trypsin were reduced to form free thiol groups. Then the trypsin containing free thiol groups was attached on the γ-MAPS hybrid monolithic column with ene-functionality via thiol-ene click chemistry to form a trypsin microreactor. The activity of the trypsin microreactor was characterized by detecting the substrate (Nα- p-tosyl- l-arginine methyl ester hydrochloride, TAME) and the product (Nα- p-tosyl- l-arginine, TA) with on-line capillary zone electrophoresis. After investigating various synthesizing conditions, it was found that the microreactor with poly(N,N′-methylenebisacrylamide) as spacer can deliver the highest activity, yielding a rapid reaction rate. After repeatedly sampling and analyzing for 100 times, the monolithic trypsin microreactor still remained 87.5% of its initial activity. It was demonstrated that thiol-ene “click” strategy for the construction of enzyme microreactor is a promising method for the highly selective immobilization of proteins under mild conditions, especially enzymes with free thiol radicals.

On-line nanoliter cycle sequencing reaction with capillary zone electrophoresis purification for DNA sequencing

An integrated system for DNA sequencing based on a nanoreactor for cycle-sequencing reaction coupled with on-line capillary zone electrophoresis (CZE) for purification and capillary gel electrophoresis (CGE) for separation is presented. Less than 100 nl of premixed reagent solution, which includes dye-labeled terminator pre-mix, bovine serum albumin and template, was hydrodynamically injected into a fused-silica capillary (75 μm I.D.) inside a laboratory-made microthermocycler for cycle sequencing reaction. In the same capillary, the reaction products were purified by CZE followed by on-line injection of the DNA fragments into another capillary for CGE. Over 540 base pairs (bp) of DNA can be separated and the bases called for single-standed DNA with 0.9% error rate. The total time was about 3.5 h, or a cycle time of 2 h with staggered operation. For double-stranded DNA, a longer reaction time was required and base calling up to 490 bp with 1.2% error rate was achieved. The whole system is readily adaptable to automated multiplex operation for DNA sequencing or polymerase chain reaction analysis.

Simultaneous stereoselective analysis of tramadol and its main phase I metabolites by on-line capillary zone electrophoresis–electrospray ionization mass spectrometry

On-line combination of partial filling capillary electrophoresis and electrospray ionization mass spectrometry was demonstrated for the simultaneous enantioseparation of tramadol and its main phase I metabolites. The partial filling technique was efficient at avoiding MS contamination by the chiral selector. Different experimental factors were investigated, including the chiral selector nature and concentration, plug length as well as the separation temperature. The best enantioseparation of the investigated compounds was achieved with a coated polyvinyl alcohol capillary and a 40 m M ammonium acetate buffer, pH 4.0, adding sulfobutyl ether β-cyclodextrin (2.5 mg/ml) as the chiral selector. The charged cyclodextrin not only allowed enantioseparation of tramadol and its metabolites, but also improved the selectivity of compounds with the same molecular mass. Finally, CE–electrospray ionisation-MS was successfully applied to the stereoselective analysis of tramadol and its main metabolites in plasma after a simple liquid–liquid extraction.

Electrospray ionization mass spectrometry and on-line capillary zone electrophoresis - mass spectrometry for the characterization of citrate synthase

The enzyme citrate synthase from E. coli is a protein with a molecular weight (Mr) of 47 885 Da (wild type). This enzyme has been studied extensively, and its amino acid sequence has been characterized. This model protein has been used in this work for development and validation of methods involving capillary electrophoresis (CE) and electrospray ionization mass spectrometry (ESI-MS). The Mr determinations were conducted using sample infusion ESI-MS, and the tryptic digestion products of wild-type citrate synthase were characterized by on-line CE-ESI-MS coupled with a sheathless interface. On-line experiments were conducted on two different mass spectrometers, a Quattro-LC triple quadrupole instrument equipped with a Z-SprayTM source (Micromass), and a reflecting time-of-flight (TOF) mass spectrometer built in-house in the Time-of-Flight Laboratory, Department of Physics, University of Manitoba. This is the first article to be written on the interfacing of a Z-SprayTM source with CE. Unmodified fused silica capillaries gold-coated sheathless interfaces were used. The on-line CE separations yielded theoretical plate numbers greater than 104 on average. Selected ion electrophorograms (SIE) of the tryptic peptides recorded on the Quattro-LC displayed S/N ratios ranging from ca. 14 to 120 on raw data. These SIE enabled identification of each peptide. The use of reflecting time-of-flight mass spectrometry (TOFMS) afforded mass resolution values of ca. 6000 (m/deltamFWHM), which enabled isotopic resolution of the peptide components. CE-ESI-MS and CE-ESI-TOFMS experiments enabled the generation of a complete tryptic map of citrate synthase.Key words: capillary electrophoresis, electrospray ionization, mass spectrometry, citrate synthase, tryptic digestion, triple quadrupole analyzer, time-of-flight analyzer.

Electrospray ionization mass spectrometry and on-line capillary zone electrophoresis - mass spectrometry for the characterization of citrate synthase

The enzyme citrate synthase from E. coli is a protein with a molecular weight (Mr) of 47 885 Da (wild type). This enzyme has been studied extensively, and its amino acid sequence has been characterized. This model protein has been used in this work for development and validation of methods involving capillary electrophoresis (CE) and electrospray ionization mass spectrometry (ESI-MS). The Mr determinations were conducted using sample infusion ESI-MS, and the tryptic digestion products of wild-type citrate synthase were characterized by on-line CE-ESI-MS coupled with a sheathless interface. On-line experiments were conducted on two different mass spectrometers, a Quattro-LC triple quadrupole instrument equipped with a Z-SprayTM source (Micromass), and a reflecting time-of-flight (TOF) mass spectrometer built in-house in the Time-of-Flight Laboratory, Department of Physics, University of Manitoba. This is the first article to be written on the interfacing of a Z-SprayTM source with CE. Unmodified fused silica capillaries gold-coated sheathless interfaces were used. The on-line CE separations yielded theoretical plate numbers greater than 104 on average. Selected ion electrophorograms (SIE) of the tryptic peptides recorded on the Quattro-LC displayed S/N ratios ranging from ca. 14 to 120 on raw data. These SIE enabled identification of each peptide. The use of reflecting time-of-flight mass spectrometry (TOFMS) afforded mass resolution values of ca. 6000 (m/deltamFWHM), which enabled isotopic resolution of the peptide components. CE-ESI-MS and CE-ESI-TOFMS experiments enabled the generation of a complete tryptic map of citrate synthase.Key words: capillary electrophoresis, electrospray ionization, mass spectrometry, citrate synthase, tryptic digestion, triple quadrupole analyzer, time-of-flight analyzer.

Sensitive high-resolution analysis of biological molecules by capillary zone electrophoresis coupled with reflecting time-of-flight mass spectrometry

Off-line and on-line capillary zone electrophoresis–electrospray ionization time-of-flight mass spectrometry (CZE–ESI-TOF-MS) experiments were conducted using uncoated fused-silica capillaries coupled to a reflecting TOF mass spectrometer via a gold-coated sheathless interface. Off-line and on-line experiments were performed on standard mixtures of proteins and peptides. Samples collected off-line electrokinetically in plastic vials were analyzed by standard ESI–TOF-MS at the pmol level. Sheathless CZE–ESI-TOF-MS was first simulated in an off-line experiment, using a test bench, in order to select a suitable running electrolyte, to find the optimal electrospray potential, and also to test the gold-coated capillary tips. This enabled an ease of transition to on-line measurements. On-line CZE–ESI-TOF-MS measurements of the total ion electropherogram (TIE) and of selected ion electropherograms (SIE) on peptide mixtures demonstrated fmol-level sensitivity, with S/ N values of 250–400 on raw data (TIE mode) and of 30–760 (SIE mode). The use of reflecting TOF-MS afforded mass resolution values R>6000 ( m/Δ m FWHM) and enabled isotopic resolution of peptide components as well as mass accuracy in the 10 ppm range. These results were comparable with values observed with the usual ESI source on the same mass spectrometer, and thus demonstrated no loss in spectral quality from using the sheathless CE interface. On-line CE separation efficiency was equivalent to that obtained off-line for the separation of a peptide mixture, with N=35 000–87 000 theoretical plates. Separations of standard proteins yielded equivalent mass spectral resolution and accuracy with separation efficiencies of N=2800–5500 and S/ N values of 110–225 on raw data. The gold-coated sheathless CE–ESI interface was found to be relatively easy to prepare with the use of gold vapour deposition. The interface produced a stable electrospray for extended periods of time and was found to be robust and reliable.

Combined liquid-liquid electroextraction and isotachophoresis as a fast on-line focusing step in capillary electrophoresis

Combined liquid-liquid electroextraction and isotachophoresis as a fast on-line focusing step in capillary electrophoresis is described. Owing to the limited conductivity of the organic phase, high electric field strengths can be applied, resulting in high migration rates. Liquid-liquid electroextraction is used to focus the analyte ions from a large organic sample into a small volume in the terminating buffer zone, just above the liquid-liquid interface. Simultaneously, isotachophoresis is used to focus the analyte ions between the terminating buffer and the leading buffer. A steady state is reached within a few minutes, as isotachophoresis starts with a small sample volume. On-line capillary zone electrophoresis is used for the separation of the analytes. Propantheline, neostigmine, salbutamol and terbutaline were used as model compounds, and crystal violet was used to visualize the process for tuning the system. Concentration detection limits of pure solutions down to 10 −9-10 −10 mol l for the model compounds were obtained using simple UV absorbance detection.

On‐line isotachophoretic sample preconcentration for enhancement of zone detectability in capillary zone electrophoresis

AbstractA simple isotachophoretic (ITP) preconcentration column for subsequent online capillary zone electrophoresis is described, and its use for enhancing detectability in capillary zone electrophoresis (CZE) is evaluated. Sample preconcentration using ITP is shown to enhance detectability in CZE by several orders of magnitude. Experimental verification of the detectability gain is demonstrated for cationic and anionic separations; an increase in detectability of more than 200‐fold was achieved. The practical applicability of this method is demonstrated using a direct analysis of thiamine in human blood.