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PubMed1998

Capillary electrophoresis and capillary electrophoresis-electrospray-mass spectroscopy for the analysis of heterocyclic amines.

Fourteen heterocyclic amines (HAs) were analyzed by capillary electrophoresis (CE) on a polyvinyl alcohol (PVA)-coated capillary column. The optimized electrolyte is composed of 20 mM ammonium acetate, pH 3.0, and 20% methanol. Similar conditions were applied in electrospray-mass spectrometry (CE-ES-MS). The CE-ES-MS optimization procedure includes the position of the capillary in the stainless steel interface, the flow of the sheath liquid and its composition.

Acetates↗

PubMed2003

Polyacrylamide gel electrophoresis followed by sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis for the study of the dimer to monomer transition of human transthyretin.

Familial amyloidotic polyneuropathy (FAP) is caused by mutations which destabilize transthyretin (TTR) and facilitate the aggregation into extracellular amyloid fibrils preferentially in peripheral nerve and heart tissues. Therapeutic and preventive trials for FAP at the plasma TTR level require a careful study of the destabilization of TTR under variable conditions. We have developed a simple double one-dimensional (D1-D) electrophoretic procedure with polyacrylamide gel electrophoresis (PAGE) followed by sodium dodecylsulfate (SDS) gradient PAGE to study the dimer to monomer transition. TTR is first isolated by PAGE from other plasma proteins. The gel strip containing the TTR fraction is incubated in 2% SDS under varying conditions of temperature, buffer composition, pH, and additives like urea and/or a sulfhydryl-reactive agent, followed by SDS-gradient PAGE for the separation of TTR dimers and monomers. We demonstrate that an unidirectional dimer to monomer transition of normal TTR is achieved at 70-80 degrees C in neutral to mild alkaline buffers or at 37 degrees C and slightly acidic pH (6-7). Addition of urea favors the transition into monomers. Amyloidogenic mutations like amyloidogenic TTR (ATTR)-V30M or ATTR-I107V favor the transition into monomers in buffer systems close to the physiological pH of human plasma. We conclude that this finding has to be considered by any hypothesis on ATTR-derived amyloidogenesis.

Amyloid↗

PubMed2005

Urtica dioica agglutinin: separation, identification, and quantitation of individual isolectins by capillary electrophoresis and capillary electrophoresis-mass spectrometry.

With benign prostatic hyperplasia (BPH) being a major health problem in ageing men, alternative therapeutic approaches (e.g., with phytopharmaceuticals) are of great interest. Based on pharmacological evidences, one of the most promising options in that respect are the lectins found in Urtica dioica (stinging nettle) roots. In this study the qualitative and quantitative analysis of individual isolectins in U. dioica extracts is described, which is the first report on using capillary electrophoresis (CE) for the analysis of lectins in plant material at all. By utilizing a 200 mM sodium acetate buffer (pH 3.75) a baseline separation and determination of four closely related isolectins was feasible within 20 min in the aqueous plant extracts. The individual compounds were identified based on reference compounds as well as data obtained from CE-mass spectrometry (MS) experiments. After modifying the optimized CE conditions to 100 mM ammonium formate buffer with pH 3.75 and a voltage of 15 kV, the isolectins were clearly assignable in positive electrospray ionization (ESI) mode. The quantitative results obtained by CE (the total lectin content varied from 0 to 0.42% in the samples) were accurate (recovery rates of spiked samples between 92.5 and 96.2%), precise (relative standard deviation < 5%) and in good agreement to those obtained by High-performance liquid chromatography (HPLC). As for peak resolution, assignable compounds and required separation time the newly developed CE method was clearly advantageous over the determination achieved by LC.

Agglutinins↗

PubMed2005

Characterization of proteins from Spirulina platensis microalga using capillary electrophoresis-ion trap-mass spectrometry and capillary electrophoresis-time of flight-mass spectrometry.

In this work, a new capillary electrophoresis-mass spectrometry (CE-MS) procedure is developed to analyze proteins in Spirulina platensis microalgae. It is demonstrated that a fine optimization of several separation parameters is essential in order to achieve suitable CE-MS analysis of these proteins in natural extracts from microalgae. Namely, optimization of the composition of the separation buffer, electrospray conditions, and washing routine between runs are required in order to obtain reliable and reproducible CE-MS analyses of the main proteins found in this microalga (namely, allophycocyanin-alpha chain, allophycocyanin-beta, c-phycocyanin-alpha, and c-phycocyanin-beta). The relative molecular mass of these biopolymers is determined using two different MS instruments coupled to CE, i.e., CE-ion trap-MS and CE-time of flight-MS (CE-TOF-MS). A comparison between the results obtained with both instruments is carried out. The high resolution of the TOF-MS enables the distinction of small modifications in proteins and, thus, a more accurate mass determination. Interestingly, molecular mass values obtained by both CE-MS procedures agree very well while these experimental values are only in partial agreement with those theoretically expected (i.e., genetically derived masses). Some protein modifications due to amino acids exchange induced by nucleotide codon mutations are proposed to explain this difference.

Bacterial Proteins↗

PubMed1984

Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis: VI. Identification of esterase D in the two-dimensional gel electrophoresis pattern of cellular proteins.

C33k polypeptide, which is a cytosol polypeptide with molecular weight of 33 000 and approximate pI value of 7.5, has three common electrophoretic phenotypes and is an abundant polypeptide in peripheral blood lymphocytes, fibroblasts and red blood cells. Family and population studies indicate that the three phenotypes of C33k polypeptide are determined by two common alleles at a single autosomal locus. The gene frequencies of the two common alleles were 0.642 and 0.358, respectively, in a Japanese population. Since esterase D has a subunit size and gene frequencies similar to those of C33k polypeptide, the phenotypes of C33k polypeptide and esterase D were compared in 18 families totaling 72 members. Perfect concordance of the phenotypes between C33k polypeptide and esterase D was observed in all 72 members. In addition, a gene dosage effect on the expression of the phenotype of C33k polypeptide was observed in the red blood cell lysate from a patient with partial 13q trisomy who was reported to have two doses of EsD1 and one dose of EsD2. These data indicate that the polymorphic C33k polypeptide is esterase D, which is assigned to chromosome 13q14. This finding is useful for the detection of proteins coding for by chromosome 13q14-linked genes in the studies on human gene mapping using somatic hybrid cell lines and two-D gel electrophoresis.

Blood Proteins↗

PubMedDate not supplied

Capillary electrophoresis and capillary electrophoresis-mass spectrometry in drug and metabolite analysis.

The structural diversity of modern therapeutic agents can lead to labour intensive method development for each drug and the structural characterization of their metabolites. In this work, we show the benefits of the high resolution capabilities of capillary electrophoresis (CE) and demonstrate that nonaqueous CE and on-line CE-mass spectrometry (CE-MS) leads to enhanced resolution and recovery of mixtures containing the prototype H2-antagonist, mifentidine, and putative metabolites. Furthermore, the usefulness of CE-tandem MS (CE-MS/MS) is also demonstrated by the structural characterization of the novel N2-hydroxylamine metabolite of mifentidine.

Electrophoresis↗

PubMed1991

Constant denaturant gel electrophoresis, a modification of denaturing gradient gel electrophoresis, in mutation detection.

Denaturing gradient gel electrophoresis (DGGE) is increasingly being utilized in mutational detection, both in characterization of variations in genomic DNA and in the generation of mutational spectra after in vitro and in vivo mutagenesis. The basis for this electrophoretic separation technique is strand dissociation of DNA fragments in discrete, sequence-dependent melting domains followed by an abrupt decrease in mobility. We have modified the DGGE by using constant denaturant gels corresponding to the specific melting domains of certain DNA fragments. This leads to increased resolution of mutants as fragments differing in as little as 1 base pair migrate with a consistently different mobility through the whole gel allowing separations of several centimeters. By using a set of constant denaturant gels it is also possible to obtain a better approximation of the location of the different mutations as each denaturant concentration will correspond to specific melting domains. We have used this technique to separate 6 out of 7 exon-3 hypoxanthine phosphoribosyltransferase (HPRT) mutants while using conventional DGGE we were only able to separate 3.

Animals↗

PubMed2005

Carrier ampholytes-based capillary electrophoresis as an alternative to capillary zone electrophoresis in classical background electrolytes.

Carrier ampholytes (CAs), originally designed for isoelectric focusing (IEF), have been used as background electrolytes (BGE) in capillary zone electrophoresis (CZE). Their main electrophoretic properties, relatively high buffering capacity and low electric conductivity allowed fast (less than 2 min) and high efficient (500,000 theoretical plates/m) separation of a test mixture of proteins under very high electric field strength (more than 1000 V/cm). The results obtained in such buffers have been compared to those obtained in more classical sodium--phosphate and sodium--N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonate) background electrolytes. High ionic strength classical buffers were necessary to achieve the separation of the proteins contained in the test mixture. This induced a significant Joule heating and temperature increase inside the capillary whereas a negligible Joule heat was produced in carrier ampholyte buffers even at the above electric field strength (higher than 1000 V/cm).

Buffers↗

PubMed2000

Determination of linear alkylbenzenesulfonates in wastewater treatment plants and coastal waters by automated solid-phase extraction followed by capillary electrophoresis-UV detection and confirmation by capillary electrophoresis-mass spectrometry.

Linear alkylbenzenesulfonates (LASs) were determined in wastewaters and coastal waters by solid-phase extraction, using two different sample preparation protocols depending on the sample treated, followed by capillary electrophoresis and ultraviolet detection (CE-UV). The linear range of the proposed method varied from 3 to 53 and from 25 to 495 microg/l, depending on the compound, with a limit of detection of 1 microg/l when 250 ml of coastal water was preconcentrated. [M-H]- ions were used for CE-MS confirmation after quantification by CE-UV. CE-MS diagnostic ions were the same ones used in LC-electrospray (ESI) MS and corresponded to m/z 297, 311, 325 and 339 for C10, C11, C12 and C13 LASs, respectively. LASs were determined in wastewater samples of the influent and effluent of three wastewater treatment plants (WWTPs), two of them using biological treatment with secondary settlement and receiving mainly domestic wastewaters whereas one of the plants was operated with physicochemical treatment and received mainly industrial wastewaters. LASs were also analyzed in two samples from coastal waters of the bay of Cadiz (Spain) receiving untreated domestic effluents. All samples were also analyzed by LC-ESI-MS and the results are compared with the CE-UV method developed in this work. The concentration levels of total LASs varied from 988 to 1309 microg/l in the influents of WWTPs, whereas in the effluents the concentrations varied from 136 to 197 microg/l. The levels of LASs in coastal wastewaters of the bay of Cadiz varied from 739 to 911 microg/l, indicating that the wastewaters discharged into the bay did not undergo any treatment at all.

Alkanesulfonic Acids↗

PubMed2000

Capillary electrophoresis and off-line capillary electrophoresis-electrospray ionization quadrupole time-of-flight tandem mass spectrometry of carbohydrates.

The use of off-line high-performance capillary electrophoresis in connection with nanospray electrospray ionization quadrupole time-of-flight tandem mass spectrometry for identification of complex carbohydrates of biological origin is presented. The method was applied to the identification of O-glycosylated amino acids and -glycopeptides from the urine of patients suffering from a hereditary disease - N-acetylhexosaminidase deficiency. Structural elements typical for O-glycosylation of proteins, like expression of core 1 and 2 type O-glycans with different numbers of N-acetyllactosaminyl repeats and different degrees of sialylation, can be directly detected.

Carbohydrate Sequence↗

PubMed2000

Open access capillary electrophoresis. A walk up capillary electrophoresis service for the synthetic chemist.

A capillary electrophoresis (CE) system has been developed to provide routine purity information to support synthetic organic chemists. A single micellar electrokinetic chromatography system that produces good selectivity with short run times was developed. The instrument operating software has been modified to run separations in a custom open access mode. No expert knowledge of CE is required to run separations.

Electrophoresis, Capillary↗

PubMed1997

Analysis of gangliosides by capillary zone electrophoresis and capillary zone electrophoresis-electrospray mass spectrometry.

Gangliosides, sialic acid(s)-containing glycosphingolipids, were separated by capillary zone electrophoresis and detected with either UV or electrospray mass spectrometry. Several electrolyte system were evaluated for the separation of underivatized gangliosides. The best result was obtained by using 50 mM borate and 50 mM phosphate buffer containing 20 mM alpha-cyclodextrin at pH 9.9. The four major ganglioside forms (GM1, GD1a, GD1b, GT1b) were successfully separated, and, moreover, each ganglioside yielded two peaks, splitting by the difference in chain length of the ceramide moiety. The resolution obtained in CE-UV could not be reproduced in CE-MS because of the incompatibility of the borate/phosphate buffer to ESI-MS. With the use of more volatile buffers, such as ammonium acetate or 2-[N-cyclohexylamino]-ethanesulfonic acid, baseline resolution was obtained for gangliosides having different number of sugars, but the two disialoganglioside isomers, GD1a and GD1b, were coeluted.

Animals↗

PubMed1999

Evaluation of volatile eluents and electrolytes for high-performance liquid chromatography-electrospray ionization mass spectrometry and capillary electrophoresis-electrospray ionization mass spectrometry of proteins. II. Capillary electrophoresis.

Peptides and proteins were separated by capillary electrophoresis (CE) in fused-silica capillaries coated with an irreversibly adsorbed monolayer of derivatized polystyrene nanoparticles. Whereas phosphate buffer, pH 3.10, enabled the highly efficient separation of basic proteins with plate counts up to 1,400,000 m-1, volatile buffer components such as formic acid or acetic acid titrated with ammonia to the desired pH had to be used for the direct coupling of CE with electrospray ionization mass spectrometry (ESI-MS). Compared to 40 mM phosphoric acid-sodium hydroxide, pH 3.10, a background electrolyte containing 125 mM formic acid-ammonia, pH 4.00, was shown to yield equivalent separation efficiency. Investigation of the influence of buffered electrolytes on the ESI-MS signal of lysozyme at pH 2.70-4.00 showed that the charge state distribution shifted to lower charge states at higher pH with a concomitant five-fold decrease in signal intensity of the most abundant signal. The presence of trifluoroacetic acid in the background electrolyte greatly increased the level of baseline noise and completely inhibited the observation of any mass signals related to proteins. Full scan spectra could be acquired from 50-500 fmol amounts of proteins during coupled CE-ESI-MS utilizing 100-125 mM formic acid-ammonia, pH 3.10. However, compared to UV detection, considerable band broadening is observed with ESI-MS detection which is mainly attributed to column overloading, band spreading in the interface, and scanning data acquisition. Finally, the major whey proteins beta-lactoglobulin A, beta-lactoglobulin B, and alpha-lactalbumin were identified in a whey drink by comparison of molecular masses determined by CE-ESI-MS to molecular masses calculated from the amino acid sequence.

Chromatography, High Pressure Liquid↗

PubMed1999

Direct assay of nonopioid analgesics and their metabolites in human urine by capillary electrophoresis and capillary electrophoresis-mass spectrometry.

Capillary electrophoresis (CE) was used for the analysis of nonopioid analgesics and their metabolites directly in urine samples. A simple, reliable screening method was developed that allows identification of the drug and/or its metabolites in urine after oral application of paracetamol, acetylsalicylic acid, antipyrine, ibuprofen, naproxen, ketoprofen and propyphenazone by their migration in CE and by their UV spectra recorded with a diode-array detector in a common CE-UV system with 50 mM borax pH 9.4 as separation buffer. For the CE-electrospray (ESI)-MS coupling a volatile 50 mM ammonium acetate buffer at pH 9.8 was used. In order to analyze the metabolic pattern in more detail different methods were developed for each drug. The separation of the metabolites of acetylsalicylic acid could be improved by injection of the urine sample at the cathodic side of the capillary. In order to identify antipyrine as neutral compound as well as its neutral metabolites-a micellar electrokinetic chromatography (MEKC) method was developed.

Analgesics, Non-Narcotic↗

PubMed2002

Capillary electrophoresis and capillary electrophoresis-ion trap multiple-stage mass spectrometry for the differentiation and identification of oxycodone and its major metabolites in human urine.

Oxycodone (OCOD) and its metabolites, including oxymorphone (OMOR), noroxycodone (NOCOD) and noroxymorphone (NOMOR), are opioids that carry an OH group at position 14. Using capillary electrophoresis (CE) with a binary phosphate buffer containing 60% ethylene glycol (pH 7.9), the migration order of OCOD and OMOR with respect to their N-demethylated analogs was found to be reversed compared to that observed for codeine, dihydrocodeine, morphine and dihydromorphine, compounds that do not have an OH group at position 14. OCOD and structurally related compounds can also be distinguished from these opioids by their absorbance spectra at low wavelengths and via a characteristic neutral H2O loss at the MS2 level. Using the binary phosphate buffer, CE with UV detection is shown to be capable of monitoring OCOD, NOCOD, OMOR (after hydrolysis only) and NOMOR (after hydrolysis and in patient urine only) in alkaline liquid-liquid extracts of urines that were collected after ingestion of 10 mg OCOD hydrochloride and in a patient urine collected at steady state (80 mg OCOD hydrochloride daily). Using an aqueous pH 9 ammonium acetate buffer, these results were confirmed by CE-MS3. Based on CE-MS, MS2 and MS3 data, the absorbance spectra measured across the CE peaks and the relative position within the electropherogram, two peaks monitored in the UV absorbance electropherograms could be assigned to the two keto-reduced metabolites 6oxycodol (60COL) and nor6oxycodol, for which no standards were available. Comparison of data obtained with urines pretreated with two different enzyme products (beta-glucuronidase and beta-glucuronidase/arylsulfatase) suggest that OCOD, NOCOD and 6OCOL are mainly glucuronidated, whereas OMOR mainly forms other conjugates. Furthermore, in a first attempt to directly measure conjugates of the compounds of interest, solid-phase extracts were analyzed by CE-MS4, which revealed the presence of the acyl glucuronides of 6OCOL and OMOR and an unidentified OMOR conjugate. The quantitation of free OCOD and NOCOD by CE-MS using deuterated internal standards is also discussed briefly.

Analgesics, Opioid↗

PubMed1996

Analysis of DNA adducts in DNA hydrolysates by capillary zone electrophoresis and capillary zone electrophoresis-electrospray mass spectrometry.

The in vitro adduct formation with phenyl glycidyl ethers (PGEs) was studied on 2'-deoxynucleotides and DNA. The modified DNA was hydrolyzed enzymatically, and the mixtures consisting of unmodified 2'-deoxynucleotide adducts were analyzed by capillary zone electrophoresis (CZE), CZE-electrospray mass spectrometry (CZE/ES-MS) and CZE-electrospray tandem mass spectrometry (CZE/ES-MS/MS) using sample stacking. For the CZE analyses, a homemade system was developed in order to enhance the reproducibility of the retention times. This modification enabled the total comparison of the electropherograms obtained for the analysis of 2'deoxynucleotides mixtures with the electropherograms obtained for the DNA hydrolysates both treated with PGEs. The assignment of adducted and nonadducted 2'-deoxynucleotide peaks was unambiguous. Analysis of the CZE/ES-MS data gave the necessary structural information and revealed the presence of mono- and dialkylated 2'-deoxynucleotides. Interpretation of the CZE/ES-MS/MS data of the monoalkylated products allowed differentiation between purine or pyrimidine alkylation and alkylation of the 5-phosphate moiety. Recording of full-scan mass spectra during CZE/ES-MS/MS analysis of 2'-deoxynucleotide reaction mixtures and DNA hydrolysates was possible, using the described CZE sample stacking technique.

Animals↗

PubMed2003

Non-equilibrium capillary electrophoresis of equilibrium mixtures--appreciation of kinetics in capillary electrophoresis.

We describe a new electrophoretic method (patent pending), Non-Equilibrium Capillary Electrophoresis of Equilibrium Mixtures (NECEEM), and demonstrate its application to the study of protein-DNA interactions. A single NECEEM experiment allows for the determination of equilibrium and kinetic parameters of protein-DNA complex formation. The equilibrium mixture is prepared by mixing protein and DNA; it contains three components: free protein, free DNA, and the protein-DNA complex. A small plug of such a mixture is injected onto a capillary and the three components are separated under non-equilibrium conditions using a run buffer that does not contain the components of the equilibrium mixture. The protein-DNA complex decays during the NECEEM separation; the resulting electropherograms contain characteristic peaks and exponential curves. A simple analysis of a single electropherogram reveals two parameters: the equilibrium dissociation constant of the protein-DNA complex and the monomolecular rate constant of complex decay. The bimolecular rate constant of complex formation can then be calculated as the ratio of the two experimentally-determined constants. NECEEM was applied to find the equilibrium and kinetic parameters of interaction between an E. coli single-stranded DNA binding protein and a fluorescently-labeled oligonucleotide. The constants determined by NECEEM are in good agreement with those obtained by other methods. The new method is simple, fast, and accurate. It can be equally applied to other non-covalent molecular complexes.

Animals↗

PubMed1998

Conformation sensitive gel electrophoresis for simple and accurate detection of mutations: comparison with denaturing gradient gel electrophoresis and nucleotide sequencing.

Previously, an assay called conformation sensitive gel electrophoresis (CSGE) was developed for scanning PCR products for the presence of single-base and larger base mismatches in DNA. The assay was based on the assumption that mildly denaturing solvents in an appropriate buffer can accentuate the conformational changes produced by single-base mismatches in double-stranded DNA and thereby increase the differential migration in electrophoretic gels of heteroduplexes and homoduplexes. Here the sensitivity of assays by CSGE was improved by limiting the maximal size of the PCR products to 450 bp and making several changes in the conditions for PAGE. With the improved conditions, CSGE detected all 76 previously identified single-base changes in a large series of PCR products from collagen genes that contain multiple exons with highly repetitive and GC-rich sequences. In a survey of 736 alleles of collagen genes, CSGE detected 223 unique single-base mismatches that were confirmed by nucleotide sequencing. CSGE has the advantage over other methods for scanning PCR products in that it is simple, requires no special preparation of PCR products, has a large capacity, and does not use radioactivity.

Base Sequence↗