bluuebunny/crossref_metadata_2025 huggingface dataset:
Capillary zone electrophoresis of inorganic anions with conductivity detection
Authors: Kaniansky Dušan , Zelenská Viera , Baluchová Daniela
Published: May 2005

A carrier electrolyte system for capillary zone electrophoresis (CZE) resolving chloride, bromide, iodide, fluoride, nitrite, nitrate, sulfate, and phosphate in a hydrodynamically closed separation compartment is described. The carrier electrolyte combines the effects of pH, polyvinylpyrrolidone (PVP) and the counterionic constituent on the effective mobilities of the anions. In 300 μm ID capillary tubes made of fluorinated ethylene‐propylene copolymer (FEP), and with detection of analytes with the aid of an alternating current conductivity detector, detection limits in the range of 3–10 ppb could be achieved for 200 nL sample volumes. The separation efficiencies were in the range of 1.5–2.5 × 105 theoretical plates per meter for an adequate sample load. The reproducibility was evaluated for two concentration levels. For concentrations close to the limits of quantitation (50–120 ppb), the RSD values ranged from 1.5–12.6%, with the highest scatter for fluoride and phosphate. The RSD values were in the range of 0.4–1.5% for 300–1200 ppb concentrations of the anions. Typical analysis times were 2–5 min, depending on the anion species. A series of water samples (drinking, river, rain) was used to assess the practical applicability of the CZE method. The method is a suitable alternative for the determination of both anionic macro‐ and microconstituents in water with a good overall selectivity.
Comparison of heparin‐binding to lactoferrin from human milk and from human granulocytes by means of affinity capillary electrophoresis
Authors: Heegaard Niels H. H. , Brimnes Jens
Published: May 2005

Human lactoferrin from two different sources: milk and secondary granules of neutrophil granulocytes, was compared with respect to heparin binding by means of affinity capillary electrophoresis. This method is based on analysis of migration pattern shifts induced by various amounts of ligand present in the electrophoresis buffer. Since lactoferrin is a basic molecule, analyses were performed at pH 8 in a capillary with a neutral hydrophilic coating. However, analyzable peaks were only obtained in the presence of heparin in the electrophoresis buffer. Proportionally to the amount of heparin present, these peaks exhibited shape changes and strong migration shifts. In the ensuing analysis it could be demonstrated that the two lactoferrins had comparable migration shifts and peak shape changes. This indicates that also in this respect the two forms of lactoferrin are identical. Affinity capillary electrophoresis is a convenient and fast method to investigate functional characteristics of low amounts of unmodified biomolecules.
Optimization of selectivity and resolution in micellar electrokinetic capillary chromatography with a mixed micellar system of sodium dodecyl sulfate and sodium cholate
Authors: Wiedmer Susanne K. , Jumppanen Juho H. , Haario Heikki , Riekkola Marja‐Liisa
Published: May 2005

Selectivity and resolution were studied for the separation of seven corticosteroids by micellar electrokinetic capillary chromatography (MEKC) using a mixed micellar solution of sodium dodecyl sulfate (SDS) and sodium cholate (SC), buffered with 3‐(N‐morpholino)propanesulfonic acid (MOPS) or 3–[(1,1‐dimethyl‐2‐hydroxyethyl)amino]‐2‐hydroxyoropane sulfonic acid (AMPSO). The changes in selectivity were compared for the AMPSO‐SDS‐SC system by varying the pH and the concentrations of AMPSO, SDS and SC. The experimental design started with the central composite design and continued in a sequential manner. The optimum selectivity for the separation of the corticosteroids was calculated from the analyte migration times and the analyte velocities, by using empirical quadratic regression models. Satisfactory regression fits and coefficients of determination for prediction were obtained with crossvalidated models. To optimize the resolution, the physical parameters of capillary length and analysis time were varied under the conditions optimal for the selectivity. In both the selectivity and the resolution, optimization the overall optimum was determined by using the desirability function technique. Analysis times were controlled by using 1,3‐diaminopropane to influence the electroosmotic flow velocity (Veo). The voltage was kept constant, which resulted in higher electric field strength in shorter capillaries. No changes in the selectivity were observed when 1,3‐diaminopropane was used to control the electroosmotic flow velocity. Such an optimization technique, where the chemical and physical factors affecting the separation are treated independently, seemed to be effective for finding the best possible resolution for the corticosteroids.
Sequencing in nanofabricated arrays: A feasibility study
Authors: Duke Thomas , Monnelly Glen , Austin Robert H. , Cox Edward C.
Published: May 2005

The feasibility of using nanofabricated arrays as electrophoretic chambers for DNA sequencing is investigated. A specific array design, consisting of rows of closely spaced posts, separated by longer open spaces, is proposed. Molecules driven through the array by an electric field get hooked over obstacles at successive rows and their progress through the device is delayed as a consequence. The dependence of the delay time on molecular size is derived. Numerical evaluation indicates that a device of modest dimensions, operating at high fields, can rapidly resolve oligonucleotides containing several hundred bases.
A rapid electrophoretic method for separating rabbit skeletal muscle myosin heavy chains at high resolution
Authors: Kubis Hans‐Peter , Gros Gerolf
Published: May 2005

An electrophoretic method using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) was developed which gives a high‐resolution separation of the known myosin heavy chains of rabbit skeletal muscle with excellent reproducibility. The gel of 10 cm total length consists of (i) a first stacking gel of 3.5% total gel concentration (T) and pH 6.8, (ii) a first separating gel of 6.6%T and pH 8.8, (iii) a second stacking gel of 6.6%T and pH 6.8, and (iv) a second separating gel of 8.8%T and pH 8.8. With this composition, a minigel system allows separation of six myosin heavy‐chain (MHC) isoforms at room temperature without cooling and within 8h. In agreement with previous reports, the isoforms appear in the sequence MHCemb, MHC IIa, MHC IId, MHCneo, MHC IIb, MHC I. A special advantage is the detectability not only of the adult but also of the embryonic and neonatal isoforms MHCemb and MHCneo.
Quantitative densitometry of proteins stained with Coomassie Blue using a Hewlett Packard scanjet scanner and Scanplot software
Authors: Vincent Sandra G. , Cunningham Peter R. , Stephens Newman L. , Halayko Andrew J. , Fisher John T.
Published: May 2005

In the present study we evaluated the performance of a software/scanner system that employed the Hewlett Packard (HP) ScanJet Plus and Scanplot Software for densitometric quantification of protein loads stained with Coomassie Brilliant Blue following sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Gels with bovine serum albumin (BSA) standards, ranging from 0.125 to 10 μg, were scanned using reflectance densitometry with 127 μm step size in both the x and y directions and a resolution of 200 dots per inch. Densitometric volume was calculated for each protein band from scanner output in the tagged image file format (TIFF) by a customized software package, Scanplot V. 4.05 (Cunningham Engineering). Protein loads between 0.125 and 10.0 μg vs. volume were fit by a second‐order regression: Volume = −0.58 × protein load2 + 16.82 × protein load + 7.87 (r = 0.991, p < 0.01). The same gels were scanned and quantified using a transmittance laser densitometer; densitometric volumes measured by both systems were highly correlated (r2 = 0.981, p < 0.01). Additional gels of BSA, smooth muscle myosin heavy chain (myosin), and actin displayed linear relationships between protein loads up to 4.0 μg and densitometric volume reflecting unique dye binding properties. We conclude that accurate and reproducible quantitative densitometry of SDS‐PAGE can be performed using the HP ScanJet Plus scanner and Scanplot software.
Deoxyribonuclease I‐facilitated electrotransfer of protein‐DNA complexes from electrophoretic gels to nitrocellulose membranes
Authors: Ivanchenko Maria , Zlatanova Jordanka
Published: May 2005

A simple and reproducible technique for efficient transfer of protein‐DNA complexes from electrophoretic gels to nitrocellulose membranes is described. Transfer of DNA‐protein complexes may be difficult, especially when the DNA is of high molecular mass. A considerable improvement in the efficiency of transfer can be achieved by directly digesting the DNA in the gel by DNase I. The method is illustrated in the example of the preferential binding of histone H1 to superhelical plasmid DNA.
Versatile low‐viscosity sieving matrices for nondenaturing DNA separations using capillary array electrophoresis
Authors: Madabhushi Ramakrishna S. , Vainer Marina , Dolnik Vladislav , Enad Shellee , Barker David L. , Harris Dennis W. , Mansfield Elaine S.
Published: May 2005

The high‐resolution separation of double‐stranded DNA (dsDNA) has important applications in physical mapping strategies and in the analysis of polymerase chain reaction (PCR) products. Although high‐resolution separations of dsDNA by capillary electrophoresis (CE) have been reported, pulsed fields were required to achieve complete resolution of DNA fragments beyond 23 kilobase pairs (kbp). Here, we report a single formulation to separate a broad range (80 bp – 40 kbp) of DNA fragments without the use of pulsed fields. We used a low‐viscosity sieving medium (ca. 5 cP, at 25°C) based on polyethyleneoxide (PEO) to separate DNA fragments up to 40 kbp. The matrix contained a mixture of 0.5% PEO (Ma 106) to separate fragments up to 1.5 kbp, combined with 0.1% PEO (Ma 8 × 106) to separate fragments between 1–40 kbp, within a single run. All PEO matrix formulations tested were compatible with a variety of intercalating dyes and with two different capillary wall coating methods. We obtained a detection limit of 25 fg of a 200 bp DNA quantitation standard using Vistra Green in the matrix. Resolution was best using short injection times (5 s or less) and low field strengths (approximately 100 V/cm). Sample runs were complete in 70 min, and use of the capillary array electrophoresis (CAE) system permitted high‐throughput DNA analysis. The size range separated is approximately 10 times greater than with conventional slab gel separations.
Measurement of antibody‐antigen dissociation constants using fast capillary electrophoresis with laser‐induced fluorescence detection
Authors: Tao Li , Kennedy Robert T.
Published: May 2005

The dissociation constant (Kd) of a monoclonal antibody with fluorescein isothiocyanate (FITC)‐labeled insulin and unlabeled insulins from several species were measured using capillary electrophoresis with laser‐induced fluorescence detection (CE‐LIF). Kd determinations were made by separating free FITC‐insulin and its complex with the antibody in equilibrated solutions in 6 s or less. The use of LIF detection allowed quantification of free and bound FITC‐insulin in the picomolar range, as is required to measure Kd's below 1 nM. The Kd of FITC‐insulin with the antibody was determined to be 0.25 nM by Scatchard analysis. The Kd's of the antibody with unlabeled insulins from several species were obtained by fitting bound over free FITC‐insulin as a function of unlabeled insulin concentration data from a series of solutions containing a fixed concentration of FITC‐insulin and antibody and variable concentrations of insulin to the expected curve derived from the equilibria and mass balance of the solutions. Kd's for the different insulins were between 0.34 and 0.64 nM.
Electrophoretic demonstration of high molecular weight fibrin degradation products persisting in chronic subdural hematomas
Authors: Toyosawa Mikiko , Kashiwagi Shiro , Pei Wang , Fujisawa Hirosuke , Ito Haruhide , Nakamura Kazuyuki
Published: May 2005

Local hyperfibrinolysis plays an important role in the pathogenesis of chronic subdural hematoma (CSH). The purpose of this study is to elucidate the nature of the local hyperfibrinolysis in CSH, by comparing the pattern of fibrin/fibrinogen degradation product (FDP) of hematomas with that of purified fibrin clots digested by plasmin in vitro. Forty‐seven hematoma samples were subjected to the analysis. FDPs of CSH and the purified fibrin clots digested by plasmin for different incubation times were identified using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and immunoblotting, and then quantified by densitometry. The effect of alpha‐2 plasmin inhibitor (A2‐PI) on the degradation process was also studied. The FDP pattern of CSH was similar to those of purified fibrin digested by plasmin for 0.75–2h. The FDP composition of CSH was closest to that of purified fibrin digested for 1h. By adding the physiological concentration of A2‐PI at the second hour, further degradation of high molecular weight FDPs was inhibited and the early FDP pattern persisted after 24h. Local hyperfibrinolysis in CSHs is characterized by incomplete fibrinolysis, which occurs only on the solid fibrin clot, and is arrested in the liquid hematoma. As a result, high molecular weight FDPs persist in CSHs for weeks or months in the hematoma. A2‐PI seems to play an important role in producing this unique FDP pattern in CSH.
Two‐dimensional electrophoresis of membrane proteins: A current challenge for immobilized pH gradients
Authors: Adessi Céline , Miege Christine , Albrieux Catherine , Rabilloud Thierry
Published: May 2005

Membrane proteins were separated by high resolution two‐dimensional (2‐D) electrophoresis. On isoelectric focusing (IEF) with immobilized pH gradients severe protein losses in the resulting 2‐D map were observed when compared with carrier ampholyte‐based IEF. This has been noticed for two different biological systems, namely the chloroplast envelope of spinach and the endocytic vesicles from Dictyostelium discoideum. The possible mechanisms of these losses on immobilized pH gradients are discussed.
Low molecular weight proteins secreted by peritoneal macrophages obtained from 2,3,7,8‐tetrabromodibenzo‐p‐dioxin‐treated NMRI mice
Authors: Schlatterer Kathrin , Esmaeli Anoosh , Chandra Angelika , Hofmann Dieter , Lerch Anja , Schlatterer Bert , Demirhan Ilhan , Chandra Prakash
Published: May 2005

The protein secretion patterns in a macrophage‐like cell line (CBrD), established from the peritoneal cells of NMRI mice treated with the dioxin analog 2,3,7,8‐tetrabromodibenzo‐p‐dioxin (TBrDD), were analyzed by high resolution two‐dimensional gel electrophoresis (2‐D PAGE), and compared to the pattern of proteins secreted by control macrophages which were intraperitoneally activated by bacterial lipopolysacchride. The most striking alterations were observed in the low molecular range. The transformed cells encode a number of low molecular mass proteins (10–20 kDa) which were not detected in control cells under identical experimental conditions. The protein pattern with respect to isoelectric point, molecular weight, optical density (OD) and area of the spot (in mm2) has been depicted by computer analysis in relation to a standardized spot outline and the spot's background (in OD). It is concluded that the transformation of murine peritoneal macrophages by TBrDD leads to an upregulation of proteins, in particular of low‐molecular‐weight proteins.
Mapping and identification of Brucella melitensis proteins by two‐dimensional electrophoresis and microsequencing
Authors: Teixeira‐Gomes Ana P. , Cloeckaert Axel , Bézard Guy , Dubray Gérard , Zygmunt Michel S.
Published: May 2005

Two‐dimensional (2‐D) gel electrophoresis was used to map Brucella melitensis proteins. The 2‐D proteins map of B. melitensis B115 revealed 595 silverstained protein spots separated by both isoelectric point and molecular mass. Twenty‐five proteins were identified either by immunoblotting using monoclonal antibodies (MAbs) or by N‐terminal microsequencing. The protein spots identified by MAbs were the 89 kDa outer membrane protein, DnaK, bacterioferritin, CP24, and BP26. Some spots were identified by N‐terminal microsequencing as proteins whose sequences had been reported previously from Brucella, such as three heat‐shock proteins, namely DnaK, GroEL and GroES; bacterioferritin; Cu‐Zn superoxide dismutase; and the 50S ribosomal protein L7/L12. Other proteins had amino acid sequences homologous with those of various proteins from other bacteria found in protein databases: ClpP; the 10K‐S protein; the ORFU phosphoprotein; succinyl‐CoA synthetase alpha subunit; an inorganic pyrophosphatase; the Fe and/or Mn superoxide dismutase; the nucleoside diphosphate kinase, an amino acid ABC type transporter, and an electron transfer flavoprotein small subunit. Seven proteins were identified with N‐terminal sequences not yet reported in databases. The 2‐D map established in this study will be the basis for comparative studies of protein expression in Brucella.
Electrophoretic analysis of cyanide depletion by Pseudomonas alcaligenes
Authors: Zaugg Spencer E. , Davidson Robert A. , Walker Jeffrey C. , Walker Edward B.
Published: May 2005

Bacterial‐facilitated depletion of cyanide is under development for remediation of heap leach operations in the gold mining industry. Capillary electrophoresis was found to be a powerful tool for quantifying cyanide depletion. Changes in cyanide concentration in aqueous suspensions of Pseudomonas alcaligenes bacteria and cyanide at eleveated pH were easily monitored by capillary electrophoresis. The resulting data can be used to study rates of cyanide depletion by this strain of bacteria. Concentrations of these bacteria at 105 cells/mL were found to reduce cyanide from 100 ppm to less than 8 ppm in four days. In addition, other ions of interest in cyanide metabolism, such as formate, can be simultaneously analyzed. Direct UV detection of cyanide at 192 nm further simplifies the analytical method for these ions.
Capillary zone electrophoresis of nitrophenols with off‐line isotachophoretic sample pretreatment
Authors: Kaniansky Dušan , Krčmová Eva , Madajová Vlasta , Masár Marián
Published: May 2005

Preparative capillary isotachophoresis (ITP) operating in a discontinuous frationation mode was studied as a sample pretreatment technique for capillary zone electrophoresis (CZE) trace analysis of a group of ten nitrophenols. Different separation mechanisms employed by these capillary electrophoresis techniques made possible a group isolation of the studied analytes by ITP while their CZE resolutions based on differences in the interactions with polyvinylpyrrolidone (PVP) provided suitable conditions for a final analytical evaluation. Experiments with model and practical water samples revealed high and reproducible recovery rates of the ITP pretreatment for nitrophenols when sample loadability limits of the preparative ITP equipment were met. A main disadvantage of the studied ITP‐CZE combination was an inefficient use of the pretreated sample as only 0.5% of the fraction containing nitrophenols was used in the final CZE step. Despite these limitations associated with the CZE sample injection, the concentration limit of detection (LOD) for nitrophenols in practical water samples could be reduced to 2–8 ppb concentrations (photometric absorbance detector operating at a 254 nm wavelength) when 200 μL sample volumes were taken for the ITP pretreatment. This was accompanied by an efficient sample clean‐up as no other trace ionic constituents originating from the samples were detected on CZE.
Determination of ethylenediaminetetraacetic acid as the nickel chelate in environmental water by solid‐phase extraction and capillary electrophoresis/tandem mass spectrometry
Authors: Sheppard Robin L. , Henion Jack
Published: May 2005

An automated extraction procedure using solid‐phase extraction (SPE) disk technology was developed for the qualitative and quantitative determination of ethylenediaminetetraacetic acid (EDTA) in water samples. The procedure involves conversion of all free and chelated EDTA present into the nickel EDTA chelate followed by extraction on strong anion exchange extraction disks. A 5 mL water sample is extracted and concentrated to 30 μL. This extract is then analyzed by capillary electrophoresis (CE) using ion spraytandem mass spectrometry (MS) for selective detection. An amine‐coated capillary column is used to separate anions using negative voltage and high electroosmotic flow. A self‐aligning liquid junction is used as the CE/MS interface. The sample is injected using field amplification for enhanced concentration detection limits down to 0.15 μg/L. This detection limit is about five times lower than any report we have found in the literature and provides the unique specificity afforded by mass spectrometry. Total sample preparation and run time was forty minutes per sample, including evaporation steps and capillary rinsing between runs. A tray of 24 vials, including samples and standards, was analyzed for these experiments and was easily finished within one day. These experiments demonstrate that CE/MS can be a rugged, quantitative technique, which, with proper sample preparation and focusing techniques, can compete with the concentration sensitivity of established analytical methods.
Very alkaline immobilized pH gradients for two‐dimensional electrophoresis of ribosomal and nuclear proteins
Authors: Görg Angelika , Obermaier Christian , Boguth Günther , Csordas Adam , Diaz Jean‐Jacques , Madjar Jean‐Jacques
Published: May 2005

Basic proteins normally lost by the cathodic drift of carrier ampholyte focusing, or separated by NEPHGE with limited reproducibility, could be well separated by two‐dimensional (2‐D) electrophoresis under equilibrium conditions using immobilized pH gradients (IPGs) 4–10 and 6–10 using a previously published protocol (Görg et al., Electrophoresis 1988, 9, 531–546). In the present study we have extended the pH gradient to pH 12 with IPGs 8–12, 9–12 and 10–12 for the analysis of very basic proteins. Different optimization steps with respect to pH engineering, gel composition and running conditions, such as substitution of acrylamide by dimethylacrylamide and addition of isopropanol with and without methylcellulose to the IPG rehydration solution (in order to suppress the reverse electroosmotic flow) were necessary to obtain highly reproducible 2‐D patterns of ribosomal proteins from HeLa cells and mouse liver. Histones from chicken erythrocyte nuclei as well as total cell extracts of erythrocytes were also successfully separated under steady‐state conditions. Due to the selectivity of isoelectric focusing in IPG 9–12, where the more acidic proteins abandon the gel, the tedious procedure of nuclei preparation prior to histone extraction can be omitted.
Towards design and comparison of World Wide Web‐accessible myocardial two‐dimensional gel electrophoresis protein databases
Authors: Plei Klaus‐Peter , Sander Stefan , Oswald Helmut , Regitz‐Zagrosek Vera , Fleck Eckart
Published: May 2005

In addition to the recently published HEART‐2DPAGE – a myocardial World Wide Web‐accessible 2‐DE gel protein database – the usage and installation of software tools are described with regard to the hard‐ and software environments. Further, access to the HEART‐2DPAGE from other two‐dimensional electrophoresis (2‐DE) databases using name or accession code of a protein is now available. Moreover, database images, published in the myocardial HSC‐2DPAGE and HEART‐2DPAGE databases are compared. Using the warping tool of the common image processing system Khoros the database images are matched and added in order to visualize the effects of warping. The application of such image processing tools is aimed at improving the comparability of protein spot patterns of different gel images available through the net.
The assay development of a molecular marker for transmissible spongiform encephalopathies
Authors: Lee Kelvin H. , Harrington Michael G.
Published: May 2005

The transmissible spongiform encephalopathies are a group of neurodegenerative diseases which include Creutzfeldt‐Jakob disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE) in cattle. Two‐dimensional electrophoresis of proteins was previously used to identify two marker proteins, 130/131, which are selectively present in the cerebrospinal fluid (CSF) of patients with CJD and not in patients with other dementias. The recent characterization of these proteins by amino acid sequencing has identified them as members of the 14‐3‐3 family of proteins. Polyclonal antibodies against 14‐3‐3 (all isoforms), 14‐3‐3γ, 14‐3‐3β, and 14‐3‐3θ are immunoreactive with a 30 kDa marker band from CJD CSF. Silver staining of two‐dimensional electrophoresis separated BSE CSF proteins does not identify a similar marker. However, 14‐3‐3 immunoreactivity is found in cattle CSF when these proteins are blotted to polyvinylidene difluoride but not when blotted to nitrocellulose.
Two‐dimensional electrophoretic analysis of human breast carcinoma proteins: Mapping of proteins that bind to the SH3 domain of mixed lineage kinase MLK2
Authors: Rasmussen Richele K. , Ji Hong , Eddes James S. , Moritz Robert L. , Reid Gavin E. , Simpson Richard J. , Dorow Donna S.
Published: May 2005

MLK2, a member of the mixed lineage kinase (MLK) family of protein kinases, first reported by Dorow et al. (Eur. J. Biochem. 1993, 213, 701–710), comprises several distinct structural domains including an src homology‐3 (SH3) domain, a kinase catalytic domain, a unique domain containing two leucine zipper motifs, a polybasic sequence, and a cdc42/rac interactive binding motif. Each of these domains has been shown in other systems to be associated with a specific type of protein interaction in the regulation of cellular signal transduction. To study the role of MLK2 in recruiting specific substrates, we constructed a recombinant cDNA encoding the N‐terminal 100 amino acids of MLK2 (MLK2N), including the SH3 domain (residues 23–77), fused to glutathione S‐transferase. This fusion protein was expressed in Escherichia coli, purified using gluthathione‐Sepharose affinity chromatography and employed in an affinity approach to isolate MLK2‐SH3 domain binding proteins from lysates of 35S‐labelled MDA‐MB231 human breast tumour cells. Electrophoretic analysis of bound proteins revealed that two low‐abundance proteins with a molecular weights (Mr) of ˜ 31 500 and ˜ 34 000, bound consistently to the MLK2N protein. To establish accurately the Mr / isoelectric point (pI) loci of these MLK2‐SH3 domain binding proteins, a number of abundant proteins in a two‐dimensional electrophoresis (2‐DE) master gel were identified to serve as triangulation marker points. Proteins were identified by (i) direct Edman degradation following electroblotting onto polyvinylidene difluoride (PVDF) membranes, (ii) Edman degradation of peptides generated by in‐gel proteolysis and fractionation by rapid (˜ 12 min)˜ microbore column (2.1 mm ID) reversed‐phase high performance liquid chromatography (HPLC), (iii) mass spectrometric methods including peptide‐mass fingerprinting and electrospray (ESI) – mass spectrometry (MS)‐MS utilizing capillary (0.2–0.3 mm ID) column chromatography, or (iv) immunoblot analysis. Using this information, a preliminary 2‐DE protein database for the human breast carcinoma cell line MDA‐MB231, comprising 21 identified proteins, has been constructed and can be accessed via the World Wide Web (URL address: www.ludwig.edu.au/www/jpsl/jpslhome.html).
Length and sequence variation in D7S22 (g3) alleles studied by high resolution length measurements and nucleotide sequencing
Authors: Andreassen Rune , Olaisen Bjørnar
Published: May 2005

In a study of DNA sequence and length variation in the repeat array of small D7S22 alleles, 100 alleles typed as the common 14 repeat allele (14R) and 92 rare ones were selected for further characterization. A polymerase chain reaction (PCR) based allele length measurement method revealed a discontinuous distribution of alleles. The 92 rare alleles were grouped by their number of repeats. All, except four 6R alleles were distributed within the 11R–19R allele groups. The 14Rs revealed no further length variation while 7 out of the 92 rare alleles showed small length deviations from the other alleles within their respective groups. Nucleotide sequencing of the repeat array was performed in 17 alleles selected from each of the nine allele groups. The micro length variation within allele groups was caused by the presence of either 33, 36 or 37 bp repeats in given positions. A comparison of three 14Rs revealed no further sequence variation between these. Nine out of the fourteen repeats in the 14R differed in sequence and/or size. Based on this difference the repeat array sequence was converted into a code of different variant repeats. The 6R showed a variant repeat code quite unlike that of the 14R, while the encoded allele structure of the other rare alleles suggested that most of them may have evolved from a 14R allele by deletion or duplication of repeat units. Nucleotide sequencing of progenitor and mutant in a D7S22 de novo mutation as well as typing in a polymorphic site near the repeat array suggested that the event was an intra‐allelic deletion of exactly three repeats. The present findings indicate that the 14R is ancestral to most rare small alleles, and that mutations in small alleles most often are intra‐allelic events leading to a change in bp size equal to an integer number of repeats.
Capillary zone electrophoresis of oligonucleotides in isoelectric buffers and against a stationary pH gradient
Authors: Stoyanov Alexander V. , Gelfi Cecilia , Righetti Pier Giorgio
Published: May 2005

Capillary zone electrophoresis of oligonucleotides in a background electrolyte of two different types of stationary buffers is proposed: single, isoelectric amphoteres and focused carrier ampholytes. In the first case, two zwitterionic molecules are evaluated: lysine and histidine. Although the former has a five times higher buffering power (β) at the pI (9.74) than the latter (pI 7.47), due to the favorable ΔpK value (1.6 vs. 3) and thus should be the preferred species, a new parameter for evaluating the performance of isoelectric buffers is proposed: the β/λ ratio, i.e., the ratio between the buffering power and its conductivity. Ideal buffers are those with the highest β/λ ratio, since this allows delivering very high voltage gradients with minimal Joule effects. Since the pI of Lys is situated in a pH region (9.74) where bulk water begins to conduct, whereas His has a pI close to neutrality, the β/λ ratio is more favorable for His than for Lys. In the second case (zone electrophoresis of oligonucleotides against a preformed pH gradient), it is shown that migration against a pH 6.5–10 Pharmalyte carrier ampholyte pH gradient offers a unique analyte resolution. This is possibly due to two effects: (i) When injected at the alkaline extreme (ca. pH 10) of the pH gradient, the oligonucleotide zones undergo a stacking effect, with consequent zone sharpening, due to modulation of their free mobility via protonation of the ‐OH group (enolate ion) in the hetero aromatic rings of G and T, which undergo a lactam‐lactim transition. (ii) As the zones migrate down the pH gradient, they transit through a pH 6.5–8.5 zone where, for Pharmalytes, the β/λ ratio reaches a maximum and is constant as well. This last condition allows high voltage gradients (typically 1000 V/cm, even in 75 μm capillaries) to be delivered, thus greatly reducing the analysis time and maintaining peak sharpness, due to limited diffusion.
Capillary electrophoresis of glycosaminoglycan‐derived disaccharides: Application to stability studies of glycosaminoglycan chitosan complexes
Authors: Denuzière Anne , Taverna Myriam , Ferrier Danielle , Domard Alain
Published: May 2005

Capillary zone electrophoresis (CZE) was used to separate the disaccharides produced by chondroitinase digestion of chondroitin sulfates. The main disaccharides formed upon depolymerization have identical charge and mass. Baseline resolution of these two compounds was achieved by selecting appropriate concentration and pH of a borate buffer. Validation of the method showed a good linearity of the response and a very satisfactory reproducibility of migration times with a relative standard deviation (RSD) of less than 0.4%. The reproducibility of peak areas was improved by using an internal standardization. The addition of cinnamic acid (internal standard) to the incubation medium allowed us to perform kinetic measurements of the depolymerization while keeping a baseline resolution of the two main disaccharides analyzed during the complete digestion course even when their concentration in the incubation medium increased. Application of this method to the comparison of the rate of hydrolysis of chondroitin sulfate and of a complex associating chondroitin sulfate with chitosan showed clearly that, at the physiological pH, chitosan protected the chondroitin sulfate from depolymerization. This phenomenon was more pronounced as the pH of the incubation medium was far from the optimum pH activity of the chondroitinase.
Sample matrix effects on glycopeptide stability by high performance capillary electrophoresis
Authors: Rush Robert S. , Boss Hollis J. , Katta Viswanatham , Rohde Michael F.
Published: May 2005

High performance capillary electrophoresis (CE) of glycoprotein digests frequently reveals extensive microheterogeneity associated with specific protein glycosylation sites. The choice of the sample matrix can influence the electrophoretic migration time, peak shape and resolution, as well as the physical stability of the product glycopeptides. Acetic acid is a frequently employed sample matrix for both capillary electrophoresis and electrospray ionization‐mass spectrometry (ESI‐MS). Acetic acid appears to enhance the spontaneous hydrolysis of sialic acids from the nonreducing termini of glycopeptides in a time‐ and concentration‐dependent manner, even at 5°C, as evidenced by changes in the electrophoretic mobility and ESI‐MS spectra of the resulting glycopeptides. The observed parallel electrophoretic mobility changes for specific glycoforms are consistent with the induction of peptide structure with time. Asialoglycopeptide mobilities were stable in acetic acid. Electrophoretic mobilities can be stabilized with propionic acid sample matrix with no apparent structural changes observed by ESI‐MS within 31 h. Migration time reproducibility was in the range of 0.1% relative standard deviation (N = 7) with excellent peak shapes and enhanced glycopeptide resolution.
Characterization of thioredoxins by sodium dodecyl sulfate‐slab gel electrophoresis and high performance capillary electrophoresis
Authors: Bunik Victoria , Shoubnikova Alexandra , Bisswanger Hans , Follmann Hartmut
Published: May 2005

Disulfide containing proteins – thioredoxins from E.coli and pig heart mitochondria – were characterized by sodium dodecyl sulfate (SDS)‐electrophoresis and high performance capillary electrophoresis (HPCE). Following the mitochondrial thioredoxin samples at different stages of purification, we found that their electrophoretic patterns vary, dependent on the redox condition of isolation, preparation of the samples for SDS‐electrophoresis, and sample storage. All these factors influenced the relative intensities of several protein bands with thioredoxin‐like mobility, whereas the sample storage also resulted in the appearance of SDS‐ and dithiothreitol (DTT)‐resistant high molecular mass forms, probably thioredoxin dimers. The multiple forms of the thioredoxin from pig heart mitochondria in SDS‐electrophoresis might be dependent on the oxidation state of the protein cysteine residues. A commercial preparation of the thioredoxin from E.coli did not exhibit any changes in mobility in SDS gels whether the sample was prepared with or without DTT. After the final purification step no correlation was found between mitochondrial thioredoxin activity, determined in the insulin assay, and its purity in SDS‐electrophoresis. A correlation was, however, found when analyzing the thioredoxin by HPCE. The latter approach demonstrated the heterogeneity of the thioredoxin samples homogeneous on SDS electrophoresis, only one of the several HPCE peaks being active in the insulin assay. Also, thioredoxin from E.coli, homogeneous on SDS‐electrophoresis, was found heterogeneous on HPCE. The peak corresponding to the insulin‐dependent thioredoxin activity was split into two by DTT treatment, suggesting that redox transformations of thioredoxin could be followed by HPCE

Presented at the “Elektrophorese Forum ”︁96” meeting of the German Electrophoresis Society, held at the Technical University Munich, October 23–25, 1996

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Isolation of rat hepatic peroxisomes by means of immune free flow electrophoresis
Authors: Völkl Alfred , Mohr Heribert , Weber Gerhard , Fahimi H. Dariush
Published: May 2005

Rat hepatic peroxisomes (PO) were separated from other cell organelles by free flow electrophoresis (FFE) in combination with immunocomplexing PO prior to FFE with an antibody directed against the cytoplasmic aspect of the peroxisomal membrane protein PMP 70. This novel approach is based on a method termed antigen‐specific electrophoretic cell separation (ASECS) which was originally introduced for the isolation of human T and B lymphocyte subpopulations by Hansen and Hannig (J. Immunol. Methods 1982, 51, 197–208). We adapted this technique to PO isolation from a crude peroxisomal fraction, streamlining it by the following modifications: (i) The sandwich‐technique recommended to further lower a negative surface charge was renounced. (ii) Instead, the pH of the electrophoresis buffer was raised from 7.2 to 8.0, thus minimizing the electrophoretic mobility of the particles immunocomplexed due to the fact that the isoelectric point (pI) of IgG molecules is close to pH 8.0. PO isolated by this modification, referred to as immune free flow electrophoresis (IFFE), are as pure, intact, and structurally well‐preserved as are highly purified PO obtained by density gradient centrifugation. The technique is currently applied for the isolation of peroxisomal subpopulations that are difficult to obtain by means of density gradient centrifugation

Presented at the “Elektrophorese Forum ”︁96” meeting of the German Electrophoresis Society, held at the Technical University Munich, October 23–25, 1996

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A new method to assign immunodetected spots in the complex two‐dimensional electrophoresis pattern
Authors: Zeindl‐Eberhart Evelyn , Jungblut Peter R. , Rabes Hartmut M.
Published: May 2005

A new, easy method for the immunodetection of specific antigens in two‐dimensional electrophoresis (2‐DE) is described. Areas of 2‐DE gels containing antigens of interest are electrophoretically transferred to polyvinylidene difluoride membranes, immunostained with specific antibodies using Fast Red or 5‐bromo‐4‐chloro‐3‐indolyl phosphate/nitro blue tetrazolium as detection systems and counterstained with Coomassie Brilliant Blue. In contrast to conventional methods, it is possible to use this procedure to exactly assign immunoreacting proteins on a single blot to their corresponding and surrounding blue‐stained protein spots.
Three approaches to enantiomer separation of β‐adrenergic antagonists by capillary electrochromatography
Authors: Nilsson Staffan , Schweitz Leif , Petersson Maria
Published: May 2005

Three different capillary electrochromatographic methods for the enantiomer separation of β‐adrenergic antagonists (acebutolol, alprenolol, atenolol, metoprolol, pindolol, prenalterol, and propranolol) were applied using different cyclodextrins (β‐cyclodextrin, carboxymethyl‐β‐cyclodextrin and dimethyl‐β‐cyclodextrin) added to the electrolyte, a cross‐linked protein‐gel (cellobiohydrolase I) and a molecularly imprinted ((R)‐enantiomer of propranolol) superporous polymer as chiral selectors. Through use of these different separation strategies, all the β‐adrenergic antagonists studied could be resolved into their enantiomers, although the three methods were carried out without extensive optimization. The protein and molecularly imprinted phases gave the highest selectivities whereas employing cyclodextrins resulted in the highest separation efficiency. Proteins and cyclodextrins are primarily natural products, albeit the cyclodextrins can be derivatized. In contrast, the molecularly imprinted chiral stationary phase can be highly customized when produced.
Histamine‐modified β‐cyclodextrins for the enantiomeric separation of dansyl‐amino acids in capillary electrophoresis
Authors: Galaverna Gianni , Corradini Roberto , Dossena Arnaldo , Marchelli Rosangela , Vecchio Graziella
Published: May 2005

Two novel monosubstituted β‐cyclodextrins (CD) bearing the histamine moiety linked to the upper CD rim, either through the amino group or the imidazole nitrogen 1N, CD‐hm and CD‐mh, were successfully used as chiral selectors in capillary electrophoresis for the enantiomeric discrimination of dansyl (Dns)‐amino acids. Good results were obtained by using low concentrations of the selectors (1–3 mM). The effect of pH on the chiral discrimination was studied in order to modulate the number and the position of the positive charges. By increasing the pH from 5 to 7.5, chiral discrimination decreased along with the deprotonation of the imidazolyl moiety. Inversion of the migration order was observed with the two CDs, depending on the relative position of the charged moieties on the upper rim. Ion pair interaction coupled to inclusion complexation seems to account for the discrimination process. The effects of the temperature, CD concentration and capillary length on chiral resolution were also examined.
Separation of synthesized enantiomers and diastereomers of thiino‐[4,3‐b]pyrrole‐2,3‐dione derivatives and 2H‐thiopyrans by capillary electrophoresis
Authors: Blitzke Thorsten , Wilde Horst , Vogt Carla
Published: May 2005

Capillary electrophoresis was used to separate diastereomers and enantiomers of synthesized thiopyrans and thiinopyrroles. Separation conditions were optimized by varying the buffer parameters, i.e., kind and concentration of chiral selector, pH, main buffering component, micelle forming additives, and content of organic solvents. The interaction of thiopyrans and thiinopyrroles with β‐ and γ‐cyclodextrins results in the separation of enantiomers and diastereomers. The magnitude of the resolution depends on the spatial requirements of the different substituents of the analytes and the dimensions of the cyclodextrin cavity. For well‐separated enantiomers or diastereomers the identification and characterization of degradation products was possible.
Enantioselective determination of 3,4‐methylene‐dioxymethamphetamine and two of its metabolites in human urine by cyclodextrin‐modified capillary zone electrophoresis
Authors: Lanz Matthias , Brenneisen Rudolf , Thormann Wolfgang
Published: May 2005

Using capillary zone electrophoresis with a phosphate buffer at pH 2.5 containing 30 mM (2‐hydroxypropyl)‐β‐cyclodextrin as chiral selector, the simultaneous separation of the enantiomers of 3,4‐methylenedioxymethamphetamine (MDMA or Ecstasy) and its two metabolites 4‐hydroxy‐3‐methoxymethamphetamine (HMMA) and 3,4‐methylenedioxyamphetamine (MDA) in human urine is reported. The assay described is based upon enzymatic hydrolysis of conjugated HMMA (major urinary metabolite) and solid‐phase extraction followed by injection of a few nL of the extract onto a 50 μm internal diameter (ID) fused‐silica capillary of 60 cm length. Solutes are detected via on‐column absorbance at 195 nm. For 375 ng/mL drug levels, intraday and interday imprecision is < 4%. With 5 mL urine samples, the detection limit is in the 20–50 ng/mL range. Via analysis of the urines of two patients, the metabolism of MDMA is demonstrated to be enantioselective, with significantly higher urinary amounts of R‐(−)‐MDMA being excreted compared to S‐(+)‐MDMA. Within 72h after drug administration one patient was determined to excrete 42.28 and 10.16% of the racemic MDMA dose (1.5 mg/kg body weight) as R‐(−) and S‐(+)‐MDMA enantiomers, respectively. Corresponding values for the second subject were found to be 28.63 and 9.34%. The metabolism of the enantiomers of the two metabolites showed interindividual differences. The first and second detected HMMA enantiomers represented 3.79 and 5.42% (first subject) and 8.51 and 4.36% (second), respectively, of the administered MDMA dose. For the MDA enantiomers, corresponding values were 2.44, 1.76, 0.75, and 0.79%, respectively.
A role for Edman degradation in proteome studies
Authors: Gooley Andrew A. , Ou Keli , Russell Jim , Wilkins Marc R. , Sanchez Jean‐Charles , Hochstrasser Denis F. , Williams Keith L.
Published: May 2005

Advances in protein database design and the software used to access the sequence data has led to progress in using protein attributes such as amino acid composition and peptide masses to identify proteins separated by two‐dimensional electrophoresis. However, Edman degradation remains the principal technique for protein identification and it presents a significant bottle‐neck in the progress towards rapid protein identification. Simple modifications to the sequencing hardware, which automate the delivery of protein spots into the sequencer, and parallel sequencing of the protein spots represent a significant advance in the use of Edman degradation to rapidly generate the powerful protein attribute, an N‐terminal sequence tag.
Models of mobility‐shift assay of complexes between dimerizing protein and DNA
Authors: Cann John R.
Published: May 2005

The theory of mass transport coupled to macromolecular interactions under chemical kinetic control forms the basis of four different models of the electrophoretic mobility‐shift assay of complexes formed between dimerizing proteins and DNA. The theory of mass action was applied to the set of simultaneous dimerization (either simple or ligand‐induced) and DNA‐binding reactions in order to fix the initial equilibrium composition of mixtures to be assayed. Theoretical mobility‐shift patterns were obtained for a range of protein concentrations at constant DNA concentration by numerical solution of the set of simultaneous transport‐reaction equations appropriate for each model. In those cases in which dimerization in solution is modeled (including heterodimerization), analysis of the peaks in the patterns provides apparent binding constants, which, when extrapolated to infinite dilution of protein, yield acceptable estimates of equilibrium constants. Those for binding of dimer are products of two or three equilibrium constants, from which the equilibrium binding constant can be extracted, privided that dimerization and, where required, ligand‐binding constants are determined by independent physicochemical methods. Dimerization of protein when bound to DNA is distinctive in that extrapolation to infinite dilution of protein is not required.