bluuebunny/crossref_metadata_2025_split huggingface dataset:
Capillary electroseparations of enkephalin‐related peptides and protein kinase A peptide substrates
Authors: Thorsteinsdóttir Margrét , Beijersten Ingegerd , Westerlund Douglas 
Published: May 2005

AbstractThe separations of enkephalin‐related peptides and protein kinase A peptide substrates, with the common structural feature ‐Arg‐Arg‐X‐Ser‐Val‐, were studied in micellar electrokinetic chromatography (MEKC) systems and compared with the capillary zone electrophoresis (CZE) mode. The influence of the magnitude and the direction of the electroosmotic flow on the selectivity was studied. Reversed electroosmosis was obtained by adding a hydrophobic amine, dimethyldodecylamine, to the background electrolyte; the amine forms cationic micelles with a low critical micelle concentration (0.3 mM). The neutral micellar agent, Brij 35, competes with the amine for adsorption sites on the capillary surface decreasing the reversed electroosmosis. In such a system, mixed cationic micelles are formed to which the peptides were not distributed at low pH, but an improved resolution was obtained due to the effects on electroosmosis. In systems containing the less hydrophobic amine dimethyloctylamine, in which probably no mixed micelles are formed, an improved separation of protein kinase A peptide substrates was obtained due to distribution to Brij 35 micelles. In separations of enkephalins, a high pH gave very low efficiencies due to surface‐analyte interactions, and the best CZE separations were obtained at low pH. Changes in migration order were observed in the pH range 2–3, possibly due to differences in peptide pKa values or conformation changes of the peptides. The enkephalins were only to a small extent distributed to the Brij 35 micelles, but this improved the separation at pH 2 compared to the CZE mode.
One‐ and two‐dimensional histone separations in acidic gels: Usefulness of methylene blue‐driven photopolymerization
Authors: Rabilloud Thierry , Girardot Valérie , Lawrence Jean‐Jacques 
Published: May 2005

AbstractWe tested the recently introduced methylene blue‐toluene sulfinate‐diphenyl‐iodonium polymerization system in order to prepare acetic acid‐urea‐Triton X‐100 gels for histone separations. When compared to standard persulfate‐based initiators, this system exhibited several advantages. First, the polymerization proceeds at a much faster rate but is easily controlled since it is light‐dependent. Second, no prerunning of the gel was required, since there is no oxidizing molecule able to introduce artifacts. Moreover, this procedure produces gels presenting cleaner backgrounds with silver staining. The ability of the procedure to carry out high resolution two‐dimensional electrophoresis of histones was also examined. High resolution two‐dimensional gels, using photopolymerized acidic gels as the first or second dimension were obtained.
Assessing the stability of cystatin/cysteine proteinase complexes using mildly‐denaturing gelatin‐polyacrylamide gel electrophoresis
Authors: Michaud Dominique , Cantin Line , Raworth David A. , Vrain Thierry C. 
Published: May 2005

AbstractA method for assessing the stability of cystatin/cysteine proteinase complexes using mildly‐denaturing gelatin‐polyacrylamide gel electrophoresis (gelatin‐PAGE) is described. As suggested by the use of well‐known cystatins (human stefins A and B, and oryzacysatins I and II) and the plant cysteine proteinase papain, the ability of cystatin/cysteine proteinase complexes to remain stable during electrophoresis is associated with the degree of affinity between the enzyme and the inhibitor (and inversely associated with the Ki values), at least with the disulfide bond‐lacking cystatins. Complexes with Ki values ≥ 10−8 M (weak interactions) are partly or completely dissociated under the conditions used, while those with lower Ki values (strong interactions) remain stable. As shown by the differential effects of two plant cystatins, oryzacystatins I and II, against a cysteine proteinase present in crude (complex) extracts from a plant pest — the two‐spotted spider mite (Tetranychus urticae Koch), the gelatin‐PAGE procedure is suitable for studying the ability of cystatins to form highly stable complexes with cysteine proteinases, without the need for prior purification steps. Considering the well‐recognized potential of proteinase inhibitors for pest and pathogen control, this analytical approach will be useful for rapidly assesing the respective potential of various cystatins for protection of plants, animals, and humans.
Analysis of ε‐N‐2‐furoylmethyl‐L‐lysine (furosine) in dried milk by capillary electrophoresis with controlled electroosmotic flow using N,N,N′,N′‐tetramethyl‐1,3‐butanediamine in the running electrolyte solution
Authors: Corradini Danilo , Cannarsa Gianfranco , Corradini Claudio , Nicoletti Isabella , Pizzoferrato Laura , Vivanti Vittorio 
Published: May 2005

AbstractThe electrophoresis of ε‐N‐2‐furoylmethyl‐L‐lysine (furosine) was studied in an attempt to develop a method for the identification and quantitation of this compound in processed food. The effect of pH and composition of electrolyte solution on both the electrophoretic migration of furosine and the electroosmotic flow in a bare fused‐silica capillary of 75 μm internal diameter was investigated. We demonstrate that the addition, to the running electrolyte solution, of N,N,N′,N′‐tetramethyl‐1,3‐butanediamine (TMBD) at concentrations ranging from 20 to 80 mM improves peak efficiency and can be used to modulate the migration time of furosine by controlling the electroosmotic flow which is reversed from cathodic to anodic. In a sample of dried milk subjected to a long period of storage under controlled conditions, furosine could be efficiently and reproducibly separated and quantitated by employing as the running electrolyte 60 mM TMBD titrated to pH 2.5. Capillary electrophoresis is a promising technique for the rapid identification and quantitation of furosine in processed food.
Ultrafast high resolution separation of large DNA fragments by pulsed‐field capillary electrophoresis
Authors: Kim Yongseong , Morris Michael D. 
Published: May 2005

AbstractPulsed‐field capillary electrophoresis (PFCE) in buffers containing ultradilute polymer solutions in used to separate long chain dsDNA in less than 4 min. Separations are shown to work with chain lengths below 10 kbp and greater than 1.5 Mbp. Several pulse protocols have been examined. If running time is to be minimized, a field inversion with higher peak amplitude in the forward direction than in the reverse, but with equal pulse durations, provides the best resolution. Other protocols can provide higher resolution, but only with longer resolution. Other protocols can provide higher resolution, but only with longer running times.
Preparative isoelectric focusing and Joule effect: A purification cell that contains a heat exchanger
Authors: Araque Arturo , Jaugey Jean , Javet Philippe 
Published: May 2005

AbstractReproducibility in protein purification by preparative isoelectric focusing depends greatly on temperature control during the separation process. A preparative apparatus is described, including a heat exchanger between compartments with isoelectric membranes. The selectivity of the isoelectric membranes was optimized as a function of isoelectric points of the separated proteins. At 2500 V and 60 W, 0.3 g of horse heart myoglobin from 0.2 g of whale skeletal muscle myoglobin could be separated in 1 h. At a total load of 2 g protein, 97% of bovine hemoglobin (2% initial concentration) was purified from bovine serum albumin (0.15%).
Biotinylated trypsin and its application as a sensitive, versatile probe for the detection and characterisation of an ovine chondrocyte serine proteinase inhibitor using Western blotting
Authors: Rodgers Kenneth John , Melrose James , Ghosh Peter 
Published: May 2005

AbstractBiotinylated trypsin (bT) was used as a probe on Western blots of 10–20% polyacrylamide gradient Tris‐Tricine gels for the detection of serine proteinase inhibitors (SPIs) isolated from extracts of ovine articular cartilage and from chondrocyte conditioned culture medium. The major cartilage SPI, a 58 kDa glycoprotein, was purified by sequential Sephacryl S–300 gel permeation, concanavalin A affinity, anion exchange and Superose 12 fast protein liquid chromatography (FPLC). A low molecular weight SPI of ∼ 6 kDa was also detectable in cartilage extracts after prolonged storage at 4°C and following affinity chromatography on immobilised chymotrypsin, suggesting that the 6 kDa inhibitor may have arisen from proteolytic modification of the 58 kDa SPI. Analysis of chondrocyte conditioned culture medium using the bT detection system revealed that the major SPI present was the 6 kDa species although a small amount of the 58 kDa SPI was also detectable. The large molecular weight ‘native’ cartilage SPI was distinct from ovine α1‐proteinase inhibitor (α1‐PI) when examined by native polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF). The 6 kDa cartilage SPI was indistinguishable from basic pancreatic trypsin inhibitor (BPTI, aprotinin) following sodium dodecyl sulfate (SDS)‐PAGE under reduced and nonreduced conditions. Amino terminal sequencing of the 6 kDa cartilage SPI revealed a strong (90%) homology with BPTL.
Distinction between genotypes of Lupinus species by sodium dodecyl sulphate‐gel electrophoresis and by capillary electrophoresis
Authors: Pollard Naomi J. , Wrigley Colin W. , Bekes Ferenc , Aumatell Anthony , Macritchie Finlay 
Published: May 2005

AbstractEffective distinction was achieved among a wide range of lupin grain samples by either sodium dodecyl sulphate (SDS)‐gel electrophoresis or capillary electrophoresis, based on grain‐protein composition. Capillary electrophoresis was faster (< 1 h) and provided slightly greater distinction between the samples. On the other hand, SDS‐gel electrophoresis could provide a greater throughput of samples in a 24 h period. Either technique could be used successfully to distinguish between lupin species and cultivars for taxonomic analysis or seed identification.
Capillary electrophoresis and electrochemical detection of underivatized oligo‐ and polysaccharides with surfactant‐controlled electroosmotic flow
Authors: Zhou Weihong , Baldwin Richard P. 
Published: May 2005

AbstractComplex polysaccharide mixtures were analyzed directly without derivatization by capillary zone electrophoresis in strongly alkaline solutions and electrochemical detection at a Cu electrode. The positively charged surfactant cetyltrimethylammonium bromide was included in the electrophoresis medium in order to reverse the electroosmotic flow and permit elution to be in order of increasing polysaccharide size. Carbohydrate samples analyzed by this approach included linear maltoses, enzymatically hydrolyzed starch, and commercially available dextrans of up to an average molecular weight of 18 300. Detection by constant‐potential oxidation at a Cu electrode was very sensitive, with detection limits for individual carbohydrates generally below the femtomole level.
Indirect UV detection of carbohydrates in capillary zone electrophoresis by using tryptophan as a marker
Authors: Lu Bing , Westerlund Douglas 
Published: May 2005

AbstractA rapid and sensitive method for the analysis of sugars was developed by capillary zone electrophoresis (CZE) with indirect UV detection. A mixture of nine mono‐ and oligosaccharides was separated in about 20 minutes on a fused silica capillary in a highly alkaline (pH > 12) background electrolyte (BGE). With tryptophan as the marker detected at 280 nm the femtomole level detection of sugars was possible. The system was optimized by studies on the buffer pH, the marker concentration, as well as the inner diameter of the capillary. The indirect detection of sugars was also performed with a cationic marker, N‐benzylcinchonidium chloride (BCDC). However, the detection sensitivity was much lower in comparison to the tryptophan marker system.
Analysis of macrolide antibiotics by capillary electrophoresis
Authors: Flurer Cheryl L. 
Published: May 2005

AbstractCapillary electrophoresis was utilized in the study of the macrolide antibiotics (i.e. pharmaceutical glycoconjugates) clarithromycin, erythromycin, oleandomycin, troleandomycin, and spiramycin. In order to assist in analyte solubilization, two buffer systems using acetonitrile were developed. The first system involved 30 mM sodium cholate and 20% acetonitrile in 80 mM sodium phosphate, pH 6. This buffer permitted the baseline resolution of all five glycoconjugated antibiotics. In addition, erythromycin was separated from its derivatives estolate and ethylsuccinate. In the absence of surfactants, a higher acetonitrile quantity, 65%, was used in the second buffer system, with 35 mM sodium phosphate, pH 6. Selectivity between oleandomycin and clarithromycin was reversed in this system compared to the cholate buffer, indicating solute interaction with the cholate micelles in the previous system. Calibration linearity and detection sensitivity were improved in the high acetonitrile buffer, due to decreased background absorbance. It was demonstrated that both buffer systems can be utilized for the visualization of minor components that may be present in bulk pharmaceuticals.
Two‐dimensional gel electrophoresis of Escherichia coli homogenates: The Escherichia coli SWISS‐2DPAGE database
Authors: Pasquali Christian , Frutiger Séverine , Wilkins Marc R. , Hughes Graham J. , Appel Ron D. , Bairoch Amos , Schaller Dominique , Sanchez Jean‐Charles , Hochstrasser Denis F. 
Published: May 2005

AbstractNumerous Escherichia coli proteins have already been characterized by two‐dimensional gel electrophoresis (2‐D PAGE), using carrier ampholytes in the first dimension (VanBogelen, R. A., Sankar, P., Clark, R. L., Bogan, J. A. and Neidhardt, F. C., Electrophoresis 1992, 13, 1014–1054). We present here a reference protein map of E. coli obtained with immobilized pH gradients (IPG) and available in a SWISS‐2DPAGE format. Out of the protein spots identified in the E. coli gene protein database by Neidhardt's group, 153 have been identified on the E.coli SWISS‐2DPAGE database map by gel comparison and most of them were confirmed either by the analysis of amino acid composition (AAC) and/or N‐terminal microsequencing. Additionally, five as yet unsequenced proteins were found. The E. coli SWISS‐2DPAGE database is part of the ExPASy molecular biology server accessible through the Word Wide Web network.
Identification of human myocardial proteins separated by two‐dimensional electrophoresis with matrix‐assisted laser desorption/ionization mass spectrometry
Authors: Thiede Bernd , Otto Albrecht , Zimny‐Arndt Ursula , Müller Eva‐Christina , Jungblut Peter 
Published: May 2005

AbstractDisease‐associated proteins separated by two‐dimensional electrophoresis (2‐DE) are often in the femtomole range. Identification of 2‐DE separated proteins by sequencing and amino acid analysis is limited to the lower picomole range. Identification down to the femtomole range can be achieved by matrix‐assisted laser desorption ionization‐mass spectrometry (MALDI‐MS). We optimized the measurement by MALDI‐MS for the analysis of proteolytic digests of 2‐DE‐separated proteins. The direct analysis of peptide mixtures can be used for rapid and sensitive protein identification. In some cases, more information about the protein can be obtained by separating the peptides by micro high‐performance liquid chromatography (HPLC) before employing MALDI‐MS analysis. More peptides are found than in the mixture, and comparison of HPLC patterns can reveal some differences to be post‐translational modifications of proteins, even in the case of identical peptide mass fingerprints. Furthermore, carboxy‐terminal sequencing by on‐target carboxypeptidase P digestion can be used to confirm the obtained result without the need for more material. The search program FRAGFIT was modified and renamed FRAGMOD to include the modifications of methionine and tryptophan oxidation and alkylation of cysteine by acrylamide into the mass search. By applying this procedure, 15 proteins were identified, among them two different putative phosphorylated forms of two proteins, a putative N‐terminal blocking group and four dilated cardiomyopathy‐associated proteins. The resulting approach for the identification may be used for large‐scale investigations of 2‐DE‐separated proteins.
The effects of polymer properties on DNA separations by capillary electrophoresis in uncross‐linked polymer solutions
Authors: Barron Annelise E. , Sunada Wade M. , Blanch Harvey W. 
Published: May 2005

AbstractLow‐viscosity, aqueous solutions of hydrophilic linear polymers have been shown to be useful for the separation of DNA restriction fragments by capillary electrophoresis (CE). However, the choice of polymer type, size, and concentration remains largely empirical, because the mechanism of high‐field electrophoretic DNA separations in polymer solutions is not well understood. To assist in elucidating the mechanism of DNA separation, we experimentally investigated the effects of polymer properties such as stiffness (persistence length), average molecular mass, polydispersity, and hydrophilicity on the separation of DNA ranging from 72 bp to 23 kbp. This was accomplished by comparing the results of DNA separations obtained by counter‐migration CE in dilute and semidilute solutions of linear polyacrylamide (PAA), hydroxyethylcellulose (HEC), and hydroxypropylcellulose (HPC) polymers of several different average molecular masses.
Direct isolation of proteins from sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and analysis by electrospray‐ionization mass spectrometry
Authors: Schuhmacher Martina , Glocker Michael O. , Wunderlin Markus , Przybylski Michael 
Published: May 2005

AbstractA new method for the isolation of proteins separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), using electroelution with a modified buffer system, is described. The method is suitable for direct characterization by electrospray‐ionization mass spectrometry (ESI‐MS), or alternatively matrix‐assisted laser desorption‐ionization (MALDI), no additional purification steps being required. Following separation by SDS‐PAGE, proteins were stained with Coomassie Blue, and isolated by electroelution using SDS‐free ammonium acetate (pH 2.5) as elution buffer. The polarity of the electroelution system was reversed, which, upon in situ dissociation of protein‐SDS complexes, resulted in migration of free proteins to the cathode under the conditions employed. Recovery rates of 25–58% were determined for model proteins. Analyses by ESI‐MS provided exact molecular weight determinations of isolated proteins and of protein mixtures not resolved by SDS‐PAGE. Essentially SDS‐free molecular ions were obtained, except for bovine serum albumin with one SDS‐adduct. Charge distribution of molecular ions were similar to those of the native proteins. The effects of β‐mercaptoethanol (β‐ME) and dithiothreitol (DTT) during electrophoresis were studied with hen egg white lysozyme, revealing the formation of mixed disulfide adducts between proteins and reducing agents. In a first bioanalytical application, hemofiltrate proteins from a patient with uremia were separated by SDS‐PAGE. An ESI‐MS analysis of the proteins isolated from the two most intensive gel bands enabled exact molecular weight determinations, and demonstrated that a gel band of ca. 17 kDa consisted of two different proteins.
Mass spectrometric characterization of glycosylated interferon‐γ variants separated by gel electrophoresis
Authors: Mørtz Ejvind , Sareneva Timo , Haebel Sophie , Julkunen Ilkka , Roepstorff Peter 
Published: May 2005

AbstractGlycosylated proteins in polyacrylamide gels were characterized by matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS) and glycosidase digestion. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of natural, human interferon‐γ (IFN‐γ) showed two glycosylated variants with apparent molecular masses of 20 and 24 kDa. MALDI‐MS of the intact IFN‐γ, electroeluted from the two bands, confirmed that these correspond to IFN‐γ molecules glycosylated at one or both of the two potential glycosylation sites, respectively. The peptide map obtained by MALDI‐MS after digestion in the gel covers 92% of the IFN‐γ sequence and revealed an N‐terminal pyroglutamate residue and one oxidized methionine residue. One glycosylated peptide was detected after treatment of the peptide mixture with neuraminidase, and the carbohydrate structure partially elucidated by sequential glycosidase digestion monitored by MALDI‐MS. A second glycosylated peptide, due to a very heterogeneous glycan structure, could only be observed after separation of the peptides by high performance liquid chromatography (HPLC).
Field and pore size dependence of the electrophoretic mobility of DNA: A combination of fluorescence recovery after photobleaching and electric birefringence measurements
Authors: Tinland Bernard , Pernodet Nadine , Weill Gilbert 
Published: May 2005

AbstractBy combining an electrophoretic cell with a setup of fluorescence recovery after photobleaching (FRAP) we can measure the electrophoretic mobility μ of double‐stranded lambda DNA in agarose gel as a function of electric field E and gel concentration C. Mobility varies linearly with the field in agreement with the biased reptation model with fluctuations. The slopes are analyzed in term of orientation and compared with birefringence results. The mobility extrapolated at zero field follows the prediction of the reptation theory; we deduced the variation of the pore size with the agarose concentration. With a special use of our setup, we measure directly the free‐mobility μ0 of the DNA.
Mechanisms of retardation of rigid spherical particles with 3 to 1,085 nm radius in capillary electrophoresis, using buffered polyacrylamide (molecular weight 5 × 106) solutions
Authors: Radko Sergey P. , Chrambach Andreas 
Published: May 2005

AbstractSubjecting particles in the size range of 3 to 1085 nm radius (R) to capillary electrophoresis in buffered solution of entangled uncrosslinked polyacrylamide (Mr 5 × 106), it was found that particle size‐dependent retardation (“molecular sieving”) becomes electric field‐ and particle size range‐dependent once the particle size exceeds 15–20 nm in radius. The field strength dependence of the retardation coefficient [KR = d(log mobility)/d(polymer concentration] and the positive or negative sign of dKR/dR suggest the existence of two different mechanisms of molecular sieving depending on the particle size range: particles with diameters less than the screening length (or blob size) of the polymer network are thought to penetrate into the available spaces within a discontinuous polymer network; particles with diameters larger than the screening length (or blob size) of the polymer network are thought to undergo size‐dependent retardation by exerting shear stress against polymer chains, and displacing them, so as to cause local deformations in a continuous polymer network. A limit in the separating capacity of molecular sieving, due to a sharp increase in the rate of band widening with polymer concentration, was found when the value of the retardation coefficient exceeded 60 (mL/g).