bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of polyanionic macromolecular carrier poly‐(N‐vinylpyrrolidone‐co‐maleic acid) and its bioconjugates by capillary electrophoresis
Authors: Györffy Erika , Pató János , Horváth Anikó , Érchegyi Judit , Teplán István , Kéri György , Idei Miklós 
Published: May 2005

AbstractAninonic carrier poly(N‐vinylpyrrolidone‐co‐maleic acid) and its conjugates, prepared with coupling of 2‐cyano‐3‐hydroxy‐5‐amino‐2‐pentenoyc(4‐trifluoromethyl anilide) or (6′,7′‐dimethyl‐1′‐quinoxalinyl)‐4‐(2′ amino) acetanilide to the carrier, were analyzed by capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) in the following buffers: 0.25N triethylammonium phosphate (TEAP); sodium dodecyl sulfate (SDS; 25–150 mM) in TEAP (pH 2.25–6.30); 0.1 M Na‐borate buffer (pH range 7–11) and SDS (25–150 mM) in Na‐borate buffer (pH range 7–11). The presence of strong carboxyl groups (dissociated even at pH 2.25) on the polymer chain was proved by the CZE method. It was also proved by potentometric titration that carboxyls with a wide range of acidity were on the polymer chain. CZE was able to differentiate among the analytes possessing carboxyl groups of different acidic strengths at pH 2.25. These components were not distinguished by CZE at high pH va use (11.0). Interaction between the analyte and SDS affected the separation at this pH, and hence good resolution was obtained by MEKC. Informative separations were achieved both for the carrier and the conjugates in TEAP buffer at pH 2.25 by the CZE method. Optimal separation was achieved in borate buffer containign 75 mM SDS at pH 11.0 for the carrier and at pH 7.7 for the conjugates in MEKC.
Wheat cultivar discrimination by capillary electrophoresis of gliadins in isoelectric buffers
Authors: Capelli Laura , Forlani Fabio , Perini Fabio , Guerrieri Nicoletta , Cerletti Paolo , Righetti Pier Giorgio 
Published: May 2005

AbstractA modified method is reported for screening of wheat cultivars: capillary zone electrophoresis of gliadins in isoelectric buffers. Previously published procedures recommended a 100 mM phosphate buffer, supplemented with 0.05% hydroxypropylmethylcellulose and 20% acetonitrile, in uncoated capillaries. Due to the very high conductivity of such a buffer (4.7 mmhos at 25°C) high speed separations (10—12 min analysis time at 800 V/cm) could only be elicited in 20 μm internal diameter (ID) capillaries, at the expense of sensitivity. In the present report, we optimized the background electrolyte as follows: 40 mM aspartic acid (pH = pI = 2.77) in the presence of 7 M urea and 0.5% short‐chain hydroxyethylcellulose (Mn 27000 Da; apparent pH 3.9 in 7 M urea). As an alternative recipe, the same isoelectric buffer can be supplemented with a mixed organic solvent composed of 4 M urea and 20% acetonitrile (apparent pH 3.66). Due to the much lower conductivity (0.7 mmhos), separations can be carried out at 1000 V/cm in only 10 min, but in larger bore capillaries (50 μm ID), ensuring a five‐times higher sensitivity. The gliadin patterns thus obtained are species‐specific and allow easy identification of all cultivars tested of both durum and bread wheat. No adsorption of proteins to the silica wall seems to occur and high reproducibility in peak areas and transit times is obtained.
Affinity capillary electrophoresis: A physical‐organic tool for studying interactions in biomolecular recognition
Authors: Colton Ian J. , Carbeck Jeffrey D. , Rao Jianghong , Whitesides George M. 
Published: May 2005

AbstractAffinity capillary electrophoresis (ACE) is a technique that is used to measure the binding affinity of receptors to neutral and charged ligands. ACE experiments are based on differences in the values of electrophoretic mobility of free and bound receptor. Scatchard analysis of the fraction of bound receptor, at equilibrium, as a function of the concentration of free ligand yields the dissociation constant of the receptor‐ligand complex. ACE experiments are most conveniently performed on fused silica capillaries using a negatively charged receptor (molecular mass < 50 kDa) and increasing concentrations of a low molecular weight, charged ligand in the running buffer. ACE experiments that involve high molecular weight receptors may require the use of running buffers containing zwitterionic additives to prevent the receptors from adsorbing appreciably to the wall of the capillary. This review emphasizes ACE experiments performed with two model systems: bovine carbonic anhydrase II (BCA II) with arylsulfonamide ligands and vancomycin (Van), a glycopeptide antibiotic, with D‐Ala‐D‐Ala (DADA)‐based peptidyl ligands. Dissociation constants determined from ACE experiments performed with charged receptors and ligands can often be rationalized using electrostatic arguments. The combination of differently charged derivatives of proteins ‐ protein charge ladders ‐ and ACE is a physical‐organic tool that is used to investigate electrostatic effects. Variations of ACE experiments have been used to estimate the charge of Van and of proteins in solution, and to determine the effect of the association of Van to Ac2KDADA on the value of pKa of its N‐terminal amino group.
Tetranucleotide frequencies in microbial genomes
Authors: Noble Peter A. , Citek Robert W. , Ogunseitan Oladele A. 
Published: May 2005

AbstractA computational strategy for determining the variability of long DNA sequences in microbial genomes is described. Composite portraits of bacterial genomes were obtained by computing tetranucleotide frequencies of sections of genomic DNA, converting the frequencies to color images and arranging the images according to their genetic position. The resulting images revealed that the tetranucleotide frequencies of genomic DNA sequences are highly conserved. Sections that were visibly different from those of the rest of the genome contained ribosomal RNA, bacteriophage, or undefined coding regions and had corresponding differences in the variances of tetranucleotide frequencies and GC content. Comparison of nine completely sequenced bacterial genomes showed that there was a nonlinear relationship between variances of the tetranucleotide frequencies and GC content, with the highest variances occurring in DNA sequences with low GC contents (less than 0.30 mol). High variances were also observed in DNA sequences having high GC contents (greater than 0.60 mol), but to a much lesser extent than DNA sequences having low GC contents. Differences in the tetranucleotide frequencies may be due to the mechanisms of intercellular genetic exchange and/or processes involved in maintaining intracellular genetic stability. Identification of sections that were different from those of the rest of the genome may provide information on the evolution and plasticity of bacterial genomes.
Illuminating the agent of syphilis: The Treponema pallidum genome project (minireview)
Authors: Norris Steven J. , Fraser Claire M. , Weinstock George M. 
Published: May 2005

AbstractAs the causative agent of the common sexually transmitted disease syphilis and a fastidious, microaerophilic obligate parasite of humans, Treponema pallidum subsp. pallidum is one of the few prominent infectious agents that has not been cultured continuously in vitro. T. pallidum therefore represents an attractive candidate for genomic sequencing. Preliminary sequence results from the 1.13 million base pair genome are consistent with the expected limited metabolic capabilities of this spirochete, but indicate that the bacterium may express toxins and surface proteins that have not been identified previously.
Electrophoresis‐related protein modification: Alkylation of carboxy residues revealed by mass spectrometry
Authors: Haebel Sophie , Albrecht Tanja , Sparbier Katrin , Walden Peter , Körner Roman , Steup Martin 
Published: May 2005

AbstractIn recent years, the combination of gel electrophoresis and mass spectrometry has developed into one of the most powerful approaches for the analysis of proteins. However, a number of gel electrophoresis‐induced protein modifications have been described. Cystein is the most endangered amino acid readily reacting with mercaptoethanol or free acrylamide. In the course of studies on glucan phosphorylases (E.C.2.4.1.1) from white potato (Solanum tuberosum L.) and the T cell receptor, we noticed that proteolytic peptides from these proteins can undergo an unexpected modification, giving rise to a mass increment of 14 Da. By post‐source decay (PSD) analysis the modification was identified as methylation of the glutamic acid side chain carboxyl group. The methylation takes place during Coomassie blue staining of proteins if both trichloroacetic acid and methanol are present in the staining solution. Replacement of methanol by ethanol under otherwise unchanged conditions results in ethylation of the peptides. The in vitro alkylation was further studied by using synthetic peptides which contain, at different positions: glutamic acid, aspartic acid or the corresponding amides. The kinetic analysis of the observed reactions revealed that glutamic acid is preferentially methylated. The three other amino acid residues can be methylated but with a velocity at least one order of magnitude lower. Although these modifications complicate the interpretation of the spectra, they provide valuable structural information.
Dual chiral recognition system involving cyclodextrin derivatives in capillary electrophoresis II. Enhancement of enantioselectivity
Authors: Jakubetz Hansjörg , Juza Markus , Schurig Volker 
Published: May 2005

AbstractThe enantiomer separation of hexobarbital was investigated by open tubular electrochromatography (OTEC) using the chiral stationary phase (CSP) CHIRASIL‐DEX (a permethylated β‐cyclodextrin covalently linked to a dimethyl‐polysiloxane) and by cyclodextrin‐electrokinetic chromatogaphy (CD‐EKC) using anionic β‐cyclodextrin‐sulfo‐n‐propyl ether (SPE‐β‐CD) and cationic β‐cyclodextrin‐2‐hydroxy‐3‐trimethylammoniumpropyl ether chloride (HTAP‐β‐CD) added to the running buffer. By employing two chiral selectors, the enantiomer separation of hexobarbital was then studied simultaneously by OTEC with CHIRASIL‐DEX and by CD‐EKC with either SPE‐β‐CD or HTAP‐β‐CD in the dual chiral recognition mode. In conjunction with CHIRASIL‐DEX, anionic SPE‐β‐CD decreased the chiral separation factor α due to compensation of enantioselectivity whereas the cationic additive HTAP‐β‐CD increased the chiral separation factor α due to enhancement of enantioselectivity. It is concluded that CHIRASIL‐DEX imparts an opposite enantioselectivity to the enantiomers of hexobarbital as compared to the charged CDs SPE‐β‐CD and HTAP‐β‐CD. Unusual peak broadening phenomena are observed in the dual chiral recognition system comprised of CHIRASIL‐DEX and HTAP‐β‐CD. The possible consequences of accidental dual chiral recognition systems caused by wall stacking effects of the mobile phase additives onto the inner surface of the capillary column are discussed.
Evidence of intra‐ and extracellular modifications of monoclonal IgG polypeptide chains generating charge heterogeneity
Authors: Mimura Yusuke , Nakamura Kazuyuki , Tanaka Tatehiko , Fujimoto Masanori 
Published: May 2005

AbstractThe heavy and light chains of IgG monoclonal antibodies (mAbs) can be shown to be heterogeneous, with respect to isoelectric points, when analyzed by two‐dimensional electrophoresis (2‐DE). The molecular basis for this charge heterogeneity has not been clearly defined but it has been suggested that it could be due, in part, to differences in glycosylation. To investigate this possibility we have compared the 2‐DE pattern of glycosylated and aglycosylated forms of the mouse IgG1 mAb (1B7‐11), produced in vitro in the presence and absence of tunicamycin. Charge heterogeneity was shown not to be a consequence of glycosylation status. Intracellular and secreted IgG mAbs were also analyzed to investigate the time course of change in charge properties of the heavy and light chains. The charge heterogeneity was found to be generated intracellularly, and alterations in charge properties could be induced during incubation under physiological conditions. Semilogarithmic plots of the density of the principal heavy and light chain spots against incubation time showed linear relationships, suggesting that the charge shifts result from a first‐order reaction. The semilogarithmic plot for the light chain correlated well with the time after IgG synthesis. These results suggest that the charge heterogeneity of an IgG mAb is due to intra‐ and extracellular modifications of the polypeptide chains which reflect “aging” of antibody molecules.
Actin‐binding proteins in mouse C2 myoblasts and myotubes: A combination of affinity chromatography and two‐dimensional gel electrophoresis
Authors: Coumans Joëlle V. F. , dos Remedios Cristobal G. 
Published: May 2005

AbstractThis paper analyzes proteins expressed in a mouse muscle precursor cell line (C2 myoblasts) and compares them with those observed in differentiated myotubes from the same cell line. We observed hundreds of proteins in myoblasts using IPG two‐dimensional gel electrophoresis but this number is greatly reduced using Mini‐Leak (divinylsulfone‐activated agarose) affinity chromatography. Two kinds of affinity columns were prepared. One contained a chemically modified monomeric actin bound to the affinity matrix. The second matrix contained a high‐affinity actin‐binding protein (DNase I) which was bound to the actin Mini‐Leak column to block specific sites on actin. Actin‐binding proteins in homogenates of myoblasts or myotubes were passed through the affinity columns and eluted under high salt conditions. The Mini‐Leak affinity medium itself appeared to have little ability to bind proteins. Our two‐dimensional (2‐D) gels identified a small number of proteins and we are currently focusing our attention on a particular protein spot which could correspond to cofilin. Comparison of myoblast and myotube proteins using affinity chromatography shows no qualitative, clearly identifiable differences but the analysis is still in progress. These findings are discussed in relation to reports in which the myoblast‐myotube transformation was associated with the up‐regulation or de novo synthesis of more than ten proteins.
Improved protein solubility in two‐dimensional electrophoresis using tributyl phosphine as reducing agent
Authors: Herbert Ben R. , Molloy Mark P. , Gooley Andrew A. , Walsh Bradley J. , Bryson Warren G. , Williams Keith L. 
Published: May 2005

AbstractIn this study, dithiothreitol was replaced by tributyl phosphine as the reducing agent in both the sample solution for the first‐dimensional isoelectric focusing and during the immobilised pH gradient (IPG) equilibration procedure. Tributyl phosphine improves protein solubility during isoelectric focusing, which results in shorter run times and increased resolution. Tributyl phosphine is nonionic and thus does not migrate in the IPG, therefore maintaining reducing conditions during the course of the first‐dimensional separation. The increased solubility provided by the maintenance of reducing conditions gives improved focusing and decreased horizontal streaking on the subsequent second‐dimension gel. The use of tributyl phosphine in the equilibration step allows the procedure to be simplified, incorporating reduction and alkylation in a single step. This is possible because, in direct contrast to dithiothreitol (DTT), tributyl phosphine does not contain a free thiol and therefore does not react with thiol‐specific alkylating reagents.
Direct cloning of polymerase chain reaction products into the pinpoint Xa1‐T vector protein expression system
Authors: Brown Louise , Yin Jian Lin , Hambly Brett 
Published: May 2005

AbstractExpression of recombinant proteins is an important method for the characterisation of the structure and function of proteins. However, many expression methods can be difficult, time‐consuming and lead to low protein yields. The Promega Pinpoint Xa1‐T vector system is a unique, one‐step cloning method that allows the direct insertion of polymerase chain reaction (PCR) fragments into the expression vector. We describe our experience of the use of this system to clone and express three proteins (8–12 kDa) directly from their PCR products. The proteins are expressed as fusion proteins with a 13 kDa biotinylated tag that can be used for detection of the expressed protein and affinity purification. In our case, the yield was greater than 20 mg per litre of culture. Expressed proteins were purified by Q‐Sepharose anion‐exchange chromatography and reverse‐phase high‐performance liquid chromatography (HPLC) instead of the conventional method of avidin‐biotin affinity chromatography. The Pinpoint vector proved to be a relatively simple and fast protein expression technique suitable for wide application for expressing recombinant proteins.
Sample handling for proteome analysis
Authors: Staudenmann Werner , Hatt Paola Dainese , Hoving Sjouke , Lehmann Anton , Kertesz Michael , James Peter 
Published: May 2005

AbstractThe main factor limiting the sensitivity range for the identification of proteins isolated by two‐dimensional (2‐D) electrophoresis is sample handling: protein detection limits on the gel, losses during extraction and digestion, as well as interference of gel contaminants and detergents with the mass spectrometry (MS) detection increasing background noise. At the one hundred picomole level, losses are fairly negligible but when the amounts drop below 1 picomole (and subfemtomole peptide detection limits have been reported recently by MS), the losses become a critical point. In order to extend proteome analysis to include very low copy number proteins, methods must be developed to minimize losses and handling steps, maximize digestion and extraction yields, as well as to lower chemical noise. We present several methods that we have developed in our laboratory to: (i) increase the amount of material available in a sodium dodecyl sulfate (SDS)‐free form which does not require staining, (ii) increase protein extraction and digestion yields and lower the contamination by autoproteolytic products, and (iii) allow direct modification of the peptide mixture to generate sequence tags.
Identification of gel‐separated proteins by liquid chromatography‐electrospray tandem mass spectrometry: Comparison of methods and their limitations
Authors: Haynes Paul A. , Fripp Natelaine , Aebersold Ruedi 
Published: May 2005

AbstractWe have compared several different experimental systems currently in use in our laboratory for protein identification by high performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC‐ESI‐MS/MS) after sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE). The efficiency of peptide recovery from trypsin‐digested gel bands or electroblotted membrane slices was examined using 35S‐labeled yeast proteins, and was found to be in excess of 80%. A dilution series of two standard proteins, bovine serum albumin (BSA) and carbonic anhydrase (CA), was analyzed by HPLC‐ESI‐MS/MS to determine what amount of protein could be loaded onto a gel and successfully identified, a measure we refer to as the practical detection limit. We were able to identify both standards at the 500 ng level in samples prepared from gel slices, using either a regular spray or a flow‐split microspray HPLC‐MS interface system. In samples prepared from membrane pieces, carbonic anhydrase was also identified at the 500 ng level, while bovine serum albumin could only be identified in samples of more than 1000 ng. In general, protein identification was slightly better in samples prepared from gels rather than membranes. A dilution series of lesser amounts of the same standard proteins was also analyzed using a gradient capillary LC system and we were able to successfully identify 50 ng of carbonic anhydrase and 100 ng of BSA.
Protein indentification using mass spectrometric information
Authors: Fenyö David , Qin Jun , Chait Brian T. 
Published: May 2005

AbstractIn an effort to gain an understanding of the value of the information in different mass spectrometric measurements for protein identification, the genome of Saccharomyces cerevisiae was studied in silico. We calculate how constraining the knowledge of the mass of a proteolytic peptide is as a function of mass and mass accuracy. We also assess the value for protein identification of additional information concerning a proteolytic peptide, including the presence or absence of a given amino acid, the number of exchangeable hydrogens, the N‐terminal sequence, and the masses of mass spectrometrically produced fragment ions. Knowledge of the relative value of these different constraints is useful in the design of efficient protein identification experiments. Finally, we describe a software tool, PepFrag, for searching protein and DNA sequence databases that can use different types of mass spectrometric information to restrict the search.
Separation and characterization of humic acids and metal humates by electrophoretic methods
Authors: Keuth Ulrich , Leinenbach Anette , Beck Horst P. , Wagner Horst 
Published: May 2005

AbstractA modified buffer for the separation of humic acids (HA) by capillary zone electrophoresis (CZE) was studied. The addition of hydroxycarboxylic acids to borate buffer makes a more efficient separation of HA possible. Two sharp peaks are obtained. Preparative isolation of these two fractions can be reached by applying the conditions used in CZE to free‐flow electrophoresis (FFE). The fractions obtained were characterized by repetition of the CZE procedure. Also metal complexes of HA were investigated by electrophoretic methods. In the presence of metal cations, HA form stable complexes of different electrophoretic mobility. These complexes can be separated by free‐flow isotachophoresis (FF‐ITP). The different fractions are analyzed by CZE, ultraviolet‐visible and atomic absorption spectrometry. Significant changes in both the ITP‐pattern of the FFE and the ‘humic hump’ in CZE demonstrate the influence of complexation on the macromolecules.
Automated free‐solution isotachophoresis: Instrumentation and fractionation of human serum proteins
Authors: Böttcher Alfred , Möllers Christoph , Lackner Karl J. , Schmitz Gerd 
Published: May 2005

AbstractAn automated free‐solution isotachophoresis system (FS‐ITP) for preparative fractionation of biopolymers is described, operated in a batch mode. The dimension of the separation chamber allows an up to 1200‐fold higher sample load compared to separation in capillaries of 180 μm inner diameter as used in analytical capillary isotachophoresis (C‐ITP). The preparative capacity of the system is within the milligram range. The method is fully compatible with analytical C‐ITP, which is essential for preparative‐scale isotachophoresis with regard to optimization of electrolyte systems and the search for suitable spacers. As a model application the fractionation of human serum proteins is reported. The collected fractions were analyzed by C‐ITP and agarose gel electrophoresis.