bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of small molecules for clinical diagnosis by capillary electrophoresis
Authors: Shihabi Zakariya K. , Friedberg Michael A.
Published: May 2005

The application of capillary electrophoresis (CE) for the analysis of small molecules in clinical research is growing steadily. Initial studies have dealt with separations of standards or compounds in clean matrices. However, later studies dealt with analysis of those compounds in serum, urine or tissues. Great progress has been accomplished in three areas of clinical interest: organic acids, amino acids and drug analysis. The analysis of these compounds by capillary electrophoresis has several distinct advantages: high resolution, simplicity, versatility and especially low operating costs. In many cases, the sample can be injected directly without complex pretreatment. Most of the described methods have been validated for their precision, linearity and accuracy. In forensic toxicology, the CE has been used for drug identification and as a complementary analytical method.
Analytical capillary isotachophoresis of human serum lipoproteins
Authors: Schmitz Gerd , Möllers Christoph , Richter Volker
Published: May 2005

An analytical free flow capillary isotachophoresis (cITP) procedure for the detailed analysis of lipoproteins on commercially available capillary electrophoresis systems has been developed. The technique is based on the specific staining of lipoproteins with the fluorescent lipophilic dye 7‐nitrobenz‐2‐oxa‐1,3‐diazole (NBD)‐ceramide before separation. Prestained lipoprotein samples are applied between leading and terminating buffer and separated into 9 well‐characterized subpopulations according to their electrophoretic mobility in the absence of any molecular sieve effect. High density lipoproteins are separated into three major subpopulations: (i) the fast migrating high density lipoprotein (HDL) subpopulation (α‐HDL, containing mainly apolipoprotein (apo) A‐I and phosphatidylcholine, (ii) the subpopulation with intermediate mobility, consisting of particles rich in cholesterol, apo A‐II, apo E and C apolipoproteins, and (iii) the slow migrating HDL subpopulation (pre‐β‐HDL), containing particles rich in apo A‐I, apo A‐IV. The majority of HDL‐associated lecithin:cholesterol acyltransferase (LCAT) activity is also associated with the last subpopulation. The apo B‐containing lipoproteins can be subdivided into three major functional groups. The first represents chylomicron derived particles and large triglyceride‐rich very low density lipoproteins (VLDL). The second group consists of small VLDL and intermediate density lipoprotein (IDL) particles, and the third group represents the low density lipoproteins (LDL). Results obtained by the isotachophoretic lipoprotein analysis revealed a good correlation in the range of HDL with routinely used techniques, like lipoprotein electrophoresis, HDL‐cholesterol analysis by a precipitation procedure or turbidi‐metric determination of apo A‐I. Similar correlations with other analytical techniques were found for the quantitation of the apo B‐containing lipoproteins. Advantages of the isotachophoretic separation compared to zone electrophoresis are the high resolution combined with small sample volumes. Moreover, lipoprotein analysis can be performed directly from whole serum, plasma, lymph and other biological fluids in a short time. With these characteristics analytical capillary isotachophoresis may be a helpful tool for a fast and reliable automated quantitation of lipoprotein subpopulations in the clinical laboratory.
Urine protein analysis by capillary electrophoresis
Authors: Friedberg Michael A. , Shihabi Zakariya K.
Published: May 2005

Increased concentration of proteins in urine as well as abnormal patterns are seen in many disorders such as various renal disorders and light chain disease. At the wavelength of 214 nm used for detection of the peptide bond, numerous compounds interfere in the analysis of urinary proteins. We show that either adsorptive filtration with a wash step or cold ethanol precipitation are two methods which can eliminate many of the interferences. The wash step is rapid, greatly reduces the interfering substances, and decreases the effect of sample matrix. Both of these methods yield results comparable to the traditional agarose method. Capillary electrophoresis (CE) is faster and more cost‐effective than agarose electrophoresis.
Clinical application of capillary electrophoresis to unconcentrated human urine proteins
Authors: Jenkins Margaret A.
Published: May 2005

Urine protein electrophoresis can be performed by capillary electrophoresis using spun unconcentrated urine diluted with running buffer. The separation which utilises the excellent sensitivity of capillary electrophoresis gives electropherograms similar to those obtained by diluting concentrated urine with buffer. A 72 cm × 50 μm internal diameter (ID) fused silica capillary was used for the analysis which took less than 15 min. Bence Jones protein can be detected from unconcentrated urine over urine protein concentrations ranging from 9.7 g/L to 0.04 g/L. Other clinical patterns, such as glomerular protein‐uria, the presence of intact immunoglobulin and Tamm Horsfall protein can also be detected. The benefit of using unconcentrated urine is the time and cost saved by not concentrating the urine.
Quantification of muscle mitochondrial oxidative phosphorylation enzymes via histochemical staining of blue native polyacrylamide gels
Authors: Zerbetto Elisabetta , Vergani Lodovica , Dabbeni‐Sala Federica
Published: May 2005

Blue native‐polyacrylamide gel electrophoresis is a powerful technique that enables the separation of intact multi‐subunit complexes. However, positive identification of particular enzymes generally requires further separation in a second dimension on a denaturing polyacrylamide gel. Histochemical staining is widely used to demonstrate enzyme activities in tissues, including oxidative phosphorylation enzymes. In this report, we demonstrate that the two techniques can be combined to quantify in situ mitochondrial enzymes, separated on nondenaturing polyacrylamide gels. The method gives quantitative results with human skeletal muscle as well as heart that contains higher mitochondrial numbers. Comparison of muscle from patients with oxidative phosphorylation enzyme deficiencies, such as those of two riboflavin‐responsive patients, before and after vitamin treatment, gives results in agreement with those obtained by analyzing the activity of the mitochondrial enzymes in muscle homogenates.
Capillary zone electrophoresis of oligonucleotides and peptides in isoelectric buffers: Theory and methodology
Authors: Righetti Pier Giorgio , Gelfi Cecilia , Perego Marilena , Stoyanov Alexandre V. , Bossi Allessandra
Published: May 2005

The use of isoelectric buffers in capillary zone electrophoresis is reviewed. Such buffers allow application of extremely high voltage gradients (up to 1000 V/cm in relatively high bore capillary, e.g. 75 to 100 μm internal diameter), permitting separations of the order of a few minutes and thus favoring high resolution due to minimal, diffusion‐driven peak spreading. The fundamental properties of ampholytes are first discussed, such as buffering power (β) as a function of ΔpK. i.e. of the distance between the pI value and neighboring protolytic groups. The highest possible relative β value (= 2) is obtained for amphoteres possessing a ΔpK = 0.6, a condition not met by existing amphoteric species. A novel parameter for ampholyte evaluation is then proposed, namely the β/λ ratio, i.e. the ratio between the β power and conductivity at the pI value. It is additionally shown that the pI is not a constant value, but depends on ampholyte concentration in solution. In addition, at constant concentration, the theoretical pI can change as a function of ΔpK. Isoelectric His and, to a lesser extent, Lys have been found to offer unique separations of oligonucleotides in sieving liquid polymers. In the absense of sieving media, isoelectric Asp, in presence of 7 M urea (apparent pH 3.77), permits unique separations of oligonucleotides having the same length but different nucleotide composition. Isoelectric Asp (pI 2.77 at 50 mM concentration) provides a medium of high resolving power for generating peptide maps. In difficult cases, of coincident titration curves, the pH can be moved up to higher values (e.g. pH 3.0 for 30 mM Asp) thus eliciting separation of unresolved peptides at pH 2.77. This was illustrated by running peptide maps of tryptic digests of human β‐globin chains. Also imino diacetic acid (pI 2.33 at 50 mM concentration) allows generation of high resolution peptide maps.
Affinity capillary electrophoresis
Authors: Rippel Geza , Corstjens Hugo , Billiet Hugo A. H. , Frank Johannes
Published: May 2005

The application of affinity capillary electrophoresis (ACE) to the study of molecular interactions is reviewed. ACE appears to be a sensitive, versatile and convenient tool to obtain reliable data on binding constants and stoichiometries of interacting systems using the Hummel‐Dreyer method and variants thereof. A powerful feature is the possibility to analyze simultaneously the affinity of a large number of compounds for the same ligand, making it a promising tool for the screening of large combinatorial libraries.
Methods for the estimation of binding constants by capillary electrophoresis
Authors: Rundlett Kimber L. , Armstrong Daniel W.
Published: May 2005

Capillary electrophoresis (CE) has developed into a particularly effective means to determine apparent equilibrium constants for molecular association in solution (e.g., to micelles, cyclodextrins, antibiotics, proteins, RNA, DNA. etc.). The various experimental, graphical and mathematical approaches for determining association constants are reviewed. In CE, association constants can be calculated because there is a relationship between substrate concentration and the measured electrophoretic mobility of the solute. Most of the approaches for obtaining association constants by CE are conceptually and mathematically related to one another. Likewise, they are analogous to many spectroscopic techniques that are used for obtaining association constants. The advantages, limitations and proper use of the various CE approaches are examined.
Integrated chip‐based capillary electrophoresis
Authors: Effenhauser Carlo S. , Bruin Gerard J. M. , Paulus Aran
Published: May 2005

Integrated capillary electrophoresis (ICE) is emerging as a new analytical tool allowing fast, automated, miniaturized and multiplexed assays, thus meeting the needs of the pharmaceutical industry in drug development. The current state‐of‐the‐art of ICE is described with an emphasis on the choice of the support material (glass or polymeric materials), electrokinetic fluid handling, and injection and detection issues. Strategies and chip designs for pre‐ or post‐column derivatization, DNA sequencing, on‐line PCR analysis, on‐chip enzymatic sample digestion, fraction isolation, and immunoassays are presented. The review concludes with a brief outlook.
Capillary electrophoresis in clinical and forensic analysis
Authors: von Heeren Franz , Thormann Wolfgang
Published: May 2005

During the past decade, capillary electrophoresis (CE) emerged as a promising, effective and economic approach for separation of a large variety of substances, including those encountered in clinical and forensic analysis. Reliable and automated CE instruments became commercially available and promoted the exploration of an increasing number of CE methods and fields of application. The widespread applicability of CE, its enormous separation power and high‐sensitivity detection schemes make this technology an attractive and promising tool. This review discusses the principles and important aspects of CE‐based assays and provides an overview of the key achievements encountered with CE in clinical and forensic analysis, including those associated with the analysis of serum proteins, hemoglobin variants, drugs and nucleic acids. Validated assays, interesting applications and future trends in clinical and forensic analysis are also discussed.
Receptor‐mediated endocytosis in kidney proximal tubules: Recent advances and hypothesis
Authors: Marshansky Vladimir , Bourgoin Sylvain , Londoño Irene , Bendayan Moïse , Maranda Bruno , Vinay Patrick
Published: May 2005

Preparation of kidney proximal tubules in suspension allows the study of receptor‐mediated endocytosis, protein reabsorption, and traffic of endosomal vesicles. The study of tubular protein transportin vitrocoupled with that of the function of endosomal preparation offers a unique opportunity to investigate a receptor‐mediated endocytosis pathway under physiological and pathological conditions. We assume that receptor‐mediated endocytosis of albumin in kidney proximal tubulesin situandin vitrocan be regulated, on the one hand, by the components of the acidification machinery (V‐type H+‐ATPase, Cl−‐channel and Na+/H+‐exchanger), giving rise to formation and dissipation of a proton gradient in endosomal vesicles, and, on the other hand, by small GTPases of the ADP‐ribosylation factor (Arf)‐family. In this paper we thus analyze the recent advances of the studies of cellular and molecular mechanisms underlying the identification, localization, and function of the acidification machinery (V‐type H+‐ATPase, Cl−‐channel) as well as Arf‐family small GTPases and phospholipase D in the endocytotic pathway of kidney proximal tubules. Also, we explore the possible functional interaction between the acidification machinery and Arf‐family small GTPases. Finally, we propose the hypothesis of the regulation of translocation of Arf‐family small GTPases by an endosomal acidification process and its role during receptor‐mediated endocytosis in kidney proximal tubules. The results of this study will not only enhance our understanding of the receptor‐mediated endocytosis pathway in kidney proximal tubules under physiological conditions but will also have important implications with respect to the functional consequences under some pathological circumstances. Furthermore, it may suggest novel targets and approaches in the prevention and treatment of various diseases (cystic fibrosis, Dent's disease, diabetes and autosomal dominant polycystic kidney disease).
Protein expression profiles in human breast ductal carcinoma and histologically normal tissue
Authors: Bini Luca , Magi Barbara , Marzocchi Barbara , Arcuri Felice , Tripodi Sergio , Cintorino Marcella , Sanchez Jean‐Charles , Frutiger SÉVerine , Hughes Graham , Pallini Vitaliano , Hochstrasser Denis F. , Tosi Piero
Published: May 2005

Reference two‐dimensional (2‐D) gels are presented for human breast ductal carcinoma and histologically normal tissue. Whole biopsy fragments were analyzed, including epithelial and nonepithelial components. Thirty‐five spots have been assigned by gel matching to the human liver SWISS‐2DPAGE reference map and/or to the human primary keratinocyte IPG map from the Danish Center for Human Genome. N‐terminal microsequencing was applied to confirm randomly chosen matching assignments and to identify six new spots. Protein expression profiles in ductal carcinoma and in normal breast tissue appeared to be similar, except for a pattern consisting of 32 spots, which were highly expressed in all carcinoma specimens, and less intense and occasionally undetectable in normal tissue. This difference was statistically significant. Assignment has been obtained for several spots, namely GRP94, GRP78, GRP75, mitochondrial HSP60, calreticulin, protein disulfide isomerase, peptidyl‐prolyl cis‐trans isomerase, collagen‐binding protein 2, fructose bisphosphate aldolase, glyceraldehyde‐3‐phosphate dehydrogenase, thioredoxin, cytochrome c oxidase VA subunit, tubulin β isoform and macrophage migration inhibitory factor (MIF). The cancer‐ and tissue‐specificity of the described pattern was assessed by matching to the Swiss‐2DPAGE human liver, hepatoma, lymphoma, erythroleukemia reference maps. The pattern of 32 spots was found to be indicative of epithelial neoplasia.
Improved detection of P53 mutations in soft tissue tumors using new gel composition for automated nonradioactive analysis of single‐strand conformation polymorphism
Authors: Schneider‐Stock Regine , Haeckel Carsten , Radig Kathrin , Pein Claus‐Dietmar , Roessner Albert
Published: May 2005

We report a new nonradioactive method to detect sequence changes, including single‐base substitutions through shifts in electrophoretic mobility using an automated fluorescence sequencer (ALFexpress, Pharmacia, Biotech) connected to external cooling equipment. Single strands were identified by incorporation of fluorescein‐labeled primers during amplification and subsequent laser detection at the bottom of the gel. The amplified polymerase chain reaction (PCR) products were heat‐denatured and loaded onto a polyacrylamide gel under nondenaturing conditions and strict control of constant low temperature. Peak shifts in the fluorogram indicated mutations. A novel gel composition improved the detection rate for mutations considerably. Automatic analysis of single‐strand conformation polymorphism (SSCP) gels saves time and costs, and is highly reproducible. The method was applied for mutation screening in exon 7 of the p53 tumor suppressor gene in DNA of freshly frozen soft tissue tumors. The mutation spectrum and frequency in exon 7 of the p53 gene are discussed with respect to oncogenesis in soft tissue sarcomas.
Peptides adsorbed on reverse‐phase chromatographic beads as targets for femtomole sequencing by post‐source decay matrix assisted laser desorption ionization‐reflectron time of flight mass spectrometry (MALDI‐RETOF‐MS)
Authors: Gevaert Kris , Demol Hans , Puype Magda , Broekaert Daniël , De Boeck Stefaan , Houthaeve Tony , Vandekerckhove Joël
Published: May 2005

We here describe a procedure for concentrating peptides from solutions by adsorbing them onto reverse‐phase beads that were added to these solutions. The beads are then transferred to the target disc of the matrix assisted laser desorption ionization‐reflectron time of flight (MALDI‐RETOF) mass spectrometer. Because of their hydrophobic nature, these beads cluster in a very small area on the target disc assuring an important concentration step. After drying, peptides are desorbed from the beads by adding a small volume of 50% acetonitrile in 0.1% trifluroacetic acid in water containing the matrix components. Hereby we focus the original amount of peptide material on the target disc on a very small surface, producing highly concentrated peptide‐matrix mixtures. This permits high yield identification and sequence tagging by post‐source‐decay analysis on peptides derived from proteins only available in the femtomole range from one‐dimensional (1‐D) or two‐dimensional (2‐D) gels. The procedure is illustrated by the identification of 38 proteins from human thrombocyte membrane skeletons.
Large‐scale identification of proteins of Haemophilus influenzae by amino acid composition analysis
Authors: Fountoulakis Michael , Juranville Jean‐François , Berndt Peter
Published: May 2005

Two‐dimensional protein maps of microorganisms are useful tools for elucidation and detection of target proteins, a process essential in the development of new pharmaceutical products. We applied amino acid composition analysis, following separation by two‐dimensional gel electrophoresis, for large‐scale identification of proteins of Haemophilus influenzae. H. influenzae is a bacterium of pharmaceutical interest of which the entire genome, comprising approximately 1700 open reading frames, has been sequenced. For amino acid analysis, we used both precolumn derivatization of amino acids followed by reversed‐phase chromatography of the derivatized residues and post‐column derivatization of the residues previously separated on an ion exchanger. The composition analyses derived from both methods allowed the identification of 110 protein spots. The proteins were identified using the AACompIdent software on the ExPASy server accessible via the World Wide Web with a success rate of 52%. In some cases, introduction of the analysis data of 12 residues was sufficient for a correct identification. Proteins which contained an unusually high percentage of one residue could be unambiguously identified. Amino acid composition analysis proved to be an error‐robust, efficient method for protein identification. The method can be practically established in every biochemical laboratory and, complementary to mass spectrometry, represents an important analytical tool for the mapping of the proteomes of organisms of interest.
Analysis of two multiplexed short tandem repeat systems using capillary electrophoresis with multiwavelength fluorescence detection
Authors: Isenberg Alice R. , Allen Ralph O. , Keys Kathleen M. , Smerick Jill B. , Budowle Bruce , McCord Bruce R.
Published: May 2005

A series of experiments was performed to analyze the utility of capillary electrophoresis (CE) with multiwavelength detection capabilities for multiplex typing of short tandem repeat loci. Characteristics of the sieving polymer, hydroxyethylcellulose, which affect resolution of single strand (ss) DNA fragments were examined. Additionally, the effects of denaturant in the polymer system, separation voltage, and analysis temperature were studied to ascertain their effects on DNA separations and capillary lifetime. The use of elevated run temperature (60°C) was found to improve sizing precision, to increase the lifetime of capillaries (100 runs or more per capillary), and to provide runtimes of under 20 min. Finally, 100 individual human DNA samples were typed successfully using CE. The average resolution obtained was 1.4 bases for a 200 base fragment with a standard deviation of sizing of 0.2 bases, allowing all alleles examined to be distinguished clarly

Presented in part at the 7th Annual Frederick Conference on Capillary Electrophoresis. Frederick, MD, USA, October 21–23, 1996

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Theoretical studies on the mobility‐shift assay of protein‐DNA complexes
Authors: Cann John R.
Published: May 2005

The theory of mass transport coupled to revesible macromolecular interactions under chemical kinetic control forms the basis for computer simulation of the electrophoretic mobility‐shift behavior of protein‐DNA complexes. Model systems include (i) specific binding of a univalent protein molecule to a single site on the DNA molecule; (ii) the putative cage effect; (iii) cooperative binding to multiple sites; (iv) formation of looped complexes of 1:1 and 2:1 stoichiometry; (v) noncooperative and cooperative, nonspecific binding modes; and (vi) binding of dimerizing transcriptional factors to response elements of target genes. Favorable comparison of simulated with experimental mobility‐shift behavior indicates that the phenomenological mechanisms, whereby observed mobility‐shift patterns are generated during electrophoresis, are embodies in the theory. These studies have provided guidelines for definitive interpretation of mobility‐shift assays and for the design of experiments to develop a detailed understanding of the particular system under investigation.
Activity staining on polyacrylamide gels of trypsin inhibitors from leaves of sweet potato (Ipomoea batatas L. Lam) varieties
Authors: Hou Wen‐Chi , Lin Yaw‐Huei
Published: May 2005

The failure of activity staining of trypsin inhibitors in crude leaf extracts of sweet potatoe varieties including Tainong 27, Tainong 34, and Tainong 57 on a 15% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gel was prevented by dipping the gels in solutions containing 10–40 mM hydrogen peroxide, 10 mM Tris buffer (pH 7.9) for 30 min before staining.
Fast detection of a (CA)18 microsatellite repeat in the IgE receptor gene by capillary electrophoresis with laser‐induced flurescence detection
Authors: Klepárník Karel , Malá Zdeňka , Haváč Zdeněk , Blažková Michaela , Hollá Lydie , Boček Petr
Published: May 2005

The optimum separation conditions of polymerase chain reaction (PCR) products have been found for high‐speed capillary electrophoresis (CE) with laser‐induced fluorescence (LIF) detection. DNA fragments obtained after PCR amplification of the region covering the (CA)18 microsatellite repeat in nitron 5 of the gene for FcERIβ, a high affinity glycoprotein receptor for IgE, located on chromosome 11 (11q13), were analyzed with the aim of investigating the repeat polymorphism. The results of polyacrylamide slab gel electrophoresis (PAGE), agarose gel electrophoresis, CE with absorbance detector and CE with LIF are compared. The CE with LIF proved to shorten analysis time by a factor of 100 when compared to slab gel electrophoresis. CE‐LIF utilizes a short capillary with an effective length of 6.3 cm and electric field strength from 100 to 550 V/cm. The respective PCR products of sizes from 116 to 210 base pairs (bp) were analyzed in 3 min.
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

Aninonic 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

A 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

Affinity 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

A 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

As 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

In 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

The 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

The 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

This 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

In 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

Expression 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

The 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

We 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

In 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

A 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

An 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.