bluuebunny/crossref_metadata_2025 huggingface dataset:
Two‐dimensional electrophoresis of human urinary proteins following concentration by dye precipitation
Authors: Marshall Thomas , Williams Katherine
Published: May 2005

The use of dye precipitation for concentration of proteins prior to 2‐DE has been demonstrated by application to human urine. The precipitation methods (T. Marshall and K. M. Williams, Clin. Chem. 1993, 39, 2314–2318; T. Marshall et al., Electrophoresis 1995, 16, 28–31) have been adapted for two‐dimensional electrophoresis (2‐DE) by modifying the reagent composition and adding sodium dodecyl sulphate (SDS) to the assay mixture. These modifications extend the precipitation range of the methods and overcome the problem of soluble glycoprotein‐dye complexes. The protein‐dye complex is recovered by centrifugation and dissolved in a small volume of sample denaturing buffer. The dye separates from the protein on 2‐DE (forming a sharp band at the anodal end of the isoelectric focusing gel) so that the positional coordinates of the polypeptides are unaffected by the treatment. Dye precipitation provides a simple, rapid and highly economic method for concentrating urines of low to intermediate protein content (0.02–0.50 g/L) prior to 2‐DE analysis.
Improved two‐dimensional electrophoretic separation of acidic proteins extracted from Norway spruce needles by using immobilized pH gradients
Authors: Davidsen Nina B.
Published: May 2005

An improved method for two‐dimensional electrophoretic separation of acidic proteins extracted from Norway spruce needles is presented. With the use of immobilized pH gradients in the first dimension, this method gave stable separations, with the proteins evently distributed over the entire gel surface. The number of spots in the electrophoretograms have increased fourfold compared to our earlier separations.
Genetic heterogeneity in acatalasemia
Authors: Góth László , Páy Anikó
Published: May 2005

203 bp long products containing exon 4 and its juntions from the catalase gene were generated by polymerase chain reaction (PCR). These products were analyzed by single strand conformational polymorphism (SSCP), heteroduplex formation and nucleotide sisters, their family members and normocatalasemic controls were analyzed. Sequence analyses did not show the G to A point mutation at position 5 of intron 4. This splicing mutation characterizes the Japanese‐type of acatalasemia.
Protein electrophoresis in polyacrylamide gels with templated pores
Authors: Rill Randolph L. , Locke Bruce R. , Liu Yingjie , Dharia Jayesh , Van Winkle David
Published: May 2005

An approach is described for the synthesis of nanostructured hydrogels with defined size channels or pores for separations of biological macromolecules. Polyacrylamide gels (15–20% acrylamide) were cast in the presence of high concentrations (5%–28%) of hydrophilic, macromolecular cosolutes either semirigid, rod‐like polyelectrolytes (short fragments of DNA or xanthan) or spherical micelles of sodium dodecyl sulfate (SDS). Polyanionic cosolutes were then removed by a combination of diffusion and electrophoresis. These processes are expected to yield gels with ‘templated’ channels having dimensions near those of the double helical polymers (diameters ≈︁ 2–3 nm, lengths of 50–200 nm) or pores near the size of the spherical SDS micelles (≈︁ 4–5 nm). This hypothesis was tested by comparing the relative electrophoretic mobilities of proteins (3400 to 43 000 Da) complexed with SDS on templated and conventional gels. Differences in electrophoretic mobilities were observed between all templated gels and control normal gels, demonstrating that the templating process altered the polyacrylamide network. No evidence was found for phase separation of cosolutes from the polyacrylamide network during polymerization. Templated pores are expected to enhance the mobilities of molecules in a particular size range relative to smaller and larger molecules. Gels templated with DNA or xanthan (8–10% final concentration) exhibited little size selectivity, but selectivity was observed for gels templated with SDS (17–20% final concentration). The degree of selectivity of gels templated with SDS varied with polyacrylamide concentration in a manner consistent with the creation of templated pores approximately the size of SDS micelles and larger than the average pore size in a surrounding polyacrylamide network.
Sodium dodecyl sulfate‐polyacrylamide gel electrophoretic method for assessing the quaternary state and comparative thermostability of avidin and streptavidin
Authors: Bayer Edward A. , Ehrlich‐Rogozinski Sarah , Wilchek Meir
Published: May 2005

Avidin, a positively charged egg‐white protein, aggregates extensively when mixed at ambient temperatures with anionic detergents, such as sodium dodecyl sulfate (SDS). The resultant aggregates fail to penetrate the stacking gel during polyacrylamide gel electrophoresis (PAGE). To prevent the formation of such aggregates, avidin was acetylated and the pI was thus reduced. Acetylated avidin was found to behave in a manner similar to that of streptavidin; under nondenaturing conditions (i.e., incubation of samples at room temperature), both proteins normally migrated mainly as tetramers with a tendency to form oligomers of the tetramer. When samples were boiled, both proteins migrated mainly as the monomer. The comparative stability properties of avidin and streptavidin were also examined using SDS‐PAGE by heating samples and determining the extent of dissociation of tetramers to monomers as a function of temperature. A distinctive transition temperature could be defined for individual samples. Using this assay, it was determined that, in the absence of biotin, the quaternary structure of streptavidin is more stable than that of avidin. Biotin appears to stabilize structures of both avidin and streptavidin to a similar degree. Acetylation of avidin thus provides a simple means to analyze the quaternary structure of the molecule using SDS‐PAGE.
Comparison of behavior of N‐substituted acrylamides and celluloses on double‐stranded DNA separations by capillary electrophoresis at 25° and 60°C
Authors: Gelfi Cecilia , Perego Marilena , Libbra Fabio , Righetti Pier Giorgio
Published: May 2005

The behavior of N‐substituted poly(acrylamides) and of cellulose in the separation of double‐stranded DNA by capillary zone electrophoresis (CZE) is evaluated. Contrary to previously held belief, which attributed best separations in poly(acrylamides) to small DNA fragments (typically in the 50–1000 bp size range) and in celluloses to large DNA fragments, we show that also celluloses can achieve fine sieving of short DNA sizes provided they are used at much higher concentrations than previously reported. For example, in the case of hydroxyethylcellulose (HEC), typically used at 0.2–0.8% concentrations, levels of 3% produce excellent patterns, at 25°C, in the 50–600 bp size range. If separations are conducted at 60°C, sieving is lost in most liquid polymers. However, if the concentration of HEC is raised to 6%, and that of hydroxypropylmethylcellulose (HPMC) above 1%, sieving is fully restored. Also, N‐substituted acrylamido derivatives, notably N‐acryloylaminoethoxyethanol (AAEE) and N‐acryloylaminopropanol (AAP) offer excellent performance at both 25° and 60°C, whereas plain poly(acrylamide) irreversibly collapses at high temperatures, possibly due to intrinsic instability of the amido bond under such harsh conditions. Among all the polymers investigated, an 8% solution of poly(AAP) offers the best performance and highest theoretical plates in the 25°–60°C interval. Separations at high temperatures are necessary when dealing with detection of point mutations in temperature‐programmed CZE and are preferred in DNA sequencing. Two additional advantages are evident when running DNA separations at 60°C: a marked reduction of analysis times and a linearization of the transit times of the larger (434–587 bp) DNA fragments, in all polymers tested.
A numerical study of persistence length effects on DNA conformation in sequencing electrophoresis
Authors: Guerry Emmanuel , Martin Olivier C. , Tricoire Hervé , Siebert Rainer , Valentin Luc
Published: May 2005

We have developed a program to evaluate the influence of DNA stiffness on molecular mobility and conformation during electrophoresis. This (currently) two‐dimensional numerical study models DNA as a chain of uniformly charged beads connected to one another by elastic segments, of finite mean size, in the presence of fixed obstacles representing gel fibers. Contrary to the standard biased reptation model (BRM), our Langevin‐type dynamics for the chain are microscopic and warrant studies of fine effects such as inner chain orientation. Using this model, we show that the introduction of a persistence length decreases the (saturated) mobility at high electric fields, providing strong evidence that the gel generates a friction force and not only a (dissipation‐free) constraint force. We also show that the persistence length leads to an increase of the chain orientation in the field direction. This suggests that DNA stiffness causes the saturation plateau value to be reached for smaller chain sizes than those predicted by the BRM model.
Simulations of the overshoot in the build‐up of orientation of long DNA during gel electropohoresis based on a distribution of oscillation times
Authors: Carlsson Christina , Larsson Anette
Published: May 2005

The preiodic extension‐contraction motion observed for long DNA molecules undergoing agarose gel electrophoresis in a constant field is believed to be important for the separation mechanism in pulsed field gel electrophoresis. These oscillations give rise to an overshoot and an undershoot in the ensemble orientation of DNA in the beginning of a field pulse, when the molecules oscillate coherently. After approximately one oscillation cycle, the coherence between the molecules is lost, and a constant, cycle‐averaged orientation is reached. In this paper we simulate this build‐up of the ensemble orientation of DNA by using a distribution of oscillation times (the time between two consecutive compressed conformations) determined by fluorescence microscopy for YOYO‐stained DNA. Six different orientation profiles, describing the orientation during one oscillation cycle, were used. The simulated orientation responses are compared with an orientation response measured by linear dichroism (LD) under the same experimental conditions as in the microscopy study. We found that the choice of orientation profile during the oscillation is important. Best agreement between the simulated and the experimental orientation response was obtained for an orientation profile based on a theoretical model by Schurr and Smith (Biopolymers 1990, 29, 1161–1165). The influence of the distribution of oscillation times and its standard deviation on the orientation response was also investigated. Furthermore, simulations at different field strengths and DNA sizes were performed and found to agree quite well with the experimentally obtained LD data.
Activation energy of the separation of DNA sequencing fragments in denaturing noncross‐linked polyacrylamide by capillary elecrophoresis
Authors: Fang Yu , Zhang Jian Zhong , Hou Joan Y. , Lu Hui , Dovichi Norman J.
Published: May 2005

We present the mobility of DNA sequencing fragments as a function of temperature in 5%T, 0%C noncross‐linked polyacrylamide and at an electric field of 150 V/cm. The mobility of the fragments follows the Arrhenius equation. The activation enegry for migration of a fragment through the polymer decreases monotonically with fragment length. The data were also analyzed in terms of the biased reptation model; the onset of biased reption with stretching occurs for longer fragments as the temperature increases. These results are quite different from those observed for separation of DNA in cross‐linked gels and represent a fundamental difference in the physics of the cross‐linked and noncross‐linked polyacrylamide.
Capillary electrophoresis of oligonucleotides in sieving liquid polymers in isoelectric buffers
Authors: Gelfi Cecilia , Perego Marilena , Righetti Pier Giorgio
Published: May 2005

Analysis of oligonucleotides (especially in regard to assessing the purity of antisense compounds) is typically performed in 18%T sieving liquid polyacrylamide, in 30% formamide and 7 M urea. Up to 600 V/cm have been reported, with transit times, for 20 to 25 oligomers, of 15–20 min. We show that the same analysis can be performed in isoelectric buffers, typically histidine (His), and in more dilute linear polyacrylamides, e.g. 10%T (at 0%C), with much reduced analysis times. A series of His concentrations has been explored, ranging from 25 to 150 mM. Best performance is obtained in 100 mM His (at pH = pI, i.e., 7.47 at 25°C), dissolved in 7 M urea, in presence of 10% sieving liquid linear polyacrylamide. Such a buffer allows delivering 800 V/cm without any loss of resolution due to Joule heating, with retention of very high resolving power down to fragments as short as tetranucleotides. Under these conditions, the analysis time for an antisense oligonucleotide containing fragments from a 10‐mer to 18‐mer is in a time window of 4–5 min. It is shown that the smallest fragment (10‐mer) migrates in the capillary at the remarkable speed of 5 cm/min.
Evaluation of capillary electrophoresis in polymer solutions with laser‐induced fluorescence detection for the automated detection of T‐cell gene rearrangements in lymphoproliferative disorders
Authors: Oda Robert P. , Wick Myra J. , Rueckert Lisa‐Marie , Lust John A. , Landers James P.
Published: May 2005

The rapid increase in the number of DNA‐based clinical diagnostic procedures, particularly polymerase chain reaction (PCR)‐based assays, has generated interest in analytical techniques that are less time‐consuming and labor‐intensive than traditional procedures such as polyacrylamide gel electrophoresis (PAGE). Capillary electrophoresis with laser‐induced fluorescence (CE‐LIF) detection, which allows for rapid and sensitive detection of DNA fragments in an automated format, is well‐suited for DNA‐based clinical assays. In this study, we demonstrate the potential of CE‐LIF for the detection of PCR products from T‐cell receptor gamma (TCRγ) gene rearrangements present in monoclonal populations of lymphocytes. The presence of monoclonal populations of T‐cells is associated (but not always synonymous) with lymphocytic malignancies. Analysis of 31 patient samples, as well as sensitivity controls, demonstrated that CE‐LIF detection of monoclonal lymphocytic populations is comparable to that of PAGE‐SYBR Green I staining, and that CE‐LIF detection can be accomplished in less than 20 min. These preliminary results illustrate the potential feasibility of a CE‐based diagnostic assay for the detection of T‐cell gene rearrangements.
DNA Typing of a polymerase chain reaction amplified D1S80/amelogenin multiplex using capillary electrophoresis and a mixed entangled polymer matrix
Authors: Isenberg Alice R. , McCord Bruce R. , Koons Barbara W. , Budowle Bruce , Allen Ralph O.
Published: May 2005

In this study, a technique was developed to separate by capillary electrophoresis (CE) the widely varying DNA fragment sizes produced by a multiplex polymerase chain reaction (PCR) amplification of the loci D1S80 and amelogenin. Experiments were performed to analyze different buffer systems and obtain optimal resolution for the separation. A matrix composed of two different molecular weights of the same polymer was constructed to separate the DNA fragments with baseline resolution, and a cubic spline fit was used to estimate the size of DNA fragments over 350 base pairs. Over 100 samples were examined to demonstrate the rapid, robust and precise characteristics of this CE system. An average relative standard deviation of 0.3% was obtained for the sizing of the D1S80 alleles in these samples. DNA from mixed body fluid samples samples subjected to environmental insult, and D1S80 sequence variants were also typed successfully. These results demonstrate that CE is a viable method for analysis of D1S80 and amelogenin forensic DNA samples.
Electrokinetic chromatography in suppressed electroosmotic flow environment: Use of a charged cyclodextrin for the separation of enantiomers and geometric isomers
Authors: Janini George M. , Muschik Gary M. , Issaq Haleem J.
Published: May 2005

Electrokinetic chromatography (EKC), with negatively‐charged cyclodextrins (NCDs) added to the buffer, was conducted in polyacrylamide‐coated columns under suppression of electroosmotic flow. The equations of migration and resolution for neutral solutes in this mode of chromatography, which for brevity we term NCD‐EKC, are presented. The chiral sulfated cyclodextrin, β‐CD‐SBE (IV), used in this study is anionic over the entire pH range accessible to capillary electrophoresis, and the coated columns are stable and provide reproducible performance in the pH range 2.5–8.8. Optimum separation was obtained in the pH range where the solutes are neutral. The incorporation of an alkyl spacer between the sulfate ion and the rim of the cyclodextrin allows an unhindered approach and inclusion of neutral solutes in the cyclodextrin cavity. Solute migration time is inversely proportional to the concentration of the chiral selector. Separation (relative migration time difference) increases with decreasing chiral selector concentration and approaches a maximum, beyond which further decreases in chiral selector concentration result in broad peaks and loss of resolution. A chiral selector concentration of 1% in a 10 mM phosphate buffer produced excellent separation of amino acids and dipeptide enantiomers. In addition to being chiral selectors, cyclodextrins are also known as shape selectors. NCD‐EKC is particularly suited for the separation of positional isomers of hydrophobic solutes. The separation of aflatoxin isomers and chlorophenol congeners is presented. In the separation of chlorophenols the more hydrophobic trichlorophenols eluted first and the least hydrophobic, phenol, eluted last.
Analysis of interaction between cadmium and metallothionein isoforms by capillary zone electrophoresis
Authors: Minami Takeshi , Matsubara Hideki , O‐higashi Masayuki , Kubo Kanenobu , Okabe Nobuo , Okazaki Yuko
Published: May 2005

The effects of cadmium on the peak area of metallothionein (MT) protein were studied by capillary zone electrophoresis with a polyacrylamide‐coated tube at neutral pH. When cadmium was added to a commericial standard MT‐1 isoform prepared from rabbit liver, the peak area of the MT‐1 isoform decreased in a time‐ and dose‐dependent manner. The MT‐2 isoform also decreased with time, but the rate of decrease was lower than that of the MT‐1 isoform. When ethylene glycol‐bis‐(β‐aminoethyl ether)‐N,N,N′N′‐tetraacetic acid (EGTA) was added to a solution containing cadmium and MT‐1, the peak area recovered with incrasing concentration of EGTA. Zinc caused a slight decrease in the peak areas of MT‐1 and MT‐2 compared with cadmium, while addition of sodium did not decrease the areas. Furthermore, the peak areas of MT isoforms of the crude extract prepared from zinc‐treated mice also decreased with increasing concentration of cadmium. These results indicate that cadmium may change the charge of MT, which may account for the observed differences in electrophoretic behavior.
Micellar electrokinetic capillary chromatography of three natural vitamin A derivatives
Authors: Profumo Aldo , Profumo Vincenzo , Vidali Giorgio
Published: May 2005

Micellar electrokinetic chromatography (MEKC) using bile salts has been employed to separate retinoids differing in structure and charge; bile salts in MEKC allows the separation of liposoluble molecules but, to the best of our knowledge, there are only few data on the above‐mentioned technique for the separation of highly hydrophobic compounds. The three natural vitamin A derivatives, retinal, retinol and retinoic acid, were successfully separated by MEKC using sodium cholate within a relatively short time (ca. 25 min), whereas the separation of these compounds was not successful using sodium dodecyl sulfate or sodium deoxycholate. Several parameters (pH and organic modifiers, in addition to bile salts concentration) have been tested to provide a system that can be extended to synthetic retinoids, which are often used in treating several diseases, including cancer prevention and therapy.
Probing the microheterogeneity of O‐specific chains from Yersinia ruckeri using capillary zone electrophoresis/electrospray mass spectrometry
Authors: Bateman Kevin P. , Banoub Joseph H. , Thibault Pierre
Published: May 2005

The analysis of underivatized oligosaccharides arising from mild acid hydrolysis of endotoxins from Yersinia ruckeri serotype 02 was achieved using on‐line capillary zone electrophoresis‐electrospray mass spectrometry (CZE‐ESMS). This technique provided unparalleled resolution of the differnt glycans obtained from purified fractions of the native endotoxins or from hydrolysis of lipopolysaccharides from Y. ruckeri. Electrophoretic conditions enabling the separation of anionic and cationic analytes were developed to determine possible sites of heterogeneity on either the core or the O‐chain glycans. Structural characterization of underivatized oligosaccharides identified in the ion electropherograms was achieved using tandem mass spectrometry under lowcollision energy conditions

NRCC 39726.

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Electrospray interface for capillary electrophoresis‐mass spectrometry with fiber‐optic UV detection close to the electrospray tip
Authors: Foret František , Kirby Daniel P. , Vouros Paul , Karger Barry L.
Published: May 2005

A miniaturized, integrated capillary electrophoresis‐ultraviolet detection‐electrospray ionization‐mass spectrometry (CE‐UV‐ESI‐MS) interface has been constructed and evaluated. The device incorporates a fiber optic detection cell close to the electrospray tip to allow UV monitoring of separated zones just prior to their admittance into the mass spectrometer. This configuration provides precise information about the time when UV‐active zones enter the electrospray and allows easy location of analyte mass information in the ion current profile. The miniaturized dimensions of the interface allow the use of short capillaries for fast separations.
Optimized continuous flow electrophoresis
Authors: Weber Gerhard , Boček Petr
Published: May 2005

Continuous flow electrophoresis (CFE) was optimized by employing (i) electrophoretic regimes with stacking properties, to eliminate electrohydrodynamic dispersion, (ii) quasi‐mixed zones to prevent precipitation of the stacked analytes, (iii) sheath liquid streams at the electrode compartment membranes to prevent penetration of the electrode reaction products into the separation chamber, (iv) proper engineering of the separation chamber to provide efficient dissipation of Joule heat, and (v) counterflow at the collection outlets to eliminate the problems of dead volumes and uneven collection of separated species. Data on direct temperature measurements in the separation chamber at various levels of the dissipated electric power are presented. Preparative runs of amyloglucosidase in the isoelectric focusing (IEF) mode and rat liver organelles in the isotochophoresis (ITP) mode demonstrate the high performance of the optimized CFE system.
Determination of peptide dissociation constants and stokes radius at different protonation stages by capillary electrophoresis
Authors: Castagnola Massimo , Rossetti Diana V. , Cassiano Loredana , Misiti Francesco , Pennacchietti Lucia , Giardina Bruno , Messana Irene
Published: May 2005

Peptide electrophoretic mobility measured by capillary zone electrophoresis can be regarded as deriving from the mean of mobilities of different protonated forms, each one participating according to its charge, Stokes radius and relative percentage. The percentage is a function of the peptide dissociation constant and solutior pH. Therefore, mobility modifications due to pH variations can be related to peptide dissociation constant, charge, and Stokes radius throughout general binding equations. Thus, not only can peptide dissociation constants be measured, but information about Stokes radius modifications linked to proton loss can also be obtained with picomoles of peptide.
DNA mobility anomalies are determined primarily by polyacrylamide gel concentration, not gel pore size
Authors: Stellwagen Nancy C.
Published: May 2005

The dependence of DNA mobility anomalies on gel pore size has been studied in polyacrylamide gels with a wide variety of compositions, using molecular weight ladders containing multiple copies of normal (12B) and anomalously slowly migrating (12A) 147‐base pair restriction fragments from plasmid pBR322 as the migrating probe molecules. If the gel pore size is increased by decreasing the total acrylamide concentration (%T) at constant cross‐linker ratio (%C), the usual method of increasing gel pore size, the mobility anomalies decrease with increasing gel pore radius as though the 12A multimers were retarded by a sieving mechanism. However, the decrease in the mobility anomalies is independent of whether the apparent gel pore radius is larger or smaller than the DNA radius of gyration, suggesting that gel pore size is not the controlling variable. If the acrylamide concentration is held constant and the gel pore size is increased by decreasing %C at constant %T, the mobility anomalies of the largest 12A multimers (6 mers and higher) decrease with increasing gel pore radius, because of sieving effects, until the effective gel pore radius becomes approximately equal to the DNA radius of gyration, after which the mobility anomalies level off and become independent of gel pore size. The mobility anomalies exhibited by 5‐mers and smaller multimers of fragment 12A are independent of gel pore radius in all gels with constant %T. Similar results are observed with a molecular weight ladder containing phased A‐tracts from the kinetoplast bending locus. Since the anomalous electrophoretic mobilities depend primarily on the total acrylamide concentration in the gel, and not on the apparent gel pore radius, increases in the magnitude of the mobility anomalies with increasing gel concentration (and decreasing gel pore radius) cannot be taken as evidence for DNA curvature.
Quantitative description of migration behavior of porphyrins based on the dynamic complexation model in a nonaqueous capillary electrophoresis system
Authors: Bowser Michael T. , Sternberg Ethan D. , Chen David D. Y.
Published: May 2005

The effect of an additive (Brij 35) on the mobilities of a group of porphyrin acids is quantitatively characterized based on a 1:1 dynamic complexation model. Varying additive concentration shifts the equilibrium and changes the viscosity of the background electrolyte. The equilibrium constant, the electrophoretic mobility of the free analyte, and the electrophoretic mobility of the complex are identified as the parameters necessary to describe the analytes' migration behavior. Several statistical methods for obtaining these parameters are discussed. The equilibrium constants and complex mobilities are calculated using three different linear regression methods. The weighted y‐reciprocal method was preferred because it gives smaller error, and the data points are evenly distributed along the concentration axis. These values are confirmed using a nonlinear regression to ensure that the proper weighting was used in the linear regression plots. The parameters are then used to predict the apparent mobilities of the analytes over the entire additive concentration range, allowing the optimum separation conditions to be identified. For disclike molecules, such as porphyrins, the mobility is determined by the orientation of the molecule in an electric field, in addition to their size and charge. The strength of binding between the porphyrins and Brij 35 depends on the number of binding sites and the solvation shell.
Effects of catholytes on the mobilization of proteins after capillary isoelectric focusing
Authors: Manabe Takashi , Miyamoto Hiromitsu , Iwasaki Aiko
Published: May 2005

Purified proteins and human plasma proteins were separated by capillary isoelectric focusing and detected by cathodic mobilization, e.g. by replacing the cathodic sodium hydroxide solution by solutions containing another anion. The effect of catholyte anions on the resolution of mobilized proteins was examined. Compared with the chloride or phosphate anion, organic anions having small dissociation constants improved protein resolution, especially in the range of acidic proteins. Among the catholytes examined, acetic acid gave the best resolution for purified proteins and human plasma proteins. Measurement of the changes of electric current during the mobilization process indicated that organic anions with low mobility move slowly towards the anode and retard changes of pH gradient in the capillary. Isoelectric focusing, cathodic mobilization, and direct pH measurement in polyacrylamide gel columns (1.3 mm internal diameter and 38 mm long) also revealed these effects of low mobility anions. In order to visualize the behavior of human plasma proteins in the mobilization step, micro two‐dimensional polyacrylamide gel electrophoresis was employed. The results indicated that the proteins migrate towards the cathode, preserving their relative positions attained during the isoelectric focusing step.
Potential of two‐dimensional electrophoresis in routine identification of closely related durum wheat lines
Authors: Picard Philippe , Bourgoin‐Grenèche Mireille , Zivy Michel
Published: May 2005

Four closely related curum wheat varieties were compared by computer‐assisted analysis of two‐dimensional electrophoretic maps of leaf proteins. A low inter‐varietal polymorphism was revealed and seven reliable qualitatively varying proteins allowed rapid visual identification of genotypes. For numerous spots, presence/absence or quantitative variations were greatly affected by a batch effect. Several criteria that should be used to discard unreliable spots or gels a priori were reviewed. Nevertheless, it was shown that, provided that the experimental design allows the integration of the batch effect, screening for discriminant markers as well as computing distances based on protein quantity variations are possible and allow variety identification. Euclidean and Mahalanobis distances allowed variety discrimination and single gel classification with a minimum risk of error, not only by taking into account the quantitative variations in discriminant proteins selected by analysis of variance, but also by taking into account all reproducible spots. The possible applications of two‐dimensional electrophoresis in variety identification are discussed.
Analysis for the stability of platinum‐containing species in soil samples using capillary electrophoresis interfaced on‐line with inductively coupled plasma mass spectrometry
Authors: Michalke Bernhard , Lustig Sönnke , Schramel Peter
Published: May 2005

Online interfacing of capillary electrophoresis (CE) with inductively coupled plasma‐mass spectrometry (ICP‐MS) has been accomplished using a laboratory‐made nebulizer. The high resolution of CE is used for the separations of Pt species of standard solutions and real samples, followed by ICP‐MS for element‐specific detection within 12 min. The samples are analyzed immediately after sample preparation or after 2–4 h of aging in order to elucidate undesired species transformations which may occur before analysis. The method was proven not to impair the species during the analytical procedure while being a necessary prerequisite to judge time‐dependent species transformations. Quality control aspects concerning species stability during the analytical procedure and stability of electrical current during nebulization were studied. A possibly interfering suction flow was estimated and found to be negligible. The results will serve as a basis for quality assurance in future investigations about Pt speciation in soil.
Stereoselective analysis of herbicides by capillary electrophoresis using sulfobutyl ether β‐cyclodextrin as chiral selector
Authors: Desiderio Claudia , Polcaro Chiara M. , Fanali Salvatore
Published: May 2005

Capillary zone electrophoresis has been used for the enantiomeric separation of several herbicides. Different β‐cyclodextrin (CD) derivatives have been investigated for chiral separations and among them the negatively charged sulfobutyl ether β‐cyclodextrin (SBE‐β‐CD) proved to be effective for the stereoselective resolutions of the investigated herbicides. The effect of CD concentration, buffer pH and organic modifier on effective mobilities, resolution and selectivity of the analytes have been studied. Addition of SBE‐β‐CD (5–50 mg/mL) to the buffer at pH 9 resulted in a general increase of migration times as well as resolution. A CD concentration as low as 5 mg/mL was effective to completely resolve napropamide and ethofumesate enantiomers. Buffer solutions containing 40 mg/mL of SBE‐β‐CD were chosen to study the effect of buffer pH (7, 8, and 9) on chiral separation of the herbicides. No great differences in resolution and effective mobilities have been found in the pH 7–9 range. The addition of different organic modifiers to the background electrolyte at pH 9, containing 20 mg/mL of SBE‐β‐CD, showed different effects. Methanol was the most effective in improving resolution but in some cases total loss in enantiomeric separation was observed. The qualitative analysis of an enantiomerically pure herbicide (flamprop isopropyl) commercial preparation is also shown.
Characterization of humic substances using capillary electrophoresis with photodiode array and laser‐induced fluorescence detection
Authors: Nordén Maria , Dabek‐Zlotorzynska Ewa
Published: May 2005

The effect of organic modifiers (organic solvents and urea) on the electrophoretic buffer was investigated in an attempt to enhance resolution in the fingerprinting of two humic substances (HS): an aquatic fulvic acid (FA) and a soil humic acid (HA). Two detection modes, photodiode array and laser‐induced fluorescence, were applied. The addition of organic solvents (acetonitrile, acetone, 2‐propanol and tetrahydrofuran) in 10 mM Na2B4O7 (pH 9.0) resulted in a clear difference in electrophoretic behavior of the studied HS compared to the absence of organic solvents. There were also marked differences in the electrophoretic profiles using a 2‐propanol‐borate buffer with urea. The study of the interaction between metals and HS showed that there was a change in the migration pattern in the presence of Al(III), indicating the specific binding sites for aluminum.
Analysis of nitramine and nitroaromatic explosives in pink water by capillar electrophoresis
Authors: Oehrle Stuart A.
Published: May 2005

Analysis of nitramine and nitroaromatic explosives is of interest for environmental applications. Analysis of 14 nitramine and nitroaromatic explosives was accomplished by micellar electrokinetic capillary chromatography (MECC). Analysis times under 14 min were possible employing a phosphate‐sodium dodecyl sulfate (SDS) electrolyte and direct UV detection at 254 nm. Analysis of various waste water samples (called “pink water”) from ammunition plants was performed.
Improved and simplified in‐gel sample application using reswelling of dry immobilized pH gradients
Authors: Sanchez Jean‐Charles , Rouge Véronique , Pisteur Michel , Ravier Florence , Tonella Luisa , Moosmayer Marlis , Wilkins Marc R. , Hochstrasser Denis F.
Published: May 2005

A simple and inexpensive methacrylate rehydration chamber was built to accomodate ten immobilized pH gradient (IPG) strips. In the chamber, entire IPG gels were used for sample application, with the protein entering the gels during their rehydration. For rehydration, commercially available or laboratory‐made strips were positioned in the grooves with the gel in contact with 500 μL of sample for 6 h or overnight. This avoided the use of sample cups, eliminated precipitation at the sample application site, thus improving resolution over the entire pH range of the gels. It also allowed precise control of protein amounts and sample volumes loaded into the IPG gels, and also lowered costs of reagents during rehydration and equilibration owing to the reduced volumes. Up to 5 mg of protein can be loaded on wide IPG gels and up to 15 mg of some samples on narrow pH range gels.
Detailed peptide characterization using PEPTIDEMASS – a World‐Wide‐Web‐accessible tool
Authors: Wilkins Marc R. , Lindskog Ingrid , Gasteiger Elisabeth , Bairoch Amos , Sanchez Jean‐Charles , Hochstrasser Denis F. , Appel Ron D.
Published: May 2005

In peptide mass fingerprinting, there are frequently peptides whose masses cannot be explained. These are usually attributed to either a missed cleavage site during the chemical or enzymatic cutting process, the lack of reduction and alkylation of a protein, protein modifications like the oxidation of methionine, or the presence of protein post‐translational modifications. However, they could equally be due to database errors, unusual splicing events, variants of a protein in a population, or artifactual protein modifications. Unfortunately the verification of each of these possibilities can be tedious and time‐consuming. To better utilize annotated protein databases for the understanding of peptide mass fingerprinting data, we have written the program “PEPTIDE‐MASS”. This program generates the theoretical peptide masses of any protein in the SWISS‐PROT database, or of any sequence specified by the user. If the sequence is derived from the SWISS‐PROT database, the program takes into account any annotations for that protein in order to generate the peptide masses. In this manner, the user can obtain the predicted masses of peptides from proteins which are known to have signal sequences, propeptides, transit peptides, simple post‐translational modifications, and disulfide bonds. Users are also warned if any peptide masses are subject to change from protein iso‐forms, database conflicts, or an mRNA splicing variation. The program is freely accessible to the scientific community via the ExPASy World Wide Web server, at the URL address: www.expasy.ch/www/tools.html.
Identification of two‐dimensional gel electrophoresis resolved yeast proteins by matrix‐assisted laser desorption ionization mass spectrometry
Authors: Larsson Thomas , Norbeck Joakim , Karlsson Hasse , Karlsson Karl‐Anders , Blomberg Anders
Published: May 2005

Protein extract from yeast cells growing exponentially in saline medium was separated by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE), with the separation in the first dimension on a wide range immobilized pH (3–10) gradient. From one preparative 2‐D gel a number of previously identified proteins were used as test material for our initial matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS) efforts on large scale rapid protein spot identification. Sample preparation via in‐gel trypsin digestion was slightly modified to be compatible to MS analysis, and via this modified procedure MS generated peptide mass profiles could, in most cases with good precision, identify the protein in question. Preferential ionization was tested on a yeast aldehyde dehydrogenase (ALD7), and it was shown that the ionization of some peptides was clearly suppressed by the presence of others. Roughly 50% of the observed peptide masses was found by the search routines in the database, and the mass measurement accuracy of the peptides was within 0.5 Da. Silver‐stained gels could be used with good results for the generation of peptides to be analyzed by MALDI‐MS. For one of the 2‐D resolved proteins, glycerol 3‐phosphatase (GPP1), the post‐source decay (PSD) spectrum proved crucial in identification.
The analysis of myocardial proteins by infrared and ultraviolet laser desorption mass spectrometry
Authors: Sutton Chris W. , Wheeler Colin H. , U Sally , Corbett Joseph M. , Cottrell John S. , Dunn Michael J.
Published: May 2005

The use of infrared (IR) and ultraviolet (UV) matrix‐assisted laser desorption (MALDI) mass spectrometry to analyse myocardial proteins separated by two‐dimensional (2‐D) polyacrylamide gel electrophoresis (PAGE) is discussed. Proteins were electroblotted onto a FluoroTrans polyvinylidene difluoride (PVDF) membrane in order to facilitate analysis by MALDI, which represented the most efficient means of extracting large numbers of proteins simultaneously. Once on a FluoroTrans membrane, IR‐MALDI was used to obtain spectra from selected protein spots, but no useful signals were obtained with UV MALDI. Spectra were generated from 46 of 50 spots analysed with protein masses from 13 to 82 kDa and isoelectric points (pI) 4.7–7.8. For those protein spots that had previously been characterised, and for which both sequence and post‐translational modification data were known, IR‐MALDI data was within plus or minus 0.5% of the expected mass. Some spots contained more than one protein signal, illustrating the increased information obtainable from MALDI, but also suggesting the limit of resolution of 2‐D gels for separating large numbers of proteins. Attempts to digest proteins with specific proteases and generate peptide mass fingerprints by MALDI analysis on the membrane were unsuccessful with either IR or UV lasers. The peptides were extracted from the membrane and readily analysed by UV MALDI for peptide spectra. Poor data was obtained for peptide digests with IR‐MALDI, probably because of matrix suppression by digest buffer. In order to obtain the maximum amount of information from blotted proteins, both IR and UV MALDI were required.
Proteome analysis of glycoforms: A review of strategies for the microcharacterisation of glycoproteins separated by two‐dimensional polyacrylamide gel electrophoresis
Authors: Packer Nicolle H. , Pawlak Anna , Kett Warren C. , Gooley Andrew A. , Redmond John W. , Williams Keith L.
Published: May 2005

Preparative two‐dimensional polyacrylamide electrophoresis (2‐D PAGE) is a method of separation which for the first time allows protein isoforms to be readily purified for subsequent analysis. The profile of the 2‐D separation of the protein complement (proteome) of eukaryotic cells and tissues typically contains obvious ‘trains’ of spots which differ in pI and/or apparent molecular mass. These are usually isoforms of the same protein and result from post‐translational modifications. There is growing evidence that alterations to the glycosylation and/or phosphorylation of a protein can be correlated with developmental and pathological changes; these changes can be visualised on the 2‐D separation. It is not clear, however, how these modifications alter the structural properties of the protein and affect their migration in this mode of separation. Strategies need to be developed to obtain a more detailed understanding of the reason for the appearance of isoforms as discrete spots on 2‐D PAGE. Standard proteins, fetuin and ovalbumin, were used to monitor the effect of the removal of glycans and phosphates on the migration of the glycoproteins in the 2‐D system. The isoforms were not simply explained by the presence or absence of a single modification. To further investigate the reasons for the different migration of the isoforms it is necessary to characterise the modifications in more detail. Unlike protein analysis, until recently the available methodology for the analysis of the glycans attached to proteins has not been sensitive enough to allow analysis of single spots in gels or blots resulting from 2‐D electrophoresis. In this paper we review current and future strategies for characterisation of protein modifications using single spots from 2‐D gels.
Comparing two‐dimensional electrophoretic gel images across the Internet
Authors: Lemkin Peter F.
Published: May 2005

Scientists around the world often work on similar data so the need to share results and compare data arises periodically. We describe a method of comparing two two‐dimensional (2‐D) protein gels of similar samples created in different laboratories to help identify or suggest protein spot identification. Now that 2‐D gels and associated databases frequently appear on the Internet, this opens up the possibility of visually comparing one's own experimental 2‐D gel image data with data from another gel in a remote Internet database. In general, there are a few ways to compare images: (i) slide one gel (autoradiograph or stained gel) over the other while back‐illuminated, or (ii) build at 2‐D gel computer database from both gels after scanning and analyzing these gels. These are impractical since in the first case the gel from the Internet database is not locally available. In the second, the costs of building a multi‐gel database solely to answer the question of whether a spot is the same spot may be excessive if only a single visual comparison is needed. We describe a distributed gel comparison program (URL: www‐lmmb.ncifcrf.gov/flicker) which runs on any World Wide Web (WWW) connected computer and is invoked from a Java‐capable web browser. One gel image is read from any Internet 2‐D gel database (e.g. SWISS‐2DPAGE) and the other may reside on the investigator's computer. Images may be more easily compared by first applying spatial warping or other transforms interactively on the user's computer. First, regions of interest are “landmarked” with several corresponding points in each gel image, then one gel image is warped to the geometry of the other. As the two gels are rapidly alternated, or flickered, in the same window, the user can slide one gel past the other to visually align corresponding spots by matching local morphology. This flicker‐comparison technique may be applied to analyzing other types of one‐dimensional and 2‐D biomedical images.
Two‐dimensional electrophoretic analysis of human milk‐fat‐globule membrane proteins with attention to apolipoprotein E patterns
Authors: Goldfarb Marcia
Published: May 2005

Two‐dimensional (2‐D; Iso Dalt method) electrophoresis was done on lipid membrane fractions from human milk samples collected in polypropylene tubes at home by the mother. The samples represent milk from two weeks to ten months post partum. Identification was made by immunoassay of Western blots. Apolipoprotein E was not easily seen on silver‐stained gels because concentration is small and spots overlap another protein. Blot assays show a more complex pattern than 34 kDa plasma apo E with molecular size isoforms at 36 kDa, 34 kDa, 20 kDa and 15 kDa. One sample was negative for apo E. Apo E plays an important role in lipid transport in tissue and in the brain. It is synthesized in a variety of tissues by specific cells. Milk may reflect several sources.