bluuebunny/crossref_metadata_2025 huggingface dataset:
An apparatus for membrane‐confined analytical electrophoresis
Authors: Ridgeway Theresa M. , Hayes David B. , Moody Thomas P. , Wilson Timothy J. , Anderson Arthur L. , Levasseur John H. , Demaine Philip D. , Kenty Burt E. , Laue Thomas M.
Published: May 2005

A membrane‐confined analytical electrophoresis apparatus for measuring the solution charge of macromolecules has been described previously (T. M. Laue et al., Anal. Biochem. 1989, 182, 377–382). Presented here is a design for this apparatus, which permits the on‐line acquisition and display of absorbance data from up to 512 positions along an analysis chamber. Concentration distributions of macromolecules in solution can be monitored in the chamber to provide steady‐state electrophoresis, electrophoretic mobility and diffusion measurements. Buffer chambers press semipermeable membranes against the open ends of a fused‐silica cuvette to form the analysis chamber. This configuration permits both the flow of buffer and the establishment of an electric field across the cuvette, while retaining macromolecules in the field of view. Though a gel may be included in the analysis chamber, none is required for gradient stabilization. The volume of sample required for analysis is 8 μL, most of which is recoverable. Experimental conditions can be varied during study by simply changing the circulating buffer and/or the electric field. The analysis and buffer chambers are held in an aluminum housing that sits in an aluminum water jacket. The water jacket provides temperature control, shielding from external electrical noise and also serves as an optical mask. Plans for the cell assembly, optical system and the computer interface for data acquisition are provided. The assembly and operation of the apparatus and the analysis of data are described.
Parallelism between width and asymmetry of peaks of rigid, spherical particles in capillary zone electrophoresis using polymer solutions
Authors: Radko Sergey P. , Chrambach Andreas
Published: May 2005

Peak width and peak asymmetry of rigid spherical particles in the size range of 3–100 nm radius (R) were measured in capillary zone electrophoresis (CZE), using buffered uncross‐linked polyacrylamide of Mr 5.0 × 106. Polymer concentration‐dependent spreading of peak width and peak asymmetry were found to parallel one another. The parallelism holds whether the particle size is within the “small” (R < 20 nm) or “large” (R > 20 nm) size ranges previously found to differ in the mechanism of particle size dependent retardation of electrophoretic migration (S. P. Radko and A. Chrambach, Electrophoresis 1996, 17, 1094–1102). In application to the “small” particle size range, the parallelism between band width and band asymmetry can be qualitatively interpreted to be consistent with the Giddings‐Weiss mechanism (G. H. Weiss et al., Electrophoresis 1996, 17, 1325–1332) of electrophoresis in polymer‐containing media which postulates a dependence of band width and band asymmetry on the equilibrium between “stationary” and “mobile” states of the particle.
Ultrathin‐layer zone electrophoresis of alcohol dehydrogenase in partly rehydrated polyacrylamide gels: An alternative to starch gel electrophoresis
Authors: Maly I. Piotr , Toranelli Mireille , Crotet Valérie , Sasse Dieter
Published: May 2005

A highly sensitive electrophoretic technique for the separation of alcohol dehydrogenase isoenzymes by zone electrophoresis in partly rehydrated polyacrylamide gels is described. Five hundred μm thin polyacrylamide gels are polymerized under standardized conditions. After polymerization the gels are washed thoroughly with distilled water to remove any unreacted monomers, catalysts or still soluble polymers. The washed gels are then impregnated with 0.5% Tween 20 and dried. Before electrophoresis the dry gels are rehydrated to a thickness of 250 μm, which makes up 50% of the original gel volume. Rehydration is carried out by use of a degassed buffer solution. This method permits the demonstration of the isoenzymes of alcohol‐dehydrogenase class I and II in man and allows quantitative determination.
Surface modification based on Si‐O and Si‐C sublayers and a series of N‐substituted acrylamide top‐layers for capillary electrophoresis
Authors: Gelfi Cecilia , Curcio Mario , Righetti Pier Giorgio , Sebastiano Roberto , Citterio Attilio , Ahmadzadeh Hossein , Dovichi Norman J.
Published: May 2005

Two approaches were used to prepare a series of surface‐modified capillaries. In the first, a sublayer was formed by coupling γ‐methacryloxypropyltrimeth‐oxysilane to the surface silanol groups forming an SI‐O bond; a top layer was then formed by polymerizing acrylamide in the capillary, which reacted with the sublayer. In the second approach, a sublayer was formed by silanol chlorination, followed by Grignard coupling of vinylmagnesium bromide to form an Si‐C bond at the surface; a top layer was formed by polymerizing either acrylamide (AA), dimethylacrylamide (DMA), N‐acryloylaminoethoxyethanol (AAEE), or N‐acryloylaminopropanol (AAP) onto the sublayer. The Si‐C‐poly(AA) capillaries were more stable and produced an approximately 10‐fold lower electroosmotic flow compared to the Si‐O‐poly(AA) capillaries. The Si‐C sublayer was used to compare the performance of all four top layers. Electroosmotic flow decreased in the order: Si‐O‐poly(AA), Si‐C‐poly(AA), Si‐C‐poly(AAEE), Si‐C‐poly(DMA), and Si‐C‐poly(AAP). Si‐C‐poly(AA) showed evidence of irreversible degradation at pH 9 already after 40–50 runs. Si‐C‐polyAAP‐coated capillaries demonstrated superior efficiency and migration time reproducibility for a number of alkaline proteins and for fluorescently labeled ovalbumin. Excellent performance was maintained, in the case of poly(AAP), for a least 300 runs (of 30 min duration) at pH 9.0.
Predictive model for capillary electrophoretic peptide mobility in 2,2,2‐trifluoroethanol‐water solution
Authors: Castagnola Massimo , Rossetti Diana Valeria , Corda Marcella , Pellegrini Mariagiuseppina , Misiti Francesco , Olianas Alessandra , Giardina Bruno , Messana Irene
Published: May 2005

Using capillary electrophoresis (CE) on a set of 21 peptides with a molecular mass ranging from about 350 to 1850 Da, the Stokes radii at different protonation stages and the acidic dissociation constants in water and in a 2,2,2‐trifluoroethanol (TFE) water mixture (30% v/v) were determined. These results permitted us to establish separately the reliability of semiempirical models utilized for the prediction of peptide size and charge at different acidic pHapp (pHapp range: 2.00–4.25). The data obtained on size and charge were utilized in order to provide suitable mobility predictions on the basis of the charge‐to‐size ratio. The best predictive conditions for size and charge were found at the most acidic range of pHapp studied (2.00–2.25), either in water or a TFE‐water mixture, and reliable predictive equations for peptide mobility were established at this pHapp.
Electrophoretic analyses in a case of monoclonal γ chain disease
Authors: Tissot Jean‐Daniel , Tridon Arlette , Ruivard Marc , Layer Andreas , Henry Hugues , Philippe Pierre , Schneider Philippe
Published: May 2005

Abnormal low molecular weight immunoglobulin heavy chain can be detected in serum samples from patients with heavy chain disease. In this paper, we report the characterization of a γ heavy chain in a patient suffering from a lymphoplasmocytic disorder, using immunofixation analysis and two‐dimensional polyacrylamide gel electrophoresis. We demonstrate that the combination of serum protein agarose electrophoresis and two‐dimensional electrophoresis (three‐dimensional electrophoresis) can be used to further characterize abnormal protein bands detected by immunofixation.
Isoelectric focusing in the vegetable seed industry
Authors: Van Den Berg Bartel M.
Published: May 2005

Different types of electrophoretic procedures were developed for hybrid purity testing based on starch gel electrophoresis (SGE), vertical polyacrylamide gel electrophoresis (PAGE), and isoelectric focusing (IEF). For the most important vegetables these methods are much faster than plant grow‐outs and relatively inexpensive. Compared to SGE and PAGE methods, horizontal IEF proved to be more efficient for large‐scale hybrid purity testing. These developments were made possible by the basic work of Harry Rilbe and improvements that were initiated as a result of Rilbe's work. The present paper describes a number of milestones during this developmental period, starting with the isoelectric focusing concept of Harry Rilbe up to the large‐scale application of IEF. Further, a comparison of IEF with DNA fingerprinting methods along with the future of both techniques is discussed with respect to hybrid purity testing in the vegetable seed industry. When it comes to a choice between the use of either IEF or a DNA‐based method, efficiency and efficacy determines the method which is best suited for hybrid purity testing. It is also concluded that in the future we will see an increased use of both IEF as well as DNA‐based methods for hybrid purity testing because expectations of growers has increased; consequently they will accept fewer inbreds in a hybrid variety, especially when growing in a greenhouse.
Detection of enzymes active on various β‐1, 3‐glucans after denaturing polyacrylamide gel electrophoresis
Authors: Trudel Jean , Grenier Jean , Asselin Alain
Published: May 2005

Enzymes were assayed for glucanase activity after denaturing sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) in gels containing β‐1,3‐glucans embedded as substrate. Lentinan, curdlan, paramylon, baker's yeast alkali‐insoluble glucan, baker's yeast alkali‐soluble glucan and carboxymethyl (CM)‐pachyman were compared to oligomeric laminarin, which is the usual substrate for assaying β‐1,3‐glucanase activities. Detecting enzyme activities by aniline blue fluorescent staining was also compared with the staining of released reducing sugars by 2,3,5‐triphenyltetrazolium chloride (TTC). For the nonreduced proteins, the Driselase extract exhibited one major band at 32.5 kDa and one less intense band at 23 kDa for most substrates with the two detection procedures. No Lyticase enzyme was detected in either detection procedures for all tested substrates. For barley enzymes, no activity was revealed after aniline blue staining while one undescribed 19 kDa glucanase activity was best shown after TTC staining with curdlan, paramylon and CM‐pachyman as substrates. In the case of reduced proteins, the Lyticase extract yielded three bands (33, 36 and 46 kDa) on several substrates with both detection procedures. This was the same for the barley leaf extract (32, 36 and 39 kDa). The Driselase extract showed one 42 kDa band. Many enzymes active on β‐1,3‐glucans are thus best revealed when proteins are denatured and reduced and when protein renaturation after SDS‐PAGE involves a pH 8.0 treatment and the inclusion of 1 mM cysteine in buffers. However, some enzymes are only detected when proteins are denatured without reduction. Finally, the use of various polymeric β‐1,3‐glucan substrates different from oligomeric laminarin is necessary to detect new types of enzymes such as the 19 kDa barley glucanase.
Expression of recombinant psoriasis‐associated fatty acid binding protein in Escherichia coli: Gel electrophoretic characterization, analysis of binding properties and comparison with human serum albumin
Authors: Vorum Henrik , Madsen Peder , Svendsen Ib , Celis Julio E. , Honoré Bent
Published: May 2005

The psoriasis‐associated fatty acid binding protein (PA‐FABP, also known as FABP5) is a novel keratinocyte protein that is highly up‐regulated in psoriatic plaques (P. Madsen, H. H. Rasmussen, H. Leffers, B. Honoré and J. E. Celis, J. Invest. Dermatol. 1992, 99, 299–305). Here we have expressed PA‐FABP in Escherichia coli as a fusion protein containing an NH2‐terminal hexa‐His tag followed by a factor Xa cleavage site. The recombinant protein was expressed at a level of about 30% of the soluble proteins and was purified to homogeneity using a simple two‐step protocol consisting of affinity chromatography on Ni2+‐nitrilotriacetic acid agarose followed by gel filtration. The recombinant protein was then digested with factor Xa and characterized by two‐dimensional gel electrophoresis. The ability of PA‐FABP to bind saturated fatty acids ranging from 6 to 16 carbons was determined directly by dialysis and compared to human serum albumin (HSA). The results showed that PA‐FABP binds multiple molecules of the fatty acids hexanoate (C6:0), octanoate (C8:0), decanoate (C10:0) and laurate (C12:0) with a K1 of about 104 m−1, and myristate (C14:0) with a K1 of 4.4 × 105 m−1. Palmitate (C16:0) also bound strongly with multiple molecules. Due to the very low solubility of palmitate its affinity to PA‐FABP was measured relatively to HSA and found to be 8.1 times lower. At ligand/protein ratios below 1, all fatty acids bound to PA‐FABP with about one to three orders of magnitude lower affinity than to HSA. The difference in the fatty acid binding properties of the two proteins may reflect differences in their three‐dimensional structures, which in the case of PA‐FABP consists mainly of β‐sheets while HSA contains predominantly α‐helices.
Rab 11a is modifiedin vivoby isoprenoid geranylgeranyl
Authors: Gromov Pavel , Celis Julio E.
Published: May 2005

Post‐translational adduction of farnesyl or geranylgeranyl moieties to a terminal cysteine residue of proteins is a characteristic feature ofras‐related GTP‐binding proteins. According to current rules of prenylation the carboxylterminal motif (CXXX or CC/CXC) as well the context of the cysteine residue dictate the extend and specificity of the isoprenoid modification. Rab 11a, a small GTP‐binding protein that is associated with pathways regulating protein traffic, terminates with isoleucine at itsC‐terminus, suggesting that it may only be geranylgeranylated. Recent finding, however, showed that rab 11a can be modifiedin vitroby different prenyl groups: farnesyl and geranylgeranyl [1]. To determine whether rab 11a is modifiedin vivoby both isoprenoids we transiently overexpressed rab 11a in COS1 cells, and analyzed the translation products by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) in combination with metabolic labeling in the presence of either [3H]farnesyl‐PP or [3H]geranylgeranyl‐PP. Contrary to thein vitroresults, our studies showed that rab 11a is post‐translationally modifiedin vivoonly by geranylgeranyl isoprenoid. The data implied thatin vivothere must exist other determinant(s) that are necessary for prenyltransferase recognition.
Rapid isolation of phage displayed antibodies to β‐actin eluted from two‐dimensional electrophoresis gel
Authors: Zhou Jian‐Nian , Linder Stig , Franzén Bo , Auer Gert , Hochstrasser Denis F. , Persson Mats A. A.
Published: May 2005

We describe a simple and efficient procedure which can be used to prepare antibodies to proteins extracted by two‐dimensional gel electrophoresis (2‐DE), using β‐actin as a model. Protein was electroeluted from a stained gel, biotinylated and used for selection of phage from a semisynthetic phage antibody library. After four rounds of selection using 50 ng β‐actin per cycle, approximately 8 × 103 phage were recovered. Antibody fragments were prepared from 21 randomly picked clones. Six of eighteen (6/18) antibody‐positive clones produced antibody fragments reacting against β‐actin in an enzyme linked immunosorbent assay (ELISA). Sequencing of the HC‐CDR3‐region showed that all six clones were independent isolates, suggesting that a large number of independent phage antibody reactivities were generated.
Analysis of Bence Jones proteinuria by high resolution two‐dimensional electrophoresis
Authors: Williams Katherine M. , Williams John , Marshall Thomas
Published: May 2005

Analysis of Bence Jones proteinuria by high resolution two‐dimensional electrophoresis (2‐DE) and immunoblotting reveals a complex pattern of light chain (LC) isoforms corresponding to the free monoclonal Bence Jones protein and its fragments. Replica blotting gives duplicate blots for LC typing (λ, ϰ) and, under the conditions employed, leaves sufficient protein for Coomassie Blue staining of the urinary protein profile and pI/Mr determination of the LC isoforms. Carrier ampholytes (CAs, in our “simplified” 2‐DE system) and immobilised pH gradients (IPGs, in the Multiphor 2‐DE system) give similar LC isoform patterns. Artifacts, including cone‐like distortions and trailing “piggyback” spots, are visualised with both 2‐DE systems. IPGs are advantageous as they allow reproducible detection of strongly basic LC isoforms by isoelectric focusing (under equilibrium conditions) without recourse to CA nonequilibrium pH gradient electrophoresis.
Identification of yeast proteins from two‐dimensional gels: Working out spot cross‐contamination
Authors: Parker Kenneth C. , Garrels James I. , Hines Wade , Butler Erin M. , McKee Andrew H. Z. , Patterson Dale , Martin Steve
Published: May 2005

With the complete sequence of the yeast genome now available, efforts by many laboratories are underway to identify each of the spots on two‐dimensional (2‐D) gels corresponding to the most abundant yeast proteins. The high mass accuracy now attainable using matrix assisted laser desorption/ionization (MALDI)‐mass spectrometry equipped with delayed extraction simplifies the process of identification, such that many spots can be unambiguously identified in a short period of time merely by using peptide mass fingerprinting and generally available database matching programs. Although it is not always possible to match spots between gels run by different laboratories, proteins generally yield the same abundant proteolytic fragments when tryptic digestions are performed. Databases containing these signature peptides not only simplify the task of reidentifying proteins from different gels, but also make it possible to identify small amounts of cross‐contaminating proteins from different spots, as well as common extraneous contaminants such as human keratins. In this paper, we present data on the identification of > 20 previously unreported yeast proteins from 2‐D gels. Some novel proteins were identified from randomly analyzed spots. Focusing on 14 spots in a narrow‐pH‐range gel, we demonstrate how organizing peak‐table data and peptide match‐list data into databases enables the identification of a larger percentage of the peaks.
An algorithm for the identification of proteins using peptides with ragged N‐ or C‐termini generated by sequential endo‐ and exopeptidase digestions
Authors: Korostensky Chantal , Staudenmann Werner , Dainese Paola , Hoving Sjouke , Gonnet Gaston , James Peter
Published: May 2005

We have developed an algorithm (MassDynSearch) for identifying proteins using a combination of peptide masses with small associated sequences (tags). Unlike the approach developed by Matthias Mann, ‘Tag searching’, in which the sequence tags are generated by gas phase fragmentation of peptides in a mass spectrometer, ‘Rag Tag’ searching uses peptide tags which are generated enzymatically or chemically. The protein is digested either chemically or with an endopeptidase and the resultant mixture is then subjected to partial exopeptidase degradation. The mixture is analyzed by matrix assisted laser desorption and ionization time of flight mass spectrometry and a list of intact peptide masses is generated, each associated with a set of degradation product masses which serve as unique tags. These ‘tagged masses’ are used as the input to an algorithm we have written, MassDynSearch, which searches protein and DNA databases for proteins which contain similar tagged motifs. The method is simple, rapid and can be fully automated. The main advantage of this approach is that the specificity of the initial digestion is unimportant since multiple peptides with tags are used to search the database. This is especially useful for proteins like membrane, cytoskeletal, and other proteins where specific endopeptidases are less efficient and lower specificity proteases such as chymotrypsin, pepsin, and elastase must be used.
A novel method for quantitative analysis of recombinant secreted proteins using two‐dimensional electrophoresis
Authors: Nygaard Sean C. , Hart Charlie , De Jongh Karen S.
Published: May 2005

We describe a two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE)‐based approach for detecting and quantifying secreted recombinant proteins in media conditioned by baby hamster kidney (BHK) cells. Seven secreted proteins were analyzed in this system: leptin, thrombopoietin, thrombin, glycoprotein 130, soluble interleukin‐2 receptor, and two novel sequences obtained from sequence database searches. BHK cells transfected with plasmids encoding each of these proteins, and cells transfected with empty plasmids (control cells), were metabolically labeled and the resulting conditioned media were analyzed by 2‐D PAGE. Gel images derived from cells expressing recombinant proteins were compared with images from control cells in order to identify spots corresponding to the expressed proteins. All seven of the test proteins were successfully detected using this method. The sensitivity of the system was evaluated by diluting samples derived from high‐expressing clones with conditioned media from control cells. The sensitivity of detection was protein‐dependent, but recombinant proteins expressed at levels as low as 10 ng/mL could be detected. Quantification of recombinant protein levels was achieved by measuring spot intensities using phosphorimager analysis. The intensity of spots corresponding to recombinant proteins were compared with the spot intensity of an endogenous BHK protein which had been calibrated to a known standard. Estimates of the levels of expressed protein determined using this technique correlated with the levels determined using standard affinity assays. We conclude that this system provides a reliable method for quantifying levels of protein expression when specific assays are unavailable.
Molecular imprint polymers as highly selective stationary phases for open tubular liquid chromatography and capillary electrochromatography
Authors: Tan Zhixin Jessica , Remcho Vincent T.
Published: May 2005

Chiral separations employing molecular imprint polymer (MIP) stationary phases in both open tubular liquid chromatography (OT‐LC) and capillary electrochromatography (OT‐CEC) are demonstrated. MIPs are highly crosslinked polymers containing spatial and functionality memory of template molecules which provide a higher degree of selectivity when used as stationary phases for chromatographic separations. Thin films of molecular imprinted polymers bonded to the inner walls of 25 μm ID fused‐silica capillaries were prepared using an in situ polymerization technique developed in our laboratory that allows the use of conventional fused‐silica capillaries with polyimide outer coatings. The success rate in preparing such open tubular columns was about 70%. Methacrylic acid and 2‐vinyl pyridine were chosen as functional monomers, and either ethylene dimethacrylate or trimethylol propane trimethacrylate was used as the crosslinker. Toluene was employed as the porogen. Effects of polymerization conditions on column preparation and chromatographic performance were studied. Enantiomeric separations of D‐ and L‐dansyl phenylalanines were achieved in both OT‐LC and OT‐CEC modes with good selectivity and efficiencies. Both types of separations may be performed on the same column using a single commercial instrument.
Development of separation systems for polynuclear aromatic hydrocarbon environmental contaminants using micellar electrokinetic chromatography with molecular micelles and free zone electrophoresis
Authors: Moy Thomas W. , Ferguson Patrick L. , Grange Andrew H. , Matchett William H. , Kelliher Virginia A. , Brumley William C. , Glassman Jack , Farley John W.
Published: May 2005

Of four systems available from the literature, based on cyclodextrins, dioctyl‐sulfosuccinate, bile salts, and molecular micelles consisting of oligomers of undecylenic acid, the most successful separation system in our hands is based on the molecular micelles, oligomers of sodium undecylenic acid (OSUA). We have employed organic additives of acetonitrile, acetone, and tetrahydrofuran in achieving separations of polyaromatic hydrocarbons (PNAs) using molecular micelles. Generally, successful separations are achieved with 20–40% composition as the organic additive in an 8 mM borate buffer. We separated 16 PNAs with 20% tetrahydrofuran in a system of 8 mM borate and 0.125 g/ 10 mL (ca. 6.25 mM) of OSUA. Typical extracts of environmental samples contain additional analytes besides the typical 16 target compounds. Among these are the nitrogen‐containing aromatics that can act as cations under conditions of low pH and additional compounds that can act as anions under basic conditions in free‐zone electrophoresis. These additional classes of analytes are separated by capillary zone electrophoresis/laser‐induced fluorescence detection using a frequency‐doubled laser operated at 257 nm.
Capillary electrophoresis chiral separations of basic compounds using cationic cyclodextrin
Authors: Wang Fang , Khaledi Morteza G.
Published: May 2005

Chiral separations of basic enantiomers were carried out by using a cationic cyclodextrin (CD), quaternary ammonium β‐cyclodextrin (QA‐β‐CD), under counter‐electroosmotic flow (counter‐EOF) conditions. The special characteristics of using a cationic CD to separate cationic enantiomers is that the EOF can be reversed and the analyte‐CD complexation is reduced. This is especially useful for chiral separation of cationic compounds, which strongly bind with neutral and anionic CDs (such as tricyclic amine compounds). The reduction in the binding constants between the CD and the cationic enantiomers makes it easier to control the optimum CD concentration. The application of the cationic CD also eliminated the peak tailing problem caused by electrodi‐spersion. The effect of pH and the concentration of QA‐βCD on chiral separation has been studied. At pH 3.02, no separation for any of the enantiomeric amines was observed. At pH 8.20, chiral separation of some tricyclic compounds was achieved at very high resolution due to the counter‐EOF setup. At pH 11.6, most enantiomers were neutral and chiral separation of some bicyclic compounds can be obtained.
Chiral separation of polychlorinated biphenyls by micellar electrokinetic chromatography with sodium cholate
Authors: Navazo Antonio Luis Crego , Marina María Luisa , Gonzalez María José
Published: May 2005

Micellar electrokinetic chromatography (MEKC) with one kind of bile salt (sodium cholate) was used to separate three chiral polychlorinated biphenyls (PCBs; 84, 95, and 176), each one in its two enantiomers. Sodium cholate was used as chiral surfactant in a 2‐(N‐cyclohexylamino) ethanesulfonic acid (CHES) buffer under alkaline (pH 10) conditions containing urea (2 M). The influence of bile salt concentration on the efficiency and the resolution between the two enantiomers of PCBs 84 and 95 was established. The chiral separation of three PCBs was successfully achieved in less than 30 min (approximately 23 min for PCB 176 and approximately 29 min for PCBs 84 and 95).
Capillary electrophoresis interfaced to inductively coupled plasma mass spectrometry for element selective detection in arsenic speciation
Authors: Michalke Bernhard , Schramel Peter
Published: May 2005

A method is presented to separate and detect six arsenic species by capillary electrophoresis (CE) interfaced to inductively coupled plasma mass spectrometry (ICP‐MS). CE was used as a highly resolving separation system, whereas ICP‐MS served as an element selective detector providing low detection limits. The special mode of operation included sample stacking and a differentiation of separation and detection. This provided separation and detection of six As species, uncharged and anionic, to be monitored within a single run. Detection limits were calculated according to IUPAC recommendation at 15 μg As/L for As (III), dimethyl arsinic acid (DA), monomethyl arsonic acid (MA) and As (V), or 65 μg As/L for arsenobetaine (AsB) and arsenocholine (AsC). Investigations were focused on possibly occurring interferences, e.g., ArCl+ interference at the monoisotope 75As. Finally, real samples from biomedical field (urine) and environmental field (sewage sludge) were analyzed.
The pH dependence of predictive models relating electrophoretic mobility to peptide chemico‐physical properties in capillary zone electrophoresis
Authors: Castagnola Massimo , Rossetti Diana Valeria , Corda Marcella , Pellegrini Mariagiuseppina , Olianas Alessandra , Misiti Francesco , Giardina Bruno , Messana Irene
Published: May 2005

We applied best fitting procedures to capillary electrophoresis (CE) mobility values, measured at varying acidic pH, of a set of 21 peptides with a molecular mass ranging from about 350 to 1850 Da. This method allowed the contemporary measurements of C‐terminus and carboxylic group of the side‐chain of aspartic and glutamic acid dissociation constants and of peptide Stokes radius at different protonation stages. Stokes radius was related to peptide molecular mass M at the power of a fractional coefficient, and best correlation was found at pH 2.25, the fractional coefficient being equal to 0.68. This value is close to that proposed by R. E. Offord (Nature 1966, 211, 591–593), who suggested a proportionality between the polymer Stokes radius and M2/3. The coefficient value decreases at higher pH, reaching a value of 0.58 at pH 4.25, corresponding to a mean peptide conformational transition towards more compact structures as a consequence of C‐terminus dissociation. The measurement of the dissociation constants of each peptide allowed us to determine the percentage error on peptide charge predictions performed utilizing mean dissociation constants. Even for the charge, the best predictive performance is obtained at the most acidic edge of the range of the pH studied, mainly at pH 2.25. Conclusively, this study shows that the best performance of predictive models for peptide CE mobility is obtainable in the very acidic pH range (2.25–2.50) and in the absence of electroosmotic flow, and that a satisfactory predictive equation of peptide electrophoretic mobility (m2V−1S−1) is given by μ = 85.4(Z/M0.68)10−8.
Changes in outer membrane protein profiles of bacteria after meropenem‐induced postantibiotic effect studied by capillary electrophoresis
Authors: Kustos Ildikó , Kocsis Béla , Kerepesi Ildikó , Kilár Ferenc
Published: May 2005

Persistent inhibition of bacterial growth, called postantibiotic effect (PAE), after a short exposure to a new carbapenem, meropenem, was determined in different strains of the Enterobacteriaceae family. Capillary electrophoresis (CE), as well as sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) were used to study the outer membrane protein (OMP) profiles before and after meropenem treatment. CE proved to be suitable for the characterization of the OMP profiles of bacteria. Significant changes in the electrophoretic patterns were observed, showing the consequential effect of meropenem on bacteria.
Understanding the mechanism of the zinc‐ion stains of biomacromolecules in electrophoresis gels: Generalization of the reverse‐staining technique
Authors: Fernandez‐Patron Carlos , Castellanos‐Serra Lila , Hardy Eugenio , Guerra Maribel , Estevez Eric , Mehl Ehrenfried , Frank Rainer W.
Published: May 2005

We have recently shown that a few nanograms of protein separated by electrophoresis in sodium dodecyl sulfate‐polyacrylamide gels can be detected by reverse‐staining, exploiting the precipitation reaction between zinc(II) and imidazole. Modifications of this method have also been generated to detect gelisolated nucleic acids and bacterial glycolipids. Because there is no recourse to chemical modifiers, the reverse‐staining technique has been valuable when micropreparing these biomacromolecules for later use or characterization. The mechanism underlying the reverse‐staining effect, however, remains incompletely understood and this has prevented a further generalization of the technique. Here, we have conducted physicochemical experiments and identified zinc imidazolate (ZnIm2) as the main component of the precipitate that forms along the surface of zinc‐imidazole reverse‐stained gels. Many staining effects observed when gels containing electrophoretically separated biopolymers are subjected to zinc‐imidazole stains have been rationalized. The reverse‐staining method has been vastly generalized, now allowing the detection of proteins and glycolipids as well as complexes of these macromolecules in native gels. We demonstrate the application of the reverse‐staining technique in situations where Coomassie blue or silver staining was inappropriate or failed to produce detection of the species of interest. The present generalization of the reverse‐staining method facilitated the characterization of biomacromolecular interaction partners in mixtures of bacterial glycolipids and human tears.
Mixed‐dye staining method for protein detection in polyacrylamide gel electrophoresis using calconcarboxylic acid and rhodamine B
Authors: Jung Da‐Woon , Yoo Gyurng‐Soo , Choi Jung‐Kap
Published: May 2005

We have developed a new mixed‐dye protein staining method that is simple, rapid, and sensitive. A freshly prepared mixture of calconcarboxylic acid (NN, 0.02%) and rhodamine B (RB, 0.04%) in 40% methanol / 7% acetic acid, was used as a staining solution. RB acts as an auxiliary agent to inhibit the binding of NN to the gel matrix, reducing the background staining and therefore enhancing the protein staining by NN. This mixed‐dye staining method reduces the total staining and destaining time to less than an hour, and increases the sensitivity to 25 ng of bovine serum albumin, which is greater than the 100 ng sensitivity limit of Coomassie Brilliant Blue R‐250 (CBBR) staining.
Mechanistic insights derived from retardation and peak broadening of particles up to 200 nm in diameter in electrophoresis in semidilute polyacrylamide solutions
Authors: Radko Sergey P. , Chrambach Andreas
Published: May 2005

Rigid spherical particles in the size range of 5–200 nm diameter were subjected to capillary zone electrophoresis (CZE) in semidilute solutions of un‐cross‐linked polyacrylamide of Mr 5, 7 and 18 × 106 (PA‐5, ‐7 and ‐18, respectively) of varying concentrations up to 1.6% and at field strengths varying from 68 to 270 V/cm. For all particles under study, the experimental Ferguson plots, log(mobility) vs. polymer concentration, permit a linear approximation. Their slope, the retardation coefficient KR = ∂log(mobility)/∂(concentration), for particles smaller than 30 nm in diameter increased with particle size in PA‐5 and −7 independently of electric field strength and polymer Mr. The KR of particles of 30 nm in diameter or more was found to be independent of particle size at the lowest field strength used but to decrease with it at the higher values of field strength. The decrease was parallel but shifted to higher values of retardation when the polymer Mr increased from 5 to 7 × 106. With a decreasing ratio of average mesh size of the polymer network, ξ, to particle radius, R, the approach to “continuity” of the polymeric medium (ξ/R < 1) with both increasing particle size and polymer concentration does not result in the retardation behavior expected according to the macroscopic (bulk) viscosity of the solution. These experimental observations were hypothetically interpreted in terms of a transition to a retardation mechanism comprising the formation of a polymer depletion layer near the particle surface ‐ polymer solution interface. Peak width exhibited an overall increase with PA‐7 concentration for all particles studied. For particles of 30 nm in diameter or less, the increase was steepest when the radius of the particle was approximately commensurate with ξ at a given polymer concentration. For the largest particle, 205 nm in diameter, peak broadening with polymer concentration was found to correlate linearly with peak asymmetry. CZE of the particles in PA‐18 solutions revealed abnormal behavior, with both mobility and peak width remaining near‐constant up to a concentration of 0.08% and sharply declining at higher concentrations. The decline of relative mobility is the same for the entire particle size range used, while peak width declines in direct relation to particle size.
A new technique for stripping of polyimide coating from fused‐silica capillaries
Authors: Lindberg Peter , Roeraade Johan
Published: May 2005

A novel method for the manufacture of detection windows in polyimide‐coated fused‐silica capillary columns is described. Concentrated fuming nitric acid at room temperature is utilized for stripping of the coating from a defined section of the capillary. The technique is particularly suitable for columns that have a heat sensitive coating on the inner wall or for columns that contain an immobilized hydrogel sieving‐matrix. A column holder for the simultaneous preparation of detection windows in an array of more than 100 capillaries is also reported.
Differential diagnosis of gammopathies by capillary electrophoresis and immunosubtraction: Analysis of serum samples problematic by agarose gel electrophoresis
Authors: Clark Raynell , Katzmann Jerry A. , Kyle Robert A. , Fleisher Martin , Landers James P.
Published: May 2005

The capabilities of capillary electrophoresis (CE) for serum protein electrophoresis and immunotyping have been demonstrated. CE‐based systems specifically designed for serum protein electrophoresis and immunotyping via immunosubtraction (IS) are now available and are being evaluated for efficiency, specificity and sensitivity by several groups. The use of CE for serum protein electrophoresis and immunotyping (IS) in the clinical laboratory compares well with agarose gel electrophoresis (AGE) and immunofixation (IF) for the detection and characterization of monoclonal proteins. In addition to routine use, this technology is useful for a subset of serum samples that are difficult to interpret with conventional technology. In this study, sera abnormalities difficult to detect/interpret by AGE‐IF are subdivided into four categories: (i) patients with polyclonal increases in immunoglobulin, (ii) point of application artifacts, (iii) abnormalities in the beta region, and (iv) patients with free light chains. CE is superior to AGE for evaluating samples characterized by the above abnormalities. Sera containing monoclonal proteins within a polyclonal increase are easier to detect by CE as well as being easier to type by IS than by IF. Point‐of‐application artifacts, periodically observed with AGE, do not exist on CE since the point of detection is remote from the point of application. Enhanced resolution in the beta region allows for increased detection of monoclonal proteins migrating in this region. Some free light chains are undetected by CE as a result of no apparent abnormalities on the CE serum protein profile and, thus, still require IF for detection. CE detects more serum electrophoretic abnormalities than AGE in this clinically important group of patients with Bence Jones proteinemia.
Structural determination of oligosaccharides from recombinant iduronidase released with peptide N‐glycanase F using fluorophore‐assisted carbohydrate electrophoresis
Authors: Hague Chuck , Masada R. Irene , Starr Christopher
Published: May 2005

The lysosomal storage disorder mucopolysaccharidoses I (MPS I) is caused by a deficiency in the production of α‐L‐iduronidase. Recently, a recombinant α‐L‐iduronidase has been produced in Chinese hamster ovary (CHO) cells. It is thought that for α‐L‐iduronidase to be correctly targeted to the lysosomal vesicle a particular oligosaccharide make‐up must be present, and characterization of the carbohydrates is critical. Oligosaccharides from α‐L‐iduronidase were analyzed using fluorophore‐assisted carbohydrate electrophoresis (FACE®). The FACE® system uses polyacrylamide gel electrophoresis to separate, quantify, and determine the sequence of oligosaccharides released from glycoproteins. Asparagine‐linked oligosaccharides were released from α‐L‐iduronidase using the enzyme peptide N‐glycanase F (PNGase F). Released oligosaccharides were labeled with a fluorophore at the reducing termini by reductive amination. A total of nine bands were sequenced from the released pool of oligosaccharides. The pool of fluorescently labeled oligosaccharides was then electrophoresed in preparative gels and each band individually excised and extracted. Isolated bands were treated with a series of exoenzymes to determine the sequence of monosaccharides that make up a particular oligosaccharide. A total of eighteen different oligosaccharides were identified from the original pool of oligosaccharides. A majority of the oligosaccharides, over 73%, were found to be of the sialylated complex type. Four of the oligosaccharides were phosphorylated, making up approximately 11% of the carbohydrate pool, and the remaining 15% were of the oligomannose type.
Electrophoretic behavior and size distribution of the acidic polysaccharides produced by the bacteria Bradyrhizobium (Chamaecytisus) strain BGA‐1 and Bradyrhizobium japonicum USDA 110
Authors: Díaz‐Marrero Ana R. , Santamaria Mónica , Poveda Ana , Jiménez‐Barbero Jesus , Corzo Javier
Published: May 2005

The electrophoretic behavior in polyacrylamide gels of the acidic polysaccharides produced by the soil bacteria Bradyrhizobium (Chamaecytisus) strain BGA1 and Bradyrhizobium japonicum USDA110 has been studied. Both polysaccharides were polydisperse, producing a ladder‐like pattern after fixation with Alcian Blue and silver staining of the gel. The polysaccharide molecules were separated according to their size, and they behaved as a collection of flexible random coils of different size and similar charge/mass ratio. The electrophoretic behavior was not affected by the presence of acetyl groups in the polysaccharide. The range of molecular weights of the exopolysaccharide produced by B. japonicum USDA110 was wider and with larger molecules than that of the polysaccharide produced by strain BGA1. The resolution was dependent on the electrophoresis buffer; the best results were achieved with Tris‐borate; in Tris‐glycine buffer, the resolution was worse, and it was not improved by the addition of sodium dodecyl sulfate (SDS).
Profiling glycoprotein N‐linked oligosaccharide by capillary electrophoresis
Authors: Chen Fu‐Tai A. , Evangelista Ramon A.
Published: May 2005

A method for analysis of N‐linked oligosaccharides derived from glycoproteins including sialic acid‐containing species is presented. It is based on the combination of specific chemical and enzymatic conversions coupled with capillary electrophoretic (CE) separation and laser‐induced fluorescence (LIF) detection. Glycoproteins were heat‐denatured in the presence of a reducing agent and the N‐linked oligosaccharides were released by peptide N‐glycosidase (PNGase F; EC3.5.1.52)‐catalyzed hydrolysis. The released N‐linked oligosaccharides were derivatized with 8‐aminopyrene‐1,3,6‐trisulfonate (APTS) under mild reductive amination conditions in which desialylation and loss of fucose residues are minimized. A model N‐linked oligosaccharide, desialylated, galactosylated biantennary, core‐substituted with fucose (A2F) was tested for APTS‐based derivatization chemistry with excellent recovery of the adduct without losing fucose and neuraminic acid residues. The profiles of heavily sialylated N‐linked oligosaccharides derived from fetuin, recombinant human erythropoietin and kallikrein are reported and the data show that the present method produces a high resoluton of the N‐linked oligosaccharide profile for fingerprinting glycans derived from glycoproteins.
Enhancement of sample loadings for the analysis of oligosaccharides isolated from Pseudomonas aeruginosa using transient isotachophoresis and capillary zone electrophoresis—electrospray—mass spectrometry
Authors: Auriola Seppo , Thibault Pierre , Sadovskaya Irina , Altman Eleonora
Published: May 2005

The analysis of underivatized core oligosaccharides arising from mild acid hydrolysis of lipopolysaccharides from Pseudomonas aeruginosa serotype O5 was achieved using a transient isotachophoretic preconcentration method coupled to capillary zone electrophoresis‐electrospray‐mass spectrometry (tCITP‐CZE‐ES‐MS). The combination of a tCITP preconcentration step provided a 10‐ to 50‐fold enhancement of sample loading and a corresponding improvement in sensitivity compared to the conventional zone electrophoresis format. Electrophoretic conditions, enabling the separation of these anionic analytes, were developed to determine possible sites of heterogeneity on either the core or the O‐chain structures. The tCITP‐CZE‐ES‐MS technique provided unparalleled resolution of the different core glycoforms and oligosaccharides obtained from the acid cleavage of the native endotoxins whether isolated following conventional gel permeation chromatography or obtained from direct hydrolysis of the bacterial isolates. These investigations also highlighted the highly phosphorylated nature of these complex cell membrane components, where the heptose residues of the core oligosaccharide can bear up to six phosphate groups

# NRCC: 42370.

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Polymerase chain reaction—single strand conformation polymorphism or how to detect reliably and efficiently each sequence variation in many samples and many genes
Authors: Jaeckel Silke , Epplen Jörg T. , Kauth Marion , Miterski Bianca , Tschentscher Frank , Epplen Cornelia
Published: May 2005

A simple and fast method with high reliability is necessary for the identification of mutations, polymorphisms and sequence variants (MPSV) within many genes and many samples, e.g. to clarify the genetic background of individuals with multifactorial diseases. We evaluated polymerase chain reaction—single strand conformation polymorphism (PCR‐SSCP) analysis to identify MPSV in several genes, which are thought to be involved in the pathogenesis of multifactorial autoimmune diseases like multiple sclerosis. The method is based on the property, that the electrophoretic mobility of single‐stranded nucleic acids depends not only on its size but also on its sequence. The target sequence was amplified, digested into fragments ranging from 50–200 bp, heat‐denatured and analyzed on native gels. The analysis of 55 PCR systems, including a total of 145 fragments demonstrates, that the detection rate of MPSV depends primarily on the fragment lengths. Appropriate dilutions of samples enhances the proportion of ssDNA compared to dsDNA. Changing the gel conditions, glycerol concentrations and/or the addition of urea may increase fragment resolution in some cases. In general, the detection of MPSV is neither influenced by their location within the fragment nor by the type of substitution, i.e. transitions or transversions. The standard PCR‐SSCP system described here provides high reliability and detection rates and allows the efficient analysis of many samples and many genes.