bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractWith 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

AbstractWe 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

AbstractWe 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

AbstractChiral 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

AbstractOf 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

AbstractChiral 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

AbstractMicellar 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

AbstractA 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

AbstractWe 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

AbstractPersistent 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

AbstractWe 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

AbstractWe 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

AbstractRigid 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

AbstractA 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

AbstractThe 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.