bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of rat brain stathmin isoforms by two‐dimensional gel electrophoresis‐matrix assisted laser desorption/ionization and electrospray ionizationion trap mass spectrometry
Authors: Zugaro Lisa M. , Reid Gavin E. , Ji Hong , Eddes James S. , Murphy Anne C. , Burgess Antony W. , Simpson Richard J. 
Published: May 2005

AbstractStathmin is a regulatory phosphoprotein that is a target for both cell cycle and cell surface receptor‐regulated phosphorylation events. There are at least 14 isoforms of stathmin that migrate on two‐dimensional electrophoresis (2‐DE): two unphosphorylated, and 12 increasingly phosphorylated proteins. Following extracellular stimuli, stathmin is phosphorylated on four serines (Ser16, Ser25, Ser38, and Ser63) by several kinases, such as mitogen‐activated protein (MAP), cdc2 kinase, protein kinase A, and Ca2+/calmodulin‐dependent kinase‐Gr. While all forms of stathmin are derived from the same protein encoded by a single mRNA, the precise nature of the post‐translational modifications has not been clear. In this study we have characterized three rat brain stathmin isoforms, #1, #3 and #4, which electrophorese on 2‐DE with apparent molecular weight (Mr)/isoelectric point (pI) values of 15 500/6.2, 15 000/6.1, and 15 000/6.0, respectively. The phosphorylation status of these isoforms was determined using a combination of peptide mapping, matrix‐assisted laser desorption/ionization mass spectrometry and electrospray‐ionization ion trap mass spectrometry. Stathmin isoform #1 was not phosphorylated, stathmin isoform #3 was phosphorylated on Ser38 only, and stathmin isoform #4 was phosphorylated on Ser38; however, the phosphorylation status of Ser63 could not be determined. In addition, three proteins which electrophorese near stathmin were identified in order to more accurately define the Mr/pI locus of this region of the 2‐DE gel map. These include: phosphatidyl ethanolamine binding protein (Mr∼18 000 /pI 6.0), synuclein forms 2 and 3 (Mr∼14 000 /pI 5.4), and synuclein form 2 (Mr∼15 000 /pI 5.0).
The identification of peptide modifications derived from gel‐separated proteins using electrospray triple quadrupole and ion trap analyses
Authors: Swiderek Kristine M. , Davis Michael T. , Lee Terry D. 
Published: May 2005

AbstractMicrospray tandem mass spectrometry (MS/MS) in combination with database search routines has become a powerful tool for the identification of proteins from femtomole amounts of material following gel electrophoresis and in‐gel digestion procedures. However, artifactual modification of susceptible residues can arise during gel electrophoresis, leading to unexpected peptide mass shifts during mass analysis. Consequently, collision‐induced dissociation (CID) spectra generated from these derivatized peptides can defy direct interpretation by automated database search routines and remain unidentified. Here, we evaluate the MS/MS spectra of peptides carrying oxidized derivatives of tryptophane and methionine residues, and various modifications of cysteine. We demonstrate that certain of these modifications generate characteristic fragmentation patterns or “fingerprints”, during CID analysis, the knowledge of which can facilitate the interpretation of the spectra. We will show that these signature fragment ions are predominantly produced during the CID analysis of singly charged ions although they can be observed in the MS/MS spectra of the doubly charged species as well. In other cases, the CID spectrum lacks a characteristic fingerprint and the modification remains silent. However, CID spectra of related peptides, differing only by their modifications, are similar and all or part of the fragment ion spectra will have shifted by a discreet mass, which facilitates the identification of the modified residue. At the same time, the comparison of related spectra can prevent misinterpretations such as the assignment of a residue mass to the wrong amino acid or a neutral loss fragment ion to a y‐ or b‐ion.
Two‐dimensional electrophoresis of human placental mitochondria and protein identification by mass spectrometry: Toward a human mitochondrial proteome
Authors: Rabilloud Thierry , Kieffer Sylvie , Procaccio Vincent , Louwagie Mathilde , Courchesne Paul L. , Patterson Scott D. , Martinez Pascal , Garin Jerome , Lunardi Joël 
Published: May 2005

AbstractOwing to the complexity of higher eukaryotic cells, characterization of a complete proteome is likely to be difficult to achieve. However, advantage can be taken of the cell compartmentalization to build organelle proteomes, which can moreover be viewed as specialized tools to study specifically the biology and “physiology” of the target organelle. Within this frame, we report here the construction of the human mitochondrial proteome, using placenta as the source tissue. Protein identification was carried out mainly by peptide mass fingerprinting, but other methods were also used (N‐terminal microsequencing, blotting). The optimization steps in two‐dimensional (2‐D) electrophoresis needed for proteome research are discussed. However, the relative paucity of data concerning mitochondrial proteins is still the major limiting factor in building the corresponding proteome, which should be a useful tool for researchers working on human mitochondria and their deficienciesThis work can be found on the Internet at the following address: http://www‐dsv.cea.fr/MitoPick/Default.html
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Correlation of acidic and basic carrier ampholyte and immobilized pH gradient two‐dimensional gel electrophoresis patterns based on mass spectrometric protein identification
Authors: Nawrocki Arkadiusz , Larsen Martin R. , Podtelejnikov Alexandre V. , Jensen Ole N. , Mann Matthias , Roepstorff Peter , Görg Angelika , Fey Stephen J. , Larsen Peter Mose 
Published: May 2005

AbstractSeparation of proteins on either carrier ampholyte‐based or immobilized pH gradient‐based two‐dimensional (2‐D) gels gives rise to electrophoretic patterns that are difficult to compare visually. In this paper we have used matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) to determine the identities of 335 protein spots in these two 2‐D gel systems, including a substantial number of basic proteins which had never been identified before. Proteins that were identified in both gel systems allowed us to cross‐reference the gel patterns. Vector analysis of these cross‐references demonstrated that there is no obvious pattern by which the mobility of a protein in one gel system can be used to predict its mobility in the other. Thus, as laboratories adopt the immobilized pH gradient‐based 2‐D gel systems, the only reliable means of translating the data gained with the carrier ampholyte‐based gel system is to positively identify the proteins in both 2‐D systems.
Continuous free‐flow electrophoresis
Authors: Křivánková Ludmila , Boček Petr 
Published: May 2005

AbstractThis review evaluates the literature on continuous free flow electrophoresis, published during the last four years. Its aim is to serve not only experts in the field but also newcomers, and, therefore, it also briefly describes the principles of the method and the techniques used, referring to fundamental papers published earlier. The actual commercial instrumentation is briefly outlined. A substantial part of this review is devoted to the optimization of the performance of this method. Finally, diverse applications of fractionations of charged species in solution, ranging from small ions to biological particles and cells, are surveyed.
Fluid‐phase marker transport in rat liver: Free‐flow electrophoresis separates distinct endosome subpopulations
Authors: Ellinger Isabella , Klapper Herbert , Fuchs Renate 
Published: May 2005

AbstractFree‐flow electrophoresis (FFE) was used to investigate the intracellular compartments involved in fluid‐phase marker, fluoresceine isothiocyanate (FITC)‐dextran, transport in the isolated perfused rat liver. One to 2 min after uptake at 37°C, FITC‐dextran was found in endosomes with the same electrophoretic mobility as early sorting endosomes labeled either by the hepatocyte‐specific marker asialoorosomucoid (ASOR) or by transferrin that enters all liver cells. Labeling at low temperature (16°C) blocked transport of ASOR and dextran in early endosomes. With increasing internalization time (3–13 min) at 37°C, FITC‐dextran‐labeled compartments co‐localized with late, ASOR‐containing endosomes. Since localization of FITC‐dextran in late transcytotic compartments was not observed upon FFE separation, it is concluded that the majority of internalized markers is directed to lysosomes. The FITC‐label did not account for the predominant lysosomal targeting of the dextran, since [3H]dextran‐labeled endosomes exhibited an identical FFE pattern. Taken together, these data indicate that the fluid‐phase marker dextran is transported through intracellular compartments with identical characteristics as endosome subcompartments of the receptor‐mediated lysosomal route.
Subcellular fractionation by organelle electrophoresis: Separation of phagosomes containing heat‐killed yeast particles
Authors: Hasan Zahra , Pieters Jean 
Published: May 2005

AbstractUptake of foreign material and its subsequent lysosomal degradation is an important function of macrophages. The mechanisms involved in the binding, uptake and delivery of such material to lysosomal organelles, are, however, poorly understood. Here we describe a method using organelle electrophoresis to study the uptake and trafficking of heat‐killed yeast particles within murine macrophages. Such yeast particles, which were fluorescently labeled, could be readily detected in intact cells as well as in subcellular fractions. Organelle electrophoresis of a homogenate from macrophages that had internalized yeast particles resulted in the separation of yeast‐containing organelles from most other subcellular membranes. In addition, this method was used to follow the kinetics of yeast particle transport within macrophages could be followed readily. Organelle electrophoresis may be a valuable tool for the analysis of phagocytosis.
Identifying sites of replication initiation in yeast chromosomes: Looking for origins in all the right places
Authors: van Brabant Anja J. , Hunt Sonia Y. , Fangman Walton L. , Brewer Bonita J. 
Published: May 2005

AbstractDNA fragments that contain an active origin of replication generate bubble‐shaped replication intermediates with diverging forks. We describe two methods that use two‐dimensional (2‐D) agarose gel electrophoresis along with DNA sequence information to identify replication origins in natural and artificial Saccharomyces cerevisiae chromosomes. The first method uses 2‐D gels of overlapping DNA fragments to locate an active chromosomal replication origin within a region known to confer autonomous replication on a plasmid. A variant form of 2‐D gels can be used to determine the direction of fork movement, and the second method uses this technique to find restriction fragments that are replicated by diverging forks, indicating that a bidirectional replication origin is located between the two fragments. Either of these two methods can be applied to the analysis of any genomic region for which there is DNA sequence information or an adequate restriction map.
Preparative electrophoresis in “sieving media” of subcellular‐sized particles
Authors: Chrambach Andreas , Chen Nong 
Published: May 2005

AbstractThe commercial gel electrophoresis apparatus with intermittent scanning of the migration path and preparative capacity (HPGE‐1000, LabIntelligence) is applicable to polymer solutions as well as gels. Unresolved rat liver microsomes can be isolated from 11–15% polyvinylpyrrolidone (PVP) solution by means of a syringe. The automated band isolation technique applied under resolving conditions in dilute polymer solutions allowed for the sequential isolation of three microsome components with 85, 76 and 75% recovery, respectively, under strict control of the dimensions of the volumetric collection module of the HPGE‐1000 apparatus. Separations of unlabeled microsomes and sea urchin egg components in dilute polymer solutions have been performed, using detection by “fluorescence reduction”. The unlabeled major component of a sea urchin egg homogenate has been isolated from electrophoresis in 1.5% PVP (Mr = 106) solution in 25–50% yield (0.24–4 μg/8 lanes of the HPGE‐1000 apparatus). However, since separations of both microsomes and sea urchin egg granules in dilute polymer solutions are restricted to a narrow range of polymer concentrations, their retardation coefficients, KR = d(log mobility)/d(polymer concentration), are not ascertained.
Electrophoretic separations using sweeping fields
Authors: Ying Feng , Mastrangelo Carlos H. , Burke David T. , Burns Mark A. 
Published: May 2005

AbstractSweeping‐field electrophoresis is investigated as a method for increasing the resolution of low‐voltage slab gel separations. In this technique a low direct current (DC) voltage is time multiplexed to an array of periodically spaced electrodes placed along the length of the slab in a manner that follows the band migration. Because the electrode spacing is smaller than the slab length, a larger field is generated, yielding an improved separation. The effect of the nonuniform electric field on band distortion is studied in some detail. Experimental band distortion results showed good agreement with theoretical predictions in a macroscopic sweeping‐field electrophoresis system. Both analytical and numerical results show that band distortion can be effectively minimized when an appropriate sweep rate is selected for a narrow band range. Using this scheme we have achieved the same number of theoretical plates as a DC‐driven system with one third of the drive voltage.
Characterization of Helicoverpa armigera gut proteinases and their interaction with proteinase inhibitors using gel X‐ray film contact print technique
Authors: Harsulkar Abhay M. , Giri Ashok P. , Gupta Vidya S. , Sainani Mohini N. , Deshpande Vasanti V. , Patankar Aparna G. , Ranjekar Prabhakar K. 
Published: May 2005

AbstractSince Helicoverpa armigera is a devastating pest, an attempt was made to separate its gut proteinases and assess their diversity. Gelatin coating present on the X‐ray film was used as a substrate to detect electrophoretically separated proteinases of H. armigera gut extract on native polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate (SDS)‐PAGE and isoelectric focusing gels. The method involves electrophoresis, followed by washing the gel with Triton X‐100 in case of SDS‐PAGE, equilibration of the gel in proteinase assay buffers, overlaying the gel on X‐ray film followed by washing the film with hot water to remove hydrolyzed gelatin revealing bands of proteinase activity. Using this protocol, at least six different proteinase isoforms were detected in H. armigera gut contents while three isoproteinases were identified in a commercial bacterial proteinase preparation. Adoption of the technique facilitated characterization of the H. armigera gut proteinases (HGP) and provided an easy tool to study the properties of the individual proteinases without purification. The approximate molecular masses of HGP as determined by SDS‐PAGE were: 172.9, 59.3, 54.9, 47.6, 44.1 and 41.6 kDa, and of bacterial proteinases: 180.7, 127.3 and 95.3 kDa. The isoelectric point (pI) values of HGP and bacterial proteinase were in the range of 5.1–7.1 and 3.5–7.7, respectively. Some of the HGP isoforms were found to be highly pH‐sensitive and showed activity only at pH 10.0. The major HGPs were inhibited by phenylmethylsulfonyl fluoride but not by (4‐amidinophenyl)‐methanesulfonyl fluoride. Incubation of HGP‐resolved electrophoretic gel strips in chickpea or winged bean proteinase inhibitor solution permitted identification of specific inhibitors of individual proteinases and revealed that the major HGPs were insensitive to chickpea inhibitors whereas winged bean inhibitors effectively inhibited all the HGPs. Our results suggest that considerable variability exists among the isoproteinases of H. armigera gut with respect to their pH optima and sensitivity towards chemical and plant proteinase inhibitors. Such diversity is of immense biological significance as it explains the polyphagous nature of the insect which imparts unique adaptability to it against the defensive proteinase inhibitors of its wide range of host plants.
A three‐wavelength labeling approach for DNA sequencing using energy transfer primers and capillary electrophoresis
Authors: Kheterpal Indu , Li Lei , Speed Terence P. , Mathies Richard A. 
Published: May 2005

AbstractCapillary electrophoresis DNA sequencing has been accomplished by using four different energy transfer primers and three fluorescence detection channels. Methods have also been developed to deconvolve the three‐color data into the four base concentrations. The nonnegative least squares and model selection method resulted in the best accuracy. The three‐color data were compared to sequencing data obtained using four detection channels and four energy transfer primers. The average accuracy rates obtained over three 500 base M13mp18 runs using three‐color coding were 96% including 18 uncallable compressions and 99.6% if these compressions are excluded. The average accuracy rate obtained using four‐color coding was 96.3% including 18 uncallable compressions and 99.9% if these compressions are excluded. Although it is unlikely that three‐color schemes will replace four‐color sequencing, these methods have exposed basic concepts that will be useful in the development of higher‐order multiplex coding methods for DNA analysis.
Multi‐capillary optical waveguides for DNA sequencing
Authors: Quesada Mark A. , Dhadwal Harbans S. , Fisk David , Studier F. William 
Published: May 2005

AbstractCylindrial capillaries can be used as optical elements in a waveguide, where refraction will confine an appropriately focused light beam to pass through the interiors of successive capillaries in a flat parallel array. Such a capillary waveguide allows efficient illumination of samples in multiple capillaries with relatively little laser power. Analytical expressions derived under paraxial and thin‐lens approximations provide guidance in selecting the capillary sizes and the refractive indices that will produce the waveguiding effect, but accurate predictions require exact ray tracing. Small reflective losses as the light passes through the capillary surfaces cause cumulative intensity decreases, but the resulting lack of uniformity can be compensated to a considerable extent by illuminating the capillary array from both sides. A 12‐capillary waveguide illuminated from both sides in air has a difference of less than 10% from the strongest to the weakest illumination. By increasing the refractive index of both the external medium and the contents of the capillaries, a 96‐capillary waveguide for DNA sequencing could be produced that would also provide nearly uniform illumination. A 12‐capillary, bi‐directionally illuminated waveguide system for DNA sequencing has been constructed. The two focused laser beams are delivered by integrated fiber optic transmitters (IFOTs), and fluorescence is collected by a set of optical fibers whose spacing exactly matches that of the capillaries in the waveguide. The system is easy to align and provides sensitive detection of fluorescence with minimal cross‐talk between channels.
Quantitative analysis of synthetic dyes in lipstick by micellar electrokinetic capillary chromatography
Authors: Desiderio Claudia , Marra Carolina , Fanali Salvatore 
Published: May 2005

AbstractThe separation of synthetic dyes, used as color additives in cosmetics, by micellar electrokinetic capillary chromatography (MEKC) is described in this study. The separation of seven dyes, namely eosine, erythrosine, cyanosine, rhodamine B, orange II, chromotrope FB and tartrazine has been achieved in about 3 min in an untreated fused silica capillary containing as background electrolyte a 25 mM tetraborate/phosphate buffer, pH 8.0, and 30 mM sodium dodecyl sulfate. The electrophoretic method exhibits precision and relatively high sensitivity. A detection limit (LOD, signal/noise = 3) in the range of 5–7.5 × 10−7 M of standard compounds was recorded. Intra‐day repeatability of all the studied dye determinations (8 runs) gave the following results (limit values), % standard deviation: 0.24–1.54% for migration time, 0.99–1.24% for corrected peak areas, 0.99–1.24% for corrected peak area ratio (analyte/internal standard) and 1.56–2.74% for peak areas. The optimized method was successfully applied to the analysis of a lipstick sample where eosine and cyanosine were present.
An exactly solvable Ogston model of gel electrophoresis IV: Sieving through periodic three‐dimensional gels
Authors: Mercier Jean‐François , Slater Gary W. 
Published: May 2005

AbstractIn this article, we extend our recently developed lattice model of gel electrophoresis to periodic three‐dimensional gels made of either isolated obstacles or infinitely long fibers. Exact mobilites are calculated using a much improved numerical method that allows us to treat very large systems. A comparison of the exact mobilities and free available volumes indicates that the main assumption of the Ogston‐Morris‐Rodbard‐Chrambach model (OMRCM), which postulates that the mobility (μ) of charged particles is directly related to the fractional gel volume available to them, is not valid. However, a study of the gel concentration and analyte size dependence of the zero‐field mobility indicates that the OMRCM and the Ferguson plots can indeed be used to obtain useful, semi‐quantitative information about the gel properties. A procedure to study more realistic three‐dimensional gel systems is discussed.
Normal and anomalous electrophoretic behavior of polymerase chain reaction‐based DNA polymorphisms in polyacrylamide gels
Authors: Lareu Victoria , Pestoni Carmela , Phillips Christopher , Barros Francisco , Carracedo Angel , Court Denise Syndercombe , Lincoln Patrick 
Published: May 2005

AbstractThe electrophoretic mobility of five single tandem repeats (STRs) and four amplified fragment length polymorphisms (AFLPs) in polyacrylamide gels was tested under denaturing and nondenaturing conditions. Relative anomalous mobility in nondenaturing conditions was found in one AT‐rich AFLP (3′ ApoB) and in two AT‐rich STRs (HUMACTBP2 and HUMF13A1). In these cases, highly anomalous electrophoretic behavior was found even when changes were made in the %T value alone. In such cases typing results were affected by the gel composition. The mobility of these systems was nevertheless normal under denaturing conditions. As a consequence of this study, we recommended that the typing of these systems should only be performed under denaturing conditions and we also recommend the further study of the electrophoretic behavior of repetitive DNA polymorphisms, especially AT‐rich systems, before they are used in forensic or genetic applications.