bluuebunny/crossref_metadata_2025_split huggingface dataset:
On‐line protein digestion and peptide mapping by capillary electrophoresis with post‐column labeling for laser‐induced fluorescence detection
Authors: Ye Mingliang , Hu Shen , Schoenherr Regine M. , Dovichi Norman J. 
Published: May 2004

AbstractA nanoliter enzyme microreactor was developed for on‐line capillary electrophoresis (CE) peptide mapping of proteins, allowing picomole quantities of proteins to be digested. The enzyme microreactor was formed by immobilizing trypsin onto a monolithic capillary column, which was prepared by in situ polymerization of glycidyl methacrylate and ethylene dimethacrylate in a capillary. Highly efficient digestion of three protein standards was demonstrated. The detection of peptide fragments in CE was enhanced by post‐column derivatization and laser‐induced fluorescence detection. The microreactor has a volume of about 30 nL and is coupled with a separation capillary via a fluid joint for on‐line digestion. The overall analysis, including digestion and separation, lasted only about 16 min. Column efficiencies > 300 000 plates/m were obtained for most peaks in the electropherogram of an on‐line peptide mapping experiment of denatured α‐lactalbumin under optimal conditions.
Blue silver: A very sensitive colloidal Coomassie G‐250 staining for proteome analysis
Authors: Candiano Giovanni , Bruschi Maurizio , Musante Luca , Santucci Laura , Ghiggeri Gian Marco , Carnemolla Barbara , Orecchia Paola , Zardi Luciano , Righetti Pier Giorgio 
Published: May 2004

AbstractA modified Neuhoff's colloidal Coomassie Blue G‐250 stain is reported, dubbed “blue silver” on account of its considerably higher sensitivity, approaching the one of conventional silver staining. The main modifications, as compared to Neuhoff's protocol, were: a 20% increment in dye concentration (from 0.1% up to 0.12%) and a much higher level of phosphoric acid in the recipe (from 2% up to 10%). The “blue silver” exhibits a much faster dye uptake (80% during the first hour of coloration, vs. none with a commercial preparation from Sigma). Even at equilibrium (24 h staining), the “blue silver” exhibits a much higher sensitivity than all other recipes, approaching (but lower than) the one of the classical silver stain. Measurements of stain sensitivity after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of bovine serum albumin (BSA) gave a detection limit (signal‐to‐noise ratio > 3) of 1 ng in a single zone. The somewhat lower sensitivity of “blue silver” as compared to classical silvering protocols in the presence of aldehydes is amply compensated for by its full compatibility with mass spectrometry of eluted polypeptide chains, after a two‐dimensional map analysis, thus confirming that no dye is covalently bound (or permanently modifies) to any residue in the proteinaceous material. It is believed that the higher level of phosphoric acid in the recipe, thus its lower final pH, helps in protonating the last dissociated residues of Asp and Glu in the polypeptide coils, thus greatly favoring ionic anchoring of dye molecules to the protein moiety. Such a binding, though, must be followed by considerable hydrophobic association with the aromatic and hydrophobic residues along the polypeptide backbone.
Intact mass measurements for unequivocal identification of hydrophobic photosynthetic photosystems I and II antenna proteins
Authors: Zolla Lello , Rinalducci Sara , Timperio Anna Maria , Huber Christian G. , Righetti Pier Giorgio 
Published: May 2004

AbstractThe purpose of this study was to determine the optimal experimental conditions necessary to allow rapid and accurate identification of highly hydrophobic proteins, such as the antenna proteins from photosystems I and II. The antenna proteins were derived from two different species, tomato and Arabidopsis, whose photosynthetic genome is well known. The separation techniques included sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) followed by immunoblotting, microamino acid sequencing, reversed‐phase high‐performance liquid chromatography (RP‐HPLC), intact mass measurements, and peptide mass fingerprinting by mass spectrometry. Immunoblotting was time‐consuming, and success was limited due to cross‐reactivity between these highly conserved sequences. The best results by far were achieved by separating intact proteins through hyphenation of reversed‐phase liquid chromatography on‐line to an electrospray ionization mass spectrometer (ESI‐MS) and consequently identifying individual proteins from their intact mass measurements (IMMs), whereas peptide mass fingerprinting was hampered by the highly hydrophobic nature of these proteins. RP‐HPLC‐ESI‐MS was superior in the quality of separation. Moreover, the high quality of mass spectra recorded during the RP‐HPLC‐ESI‐MS analysis meant that the relative deviations of the molecular masses determined with a quadrupole ion trap mass analyzer ranged between 100 and 300 ppm. Thus, the correspondence between the intact mass values measured with those deduced from the DNA sequences allowed the different types of antenna proteins to be identified and assigned to their corresponding gene families. By utilizing this correlation, it was possible to spot gene products of previously cloned genes.
Comparison of two glutaraldehyde immobilization techniques for solid‐phase tryptic peptide mapping of human hemoglobin by capillary zone electrophoresis and mass spectrometry
Authors: Migneault Isabelle , Dartiguenave Catherine , Vinh Joëlle , Bertrand Michel J. , Waldron Karen C. 
Published: May 2004

AbstractStabilization of proteolytic enzymes, especially by immobilization, is of considerable interest because of their potential applications in medicine and the chemical and pharmaceutical industries. We report here a detailed comparison of two procedures for trypsin immobilization using the same homobifunctional agent, glutaraldehyde, for the purpose of peptide mapping. These methods include covalent coupling either to controlled pore glass (solid support) or via a cross‐linking reaction (without any solid support). The immobilized trypsin preparations were characterized by the determination of immobilization efficiency, which ranged from 68 to > 95%, and measurement of apparent kinetic parameters toward a synthetic peptide‐like substrate. Batch digestions of whole denaturated human normal adult hemoglobin (HbA) were performed to obtain peptide maps by capillary zone electrophoresis (CZE). Migration time reproducibility of the CZE maps was excellent, with a mean relative standard deviation of 1.5%. Moreover, the two immobilized enzyme preparations showed excellent reproducibility for repeated digestions. Matrix‐assisted laser desorption/ionization (MALDI)‐mass spectrometry was also used for peptide mass mapping of denaturated HbA digested using the two immobilized trypsin preparations. Even though the two immobilized trypsin preparations do not behave identically, similar sequence coverages of 57% and 61% (for the two HbA chains merged) were achieved for the support‐based and cross‐linked trypsin preparations, respectively.
Peptide separations by slab gel electrophoresis in Pluronic F127 polymer liquid crystals
Authors: Rill Randolph L. , Al‐Sayah Mohammad A. 
Published: May 2004

AbstractProteomics and peptidomics could benefit from simple methods for high‐resolution separation of oligopeptides analogous to slab gel electrophoresis of proteins. Gels of Pluronic F127 copolymer surfactant were investigated as media for slab gel electrophoresis of oligopeptides using a trypsin digest of myoglobin. Concentrated solutions of Pluronic F127 are fluid at low temperatures (≤ 5°C), but become a gel‐like micellar liquid crystal upon warming. Nucleic acids are well separated by electrophoresis in these gels as previously shown by Rill and Liu. Good separations of myoglobin tryptic peptides were accomplished by electrophoresis on slab gels of 24% Pluronic F127 or 15% polyacrylamide using the alkaline Laemmli buffer system (without sodium dodecyl sulfate). Labeling of peptides with the succinimidyl ester of Cascade Yellow (CY) prior to electrophoresis allowed sensitive detection with blacklight illumination at 365 nm. Labeled tryptic peptides were identified by matrix‐assisted laser desorption/ionization‐time of flight (MALDI‐TOF)‐mass spectrometry. An inverse dependence of electrophoretic mobility on mass of peptides with charge Z = −1 was observed in both media. Two‐dimensional (2‐D) electrophoresis of myoglobin peptides on polyacrylamide, then on Pluronic media, at pH 8.3 indicated that the primary separation mechanism of most peptides was the same in both media. A few off‐diagonal spots indicated that some peptides were preferentially retarded in Pluronic gels, perhaps due to hydrophobic effects. The ease of gel preparation and peptide recovery are advantages of Pluronic F127 gels for oligopeptide electrophoresis. The two media can be combined conveniently for 2‐D electrophoresis, providing means to facilitate protein identification and peptidomics.
Capillary electrophoresis of single mammalian cells
Authors: Woods Lori A. , Roddy Thomas P. , Ewing Andrew G. 
Published: May 2004

AbstractCapillary electrophoresis (CE) has been established as powerful tool for single cell analysis. Newly developed sampling, separation and detection methods have allowed the investigation of single mammalian cells with CE despite their small size and complex composition. Advances in sample injection techniques include several novel methods for the injection of whole cells and sampling techniques for the study of cellular secretion. CE of single mammalian cells has been applied in a wide range of fields including protein analysis, neuroscience, and oncology. The development of new detection schemes in the analysis of single mammalian cells with CE has included studies of protein expression and the utilization of mass spectrometric and electrochemical detection. Subcellular mammalian cell analysis with CE also has been investigated.
Selectivity of quadruplex DNA stationary phases toward amino acids in homodipeptides and alanyl dipeptides
Authors: Vo Trang U. , McGown Linda B. 
Published: May 2004

AbstractSeries of dipeptides, including homodipeptides and alanyl dipeptides, were separated using quadruplex (G‐quartet) DNA stationary phases in open‐tubular capillary electrochromatography (OTCEC). The stationary phases were constructed by covalently attaching the DNA oligonucleotides to the inner capillary surface. Three different G‐quartet forming oligonucleotides were investigated: the two‐plane G‐quartet forming thrombin‐binding aptamer, the four‐plane analogue of the thrombin‐binding aptamer, and a two‐plane oligonucleotide identical to the thrombin‐binding aptamer except for the replacement of the guanine by thymine in the central bridging loop of the G‐quartet structure. Results were compared with results obtained using capillary electrophoresis on a bare capillary and OTCEC using an oligonucleotide with the same base composition as the thrombin‐binding aptamer but in a different sequence that does not allow G‐quartet formation as the stationary phase.
An investigation into the human serum “interactome”
Authors: Zhou Ming , Lucas David A. , Chan King C. , Issaq Haleem J. , Petricoin Emanuel F. , Liotta Lance A. , Veenstra Timothy D. , Conrads Thomas P. 
Published: May 2004

AbstractThe protein content of human serum is composed of a millieu of proteins from almost every type of cell and tissue within the body. The serum proteome has been shown to contain information that directly reflects pathophysiological states and represents an invaluable source of diagnostic information for a variety of different diseases. Unfortunately, the dynamic range of protein abundance, ranging from ≫ mg/mL level to ≪ pg/mL level, renders complete characterization of this proteome nearly impossible with current analytical methods. To study low‐abundance proteins, which have potential value for clinical diagnosis, the high‐abundant species, such as immunoglobulins and albumin, are generally eliminated as the first step in many analytical protocols. This step, however, is hypothesized to concomitantly remove proteins/peptides associated with the high‐abundant proteins targeted for depletion. In this study, immunoprecipitation was combined with microcapillary reversed‐phase liquid chromatography (μRPLC) coupled on‐line with tandem mass spectrometry (MS/MS) to investigate the low‐molecular‐weight proteins/peptides that associate with the most abundant species in serum. By this targeted isolation of select highly abundant serum proteins, the associated proteins/peptides can be enriched and effectively identified by μRPLC‐MS/MS. Among the 210 proteins identified, 73% and 67% were not found in previous studies of the low‐molecular‐weight or whole‐serum proteome, respectively.
Membrane‐mediated capillary electrophoresis: Interaction of cationic peptides with bicelles
Authors: Mills John O. , Holland Lisa A. 
Published: May 2004

AbstractElectrokinetic capillary chromatography is applied to determine the membrane affinity of peptides using both 1,2‐dihexanoyl‐sn‐glycero‐3‐phosphocholine (DHPC) micelles and DHPC/1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC) bicelles under controlled conditions. The effect of temperature and the bicelle q value in surface association with cationic peptides is studied. The cationic peptides selected have a well‐defined membrane structure (indolicidin), induced secondary structure (melittin, magainin 2), or do not possess classical secondary structure (atrial natriuretic peptide (ANP) 1‐28, 4‐28, 5‐27). Electrokinetic capillary chromatography facilitated by DMPC and DHPC additives provides a rapid means of estimating lipophilicity and screening for peptides that have membrane affinity.
Proteomic analysis of carbonylated proteins in two‐dimensional gel electrophoresis using avidin‐fluorescein affinity staining
Authors: Yoo Byoung‐Sam , Regnier Fred E. 
Published: May 2004

AbstractA method for detecting carbonylated proteins in two‐dimensional electrophoresis (2‐DE) was developed using biotinylation and avidin‐fluorescein isothiocyanate (FITC) affinity staining. The method was used to examine oxidatively modified proteins associated with oxidative stress. Carbonyl formation in proteins was first examined in a model system by subjecting bovine serum albumin (BSA) and ribonuclease A (RNase A) to metal‐catalyzed oxidation (MCO). Carbonyl group formation was found to occur at multiple sites along with a small amount of polypeptide chain cleavage. In vivo studies were conducted in yeast cell cultures using 5 mM hydrogen peroxide to induce oxidative stress. Biotinylation of yeast protein was accomplished during extraction at 4°C in a lysis buffer containing 5 mM biotin‐hydrazide. Biotin‐hydrazide forms a Schiff base with a carbonyl group on an oxidized protein that is subsequently reduced before electrophoresis. Proteins were separated by either 2‐DE or sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Biotinylated species were detected using avidin‐FITC affinity staining. Detection sensitivity with biotinylated proteins was five times higher than achieved by silver staining. The limit of detection with avidin‐FITC staining approached 0.64 pmol of protein‐associated carbonyls. Twenty carbonylated proteins were identified in the proteome of yeast following oxidative stress with hydrogen peroxide. Matrix‐assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS) analysis of tryptic peptides was used to identify peptides extracted from gels. Aconitase, heat shock protein SSA1 and SSC1, pyruvate decarboxylase isozyme 1, pyruvate kinase 1, enolase 1 and 2, phosphoglycerate kinase, fructose‐bisphosphate aldorase, and glyceraldehyde‐3‐phosphate dehydrogenase were among the major targets of oxidative stress.
Full scale experiments for evaluating theoretical fire wall models
Authors: Clancy P. , Young S. A. 
Published: May 2004

AbstractThe aim of the research described in this paper was to provide experimental results for the evaluation of theoretical models for predicting the behaviour and time‐to‐failure of loadbearing and non‐loadbearing wood framed walls in fire. References for thermal and mechanical properties of wood and gypsum board are given to provide comprehensive input for the evaluation of theoretical wall models. The scope of the research involved full‐scale uninsulated cavity walls with well‐controlled clearly known conditions including initial ambient vertical load capacity for benchmarking the reduction in capacity and stiffness, rotational stiffness of supports, eccentricity of vertical load, elastic moduli of wood and gypsum board in compression, stiffness of slip between gypsum board and studs and end stud effects. The experiments were repeated and they demonstrated that the controls led to high consistency in the results despite the inherent large variability of the mechanical properties of wood. The results include temperature distributions, initial vertical load capacity, load‐deflection plots and times‐to‐failure. The results show that the temperatures in the studs are approximately uniform until all the moisture is vaporized. Thermal properties of wood will not vary significantly for consistent density, moisture content and species of wood. The main structural actions that should be modelled for different loading regimes are deduced. Copyright © 2004 John Wiley & Sons, Ltd.
High‐order accurate numerical solutions of incompressible flows with the artificial compressibility method
Authors: Ekaterinaris John A. 
Published: May 2004

AbstractA high‐order accurate, finite‐difference method for the numerical solution of incompressible flows is presented. This method is based on the artificial compressibility formulation of the incompressible Navier–Stokes equations. Fourth‐ or sixth‐order accurate discretizations of the metric terms and the convective fluxes are obtained using compact, centred schemes. The viscous terms are also discretized using fourth‐order accurate, centred finite differences. Implicit time marching is performed for both steady‐state and time‐accurate numerical solutions. High‐order, spectral‐type, low‐pass, compact filters are used to regularize the numerical solution and remove spurious modes arising from unresolved scales, non‐linearities, and inaccuracies in the application of boundary conditions. The accuracy and efficiency of the proposed method is demonstrated for test problems. Copyright © 2004 John Wiley & Sons, Ltd.
Simulation of single‐ and two‐phase flows on sliding unstructured meshes using finite volume method
Authors: Basara B. , Alajbegovic A. , Beader D. 
Published: May 2004

AbstractThe paper presents an efficient finite volume method for unstructured grids with rotating sliding parts composed of arbitrary polyhedral elements for both single‐ and two‐phase flows. Mathematical model used in computations is based on the ensemble averaged conservation equations. These equations are solved for each phase and in case of single‐phase flow reduce to the transient Reynolds‐averaged Navier–Stokes (TRANS) equations. Transient flow induced by rotating impellers is thus resolved in time. The use of unstructured grids allows an easy and flexible meshing for the entire flow domain. Polyhedral cell volumes are created on the arbitrary mesh interface placed between rotating and static parts. Cells within the rotating parts move each time step and the new faces are created on the arbitrary interfaces only, while the rest of the domain remain ‘topologically’ unchanged. Implicit discretization scheme allows a wide range of time‐step sizes, which further reduce the computational effort. Special attention is given to the interpolation practices used for the reconstruction of the face quantities. Mass fluxes are recalculated at the beginning of each time step by using an interpolation scheme, which enhances the coupling between the pressure and velocity fields. The model has been implemented into the commercially available CFD code AVL SWIFT (AVL AST, SWIFT Manual 3.1, AVL List GmbH, Graz, Austria, 2002). Single‐phase flow in a mixing vessel stirred by a six‐bladed Rushton‐type turbine and two‐phase flow in aerated stirred vessel with the four‐blade Rushton impeller are simulated. The results are compared with the available experimental data, and good agreement is observed. The proposed algorithm is proved to be both stable and accurate for single‐phase as well as for the two‐phase flows calculations. Copyright 2004 John Wiley & Sons, Ltd.
Assessment of sex‐specific genetic and environmental effects on bone mineral density
Authors: Brown Lillian B. , Streeten Elizabeth A. , Shuldiner Alan R. , Almasy Laura A. , Peyser Patricia A. , Mitchell Braxton D. 
Published: May 2004

AbstractAlthough it is widely accepted that genes contribute significantly to the variation in bone mineral density (BMD), the nature of the genetic contribution is poorly defined. There are large gender differences in BMD, although whether sex‐specific genetic effects influencing variation in BMD contribute to these differences is not known. To address this issue, we studied 929 subjects from large families participating in the Amish Family Osteoporosis Study. Bone mineral density was measured at the hip and spine by dual energy x‐ray absorptiometry (DXA). We used variance decomposition procedures to partition variation in BMD into genetic and environmental effects common to both sexes and to men and women separately. After accounting for covariate effects, the heritability of BMD ranged from 0.63 to 0.72 in men and 0.80 to 0.87 in women. The residual environmental variance in BMD at the spine, but not hip, was significantly higher in men than in women (P < 0.05), reflecting a greater variance in BMD due to unexplained non‐genetic factors in men. In contrast, there were no significant differences between men and women in the magnitude of the genetic variance in BMD, nor did the genetic correlation in BMD between men and women differ significantly from one. Overall, these analyses do not provide evidence for sex‐specific genetic effects, suggesting that many of the genes influencing variation in BMD should be detectable in both men and women. © 2004 Wiley‐Liss, Inc.
End‐to‐side venous anastomosis with the internal jugular vein stump: A preliminary report
Authors: Graham Brian B. , Varvares Mark A. 
Published: May 2004

AbstractBackground.Failure of free flaps is frequently because of failure of the venous vascular anastomosis. A new venous anastomosis technique that uses the stump of the internal jugular vein (IJV) is described, with preliminary data on operative outcomes.Methods.Retrospective analysis of eight head and neck free flap operations in which a venous anastomosis to the stump of an IJV was used compared with 222 other free flap operations in the same period (1995–2001) at a single institution.Results.None of the eight free flaps that used venous end‐to‐side anastomoses to the stump of an IJV failed. There were 15 failures associated with the other 222 venous microvascular anastomoses (6.8%).Conclusions.This new technique resulted in no failures in eight operations. Advantages of this technique include avoiding creating an anastomosis under tension or requiring a saphenous vein graft and reducing pedicle torsion at the hilum of the flap. © 2004 Wiley Periodicals, Inc. Head Neck 26: 537–540, 2004