bluuebunny/crossref_metadata_2025 huggingface dataset:
Gel and free solution electrophoresis of variably charged polymers
Authors: Hoagland David A. , Smisek David L. , Chen Derek Y.
Published: May 2005

To assess the role of charge density on polyelectrolyte mobility, both gel and free solution electrophoresis experiments are performed on poly(acrylic acid) and acrylic acid/acrylamide copolymers. Control of charge density for poly(acrylic acid) is achieved through solution pH, while control for acrylic acid/acrylamide copolymers is obtained through chain composition. In either approach, the effective fraction of charged repeat units can be varied from 0 to 100% without a major interruption of solvent quality. Polyelectrolyte mobility in the presence of a monovalent counterion is observed to rise linearly with charge density when this density is low. A transition to charge density independence then occurs over a surprisingly narrow window of charge density. For vinyl polymers of the sort examined here, the transition occurs when 35–40% of the repeat units are charged. These observations are qualitatively consistent with the free solution electrophoresis model proposed by Manning and several previous data sets. An unexpected overlap of normalized gel and free solution data reveals that the charge density exerts a comparable influence in either environment. Results from the present study help define the experimental conditions in which electrophoresis can provide polymer separation by charge density and those in which the method can provide polymer separation by molecular weight.
Electrophoretic mobility of composite objects in free solution: Application to DNA separation
Authors: Long Didier , Ajdari Armand
Published: May 2005

We propose here a simple procedure to estimate the electrophoretic mobility of composite objects obtained by linking two charged subunits, stressing that this electrophoretic mobility is in general different from the ratio of the total charge to the total friction coefficient. We focus especially on the situation where at least one of the subunits is a polyelectrolyte. Our remarks in particular correct the existing theoretical analysis of separation capabilities of devices in which a buoy (sphere, protein, polymer) is attached to a DNA chain. We also predict that, in some cases, the direction of electrophoretic motion can be reversed by increasing the electric field amplitude.
Theory of electrophoretic mobility of a polyelectrolyte in semidilute solutions of neutral polymers
Authors: Muthukumar Murugappan
Published: May 2005

An explicit formula is derived for the electrophoretic mobility of a polyelectrolyte molecule in a semidilute solution of neutral polymers in terms of the molecular weight N of a polyelectrolyte, concentration c of neutral polymers, Debye length and solvent quality. The nature of the crossover between different regimes, as c, N, and the salt concentration are varied, is presented. The calculations show that one progressively enters into regimes of no‐separation, separation, and no‐separation in the electrophoresis of polyelectrolytes as c is increased. If c is very high, one returns to the mechanism of separation in gel electrophoresis.
Use of neutral surfactants for the capillary electrophoretic separation of hydrophobically modified poly(acrylic acids)
Authors: Collet Jérǒme , Tribet Christophe , Gareil Pierre
Published: May 2005

Hydrophobically modified poly(acrylic acids) (HMPAs) are random copolymers of sodium acrylate and dodecyl acrylamide, containing 0–10% mol/mol of dodecyl grafts. The hydrophobic character of different HMPAs of average molecular weight 150 000 was studied by capillary electrophoresis (CE), using neutral surfactants as buffer additives. The differentiation of the electrophoretic mobilities of HMPAs with their hydrophobicity was achieved through the use of nonionic Brij 35 and zwitterionic DAPS surfactants. A nearly baseline separation of the precursor and three HMPAs derivatives was obtained in a poly(ethylene glycol)‐coated capillary with a background electrolyte containing 10 mM N‐dodecyl‐N,N‐dimethyl‐3‐ammonio‐1‐propanesulfonate (DAPS) and 10 mM borax (pH 9.2). In addition to CE experiments, the polymer‐surfactant interactions were also investigated by means of quasi‐elastic light scattering (QELS) and viscosimetric measurements. According to the latter results, the separation mechanism was interpreted as an expansion of the polymer coil in the presence of micelles and subsequent change of its frictional properties. A true micellar electrokinetic capillary chromatography (MEKC) partitioning model was discarded on the basis of the relative sizes of the macromolecule and the micelles.
Determination of denatured serum proteins in the casein fraction of heat‐treated milk by capillary zone electrophoresis
Authors: Recio Isidra , Olieman Cornelis
Published: May 2005

The amount of heat‐denatured serum proteins in heat‐treated milk could be estimated by analyzing the casein fraction, obtained by isoelectric precipitation at pH 4.6, by capillary zone electrophoresis. A hydrophilically coated capillary was used in combination with 6 M urea in a citrate buffer at pH 3. Optimization of the sample and running buffer minimized adsorption of serum proteins, especially that of bovine serum albumin (BSA). This afforded a detection limit down to ca. 5–65 μg/mL of the three main serum proteins in milk. The detector response (UV at 214 nm) was linear in the range of 0.05–0.35 and 0.05–0.85 mg/mL for α‐lactalbumin and β‐lactoglobulin, respectively. BSA showed a slightly less linear behavior, due to residual adsorption to the capillary wall. The recovery of serum proteins was in the range of 89–107%. The method was evaluated by analyzing Dutch commercial milks and cheese milk, which had received increasing heat loads. The additions of milk powder to pasteurized milk could be detected by this method as well as the serum protein to casein ratio in various products.
Computer optimization of background electrolyte composition in the separation of metal ions by capillary electrophoresis
Authors: Billiet Hugo A. H. , Andersson Per E. , Haddad Paul R.
Published: May 2005

The iterative regression design developed earlier for high performance liquid chromatography (HPLC) has been applied successfully to the optimization of the separation of 11 metal ions by capillary electrophoresis. The parameters used for optimization were pH (over the range 3.5–5.0) and the concentration of 2‐hydroxyisobutyric acid (HIBA) (2–20 mM) used as the complex‐forming ligand added to the background electrolyte. Five initial experiments were performed and the response surface was calculated using the relative resolution product as criterion. Three additional experiments were sufficient to establish a stable optimum, with which all 11 metal ions were separated and well spread in the electropherogram. The total analysis time was less than 5 min.
Fish species identification by isoelectric focusing of parvalbumins in immobilized pH gradients
Authors: Esteve‐Romero Josep S. , Yman Ingrid Malmheden , Bossi Alessandra , Righetti Pier Giorgio
Published: May 2005

Isoelectric focusing in immobilized pH gradients (IPG) is reported for unequivocal identification of fish species. Three orders of fishes have been analyzed: salmons, flat and cod fishes. In each family, four closely related species have been analyzed. For clear‐cut species identification, two strategies have been adopted: (i) to perform IPGs in very narrow (1 pH unit and less) acidic gradients, typically spanning the pH 4–5 range, where fewer proteins are present and the pattern is much clearer; (ii) to focus the analysis on the parvalbumins, since this protein class is highly species‐specific and resistant to heat. Thus, not only fresh muscle could be analyzed, but also boiled fish samples. In all cases unambiguous determination of each species could be performed, either by simple visual band inspection or, in the most difficult cases, by densitometric evaluation of the Coomassie‐blue stained profiles. The analysis was performed in extracts of single species and also in mixtures of the most closely related species.
The construction of the World Wide Web‐accessible myocardial two‐dimensional gel electrophoresis protein database “HEART‐2DPAGE”: A practical approach
Authors: Pleißner Klaus‐Peter , Sander Stefan , Oswald Helmut , Regitz‐Zagrosek Vera , Fleck Eckart
Published: May 2005

The construction of the two‐dimensional electrophoresis (2‐DE) gel protein database “HEART‐2DPAGE” for human heart proteins, accessible via the World Wide Web (WWW), is described. This database provides the opportunity to retrieve descriptive information interactively by mouse clicking on a protein spot within a 2‐DE gel image or to retrieve the position of a protein if its protein name or a search expression is given. The realization of searching for proteins, the creation of clickable spots, marked by crosses or textual information, and interactive viewing opportunities are discussed from a softwareoriented point of view. The database takes into account that some human heart proteins are chamber specific. Several Common Gateway Interface (CGI) scripts are described and some practical experience is discussed.
High‐performance field inversion capillary electrophoresis of 0.1–23 kbp DNA fragments with low‐gelling, replaceable agarose gels
Authors: Chen Nong , Wu Lijuan , Palm Anders , Srichaiyo Tasanee , Hjertén Stellan
Published: May 2005

Field inversion capillary gel electrophoresis (FICGE) has been used for the separation of 0.1–23 kbp DNA fragments in a low‐melting, low‐gelling agarose gel. The influence of the amplitude of the voltage pulses, the pulse times and gel concentrations on the separation factor has been studied and found to be similar to that of polyacrylamide gels. Very high resolution can be obtained by altering continuously the pulse times and/or voltage according to a program tailor‐made for the size of the DNA molecules to be separated (programmed FICGE). The advantage of these agarose gels in comparsion with polyacrylamide gels is that they are nontoxic, easy to prepare and they have high UV transmission. They give a resolution that is equivalent to or better than that of polyacrylamide gels, and the risk of bubble formation in the gel is small. In addition, they are replaceable and, therefore, very convenient for automated analyses.
Capture in the gel: Intermoleculare triplex formation during gel electrophoresis
Authors: Belotserkovskii Boris P. , Johnston Brian H.
Published: May 2005

Analysis of unusual gel mobility patterns formed by certain DNA triplexes has revealed that intermolecular triplex formation can occur during gel electrophoresis when a faster migrating single strand overtakes a slower migrating band containing a duplex of appropriate sequence. Control experiments showed that this capture of the third strand occurs by sequence‐specific hybridization rather than some nonspecific retardation. This phenomenon can be used to detect triplexes by a gel‐shift assay even if their lifetime is much shorter than the time of gel electrophoresis.
Quantitation of glycated hemoglobins in human adult blood by capillary isoelectric focusing
Authors: Conti Monica , Gelfi Cecilia , Bosisio Adriana Bianchi , Righetti Pier Giorgio
Published: May 2005

A precise and reproducible method for assessment of glycated hemoglobin in human adult red blood cells is reported, based on capillary isoelectric focusing (IEF). In order to obtan baseline resolution between adult hemoglobin (Hb A) and its glycated form (Hb A1c), two species which differ by minute ΔpI values, < 0.03 pH units, the following procedure was adopted: the focusing mixture consisted of 5% Ampholine, pH 6–8, 0.5% Pharmalyte, pH 3–10, 3% short‐chain liquid polyacrylamide and an equimolar mixture of two “separators”, 0.33 M β‐alanine and 0.33 M 6‐aminocaproic acid. The last two compounds flatten the pH gradient in the pI region of the two Hbs, thus allowing full separation. Additionally, the Hb samples, instead of being pulse‐loaded, are uniformly distributed in the background electrolyte. A longer capillary lifetime is obtained if all nonbuffering ions are eliminated; thus, as catholyte, 50 mM Lys (pH 9.7) is utilized and as anolyte 50 mM acetic acid (pH 3.5) is adopted. The percentages of Hb A1c, as obtained by capillary IEF, are in good agreement (±6%) with data obtained by one of the standard zone electrophoretic methods in clinical chemistry, i.e., the Helena REP Glyco gel system.
Selecting and amplifying one fragment from a DNA fragment mixture by polymerase chain reaction with a pair of selective primers
Authors: Matsunaga Hiroko , Kohara Yoshinobu , Okano Kazunori , Kambara Hideki
Published: May 2005

A new method for selecting and amplifying a single DNA fragment from a mixture is proposed. This method is applicable for the rapid classification of DNA fragments from a mixture and for preparation of sequencing templates. DNAs of several to tens of kilobases (kb) are digested with a four‐base recognition restriction enzyme to produce smaller fragments. The complementary strand extension reactions are then carried out to produce fluorophore‐labeled DNA fragments from the digestion products. These fragments can be rapidly classified accrording to their terminal‐base sequences and their sizes are analyzed by capillary‐array gel electrophoresis (CAGE). Electropherograms are used to characterize the fragments and to select polymerase chain reaction (PCR) primers. Any fragment in a digestion mixture can be amplified by PCR with a pair of primers selected from a primer pool by referring to the electropherograms of the fragments. This method was successfully used to compare the electropherograms of two different DNA strands and to sequence a several‐kb DNA fragment without subcloning. Combined with CAGE, this method could be used to dramatically simplify DNA fragment analysis.
DNA sequencing using a four‐color confocal fluorescence capillary array scanner
Authors: Kheterpal Indu , Scherer James R. , Clark Steven M. , Radhakrishnan Arun , Ju Jingyue , Ginther Charles L. , Sensabaugh George F. , Mathies Richard A.
Published: May 2005

The design, construction and operation of a four‐color capillary array electrophoresis scanner are presented. The use of sensitive energy transfer primers facilitates four‐color detection of the DNA sequencing fragments following excitation at a single laser wavelength (488 nm). This scanner collects fluorescence data from up to 25 capillaries in parallel. The resulting four‐color image files are automatically reduced to four‐color line plots, and a base‐calling program (Sax) is used to call the sequence. The performance of this system for DNA sequencing is demonstrated by examining twelve different motifs of the hypervariable region I of human mitochondrial (mt) DNA obtained from a Sierra Leone population.
Factors that affect the stability of protein‐DNA complexes during gel electrophoresis
Authors: Fried Michael G. , Bromberg Jennifer L.
Published: May 2005

The gel electrophoresis mobility shift assay is widely used for qualitative and quantitative characterization of protein complexes with nucleic acids. Often it is found that complexes persist within electrophoresis gels for much longer than expected on the basis of their free‐solution lifetimes. Volume exclusion, direct interaction with gel matrices and the reduction of water activity by the gel have been proposed as mechanisms enhancing the stability of complexes during electrophoresis. We have used the well‐characterized interaction of the E. coli cyclic AMP receptor protein (CAP) with lactose promoter DNA to test these proposals. We found that the activity of water within polyacrylamide gels differs little from that of the buffer in which they were cast and that the dependence of the dissociation rate constant on water activity is too small for osmotic stabilization to contribute significantly to the lifetimes of CAP‐DNA complexes. In addition, we found that a cross‐linked gel matrix is not required for the stabilization of CAP‐DNA complexes, that comparable stabilization is produced by three dissimilar polymers (linear polyacrylamide, dextran and polyethylene glycol), and that these polymers stabilize complexes more effectively than equivalent weight concentrations of their cognate monomers. While these results challenge the notion that direct interaction with the gel matrix contributes to the stability of protein‐DNA complexes, they are all features expected of excluded volume mechanisms.
Detection of conformation bias between the complementary strands of deoxynucleotide repeat (TG/AC)n
Authors: Dobi Albert , Mahan Mark A. , Agoston Denes V.
Published: May 2005

Antiparallel complementary strands of DNA contribute symmetrically to the helical structure and its stability; rough conformational differences between complementary strands would interfere with this symmetric contribution and the stability. To detect conformational differences between complementary strands, we challenged the complementary strands to different “pore size” of acrylamide gels and measured differences in migration during electrophoretic mobility analysis. Changes in migration differences revealed a significant structural bias between the complementary strands of the dinucleotide repeat deoxy‐(TG/AC)n.
Gel electrophoretic mobility of charged particles in a medium with curved channels
Authors: Fridrikh Serguei , Gotlib Yulii , Belenkij Boris
Published: May 2005

A model of electrophoretic mobility of small charged particles (for example short DNA fragments) in media with curved channels is proposed. The medium is represented by a dense material embedding the curved channels accessible to the charged particles. The steady flow method is used to obtain an analytical expression of the particles' electrophoretic mobility as a function of the channel's shape. The analogy between the statistical properties of the channels and polymer chains (free‐jointed and with the persistent mobility mechanism) is used.
Rapid and enhanced detection of mitochondrial DNA variation using single‐strand conformation analysis of superposed restriction enzyme fragments from polymerase chain reaction‐amplified products
Authors: Barros Francisco , Lareu María Victoria , Salas Antonio , Carracedo Angel
Published: May 2005

A strategy is described for detecting mitochondrial (mt) DNA variation which permits rapid and straightforward screening for forensic purposes. The method is based on the selection of fragments with adequate length for performing single strand conformation polymorphism (SSCP) analysis selecting a set of restriction enzymes (RE) which yield fragments with prefixed lengths. After digestion of mtDNA by the appropriate enzyme or set of enzymes, SSCP analysis is performed in a semiautomatic electrophoretic system using a silver staining detection method. The conformational changes due to single mutations were therefore found not to change the electrophoretic protocol but to change the relative position of the mutations within the fragment. The discrimination power of this method is estimated to be 90% when two restriction enzymes (MspI and Hinfl) are used, but it is considerably higher when other enzymes are added.
Cytosolic proteins from neutrophilic granulocytes: A comparison between patients with severe chronic neutropenia and healthy donors
Authors: Kasper Brigitte , Thole Hubert H. , Patterson Scott D. , Welte Karl
Published: May 2005

A modified technique of two‐dimensional gel electrophoresis (2‐DE) was used to investigate differences in the pattern of cytosolic proteins of neutrophilic granulocytes from patients with severe congenital neutropenia, cyclic neutropenia, and idiopathic neutropenia in comparison with healthy donors. At the time of study, all patients tested received treatment with a recombinant human granulocyte colony‐stimulating factor (r‐metHuG‐CSF; Filgrastim, Amgen). Using the Investigator 2‐D Electrophoresis System™ (Millipore) we were able to detect more than 1000 protein spots in the cytosol of neutrophilic granulocytes from both patients and healthy controls. We investigated six patients with severe congenital neutropenia, five patients with cyclic neutropenia, four patients with idiopathic neutropenia, and 13 healthy donors. In the cytosol of neutrophilic granulocytes from patients we found an additional protein spot. This protein spot (molecular mass approximately 32.4 kDa, pI about 5.5) could be identified by internal sequencing after in‐gel digestion with endoproteinase Lys‐C as tropomyosin. The importance of the overexpression of tropomyosin in neutrophilic granulocytes from patients with severe chronic neutropenia is not yet understood.
Analysis of host‐induced response in the rice blast fungus Magnaporthe grisea using two‐dimensional polyacrylamide gel electrophoresis
Authors: Kachroo Pradeep , Lee Kelvin H. , Schwerdel Cornelia , Bailey James E. , Chattoo Bharat B.
Published: May 2005

Two‐dimensional (2‐D) polyacrylamide gel electrophoresis of proteins was used to study the response of the rice blast fungus to extracts prepared from resistant and susceptible rice cultivars. A protein of molecular mass 31 kDa was induced by a susceptible host extract, while the fungus exposed to extract from the resistant cultivar and the untreated samples did not show the presence of this protein. Levels of this 31 kDa protein increased 30‐fold, 72h after treatment with plant extracts, with the concomitant appearance of at least sixteen other novel proteins. Fungus treated with extracts of resistant host or the untreated samples did not show any of these proteins while the proteins specific to different growth stages appeared as expected. Analysis of the extracellular samples showed induction of a 17 kDa protein after 72h in the culture treated with susceptible host extract. Since the resistant host extract does not cause induction of any protein it is likely that the proteins induced in response to the susceptible host are expressed during the disease process and/or its establishment. Our study demonstrates usefulness of 2‐D analysis in understanding host‐pathogen interactions.
Micellar electrokinetic capillary chromatography as a method for the determination of n‐octanol/water partition coefficients of pesticides
Authors: Dinelli Giovani , Mallegni Roberto , Vicari Alberto
Published: May 2005

Micellar electrokinetic capillary chromatography (MEKC) was used to determine the n‐octanol/water partition coefficient (Kow) of nonionic and ionic organic chemicals, mainly herbicides. Three electrolyte buffer systems with different organic modifiers were tested to evaluate their effect on the linear relationship between log Kow and log ḱ. The effect of the pH value of injected samples on linear log Kow/log ḱ relationship of ionic chemicals was also evaluated. Since a good correlation was found between log Kow values and the logarithms of the capacity factor (ḱ) of each tested organic solvent, other parameters were considered to evaluate the efficacy of the method, such as the rate of electroosmotic flow, the efficiency and reproducibility of the separation, and the separation number (n). The method was found to be applicable over a range of log Kow values from 0.72 to 5.1 and demonstrated a good predictive capability, giving similar results to direct experimental measures, at least within classes of compounds with similar physico‐chemical properties.
Phosphated surfactants as pseudostationary phase for micellar electrokinetic chromatography: Separation of polycyclic aromatic hydrocarbons
Authors: Akbay Cevdet , Shamsi Shahab A. , Warner Isiah M.
Published: May 2005

A double alkyl chain di(2‐ethylhexyl)phosphate (DEHP) is introduced as a potential anionic micellar pseudophase for a wide range of benzene derivatives and/or polycylic aromatic hydrocarbons (PAHs). Several parameters such as concentration of phosphated surfactant, type and concentration of organic solvents (acetonitrile, isopropanol, and methanol), as well as capillary electrophoresis separation voltage were optimized to enhance resolution, efficiency and selectivity as well as to maximize peak capacities. The migration times and selectivity order for a number of PAHs differ significantly, depending on the type of organic solvent added to the DEHP surfactant. Acetonitrile at a concentration of 30% V/V in combination with 100 mM DEHP gave optimum separation for a mixture of 21 benzene derivatives and PAHs in under 16 min.
pH Changes in Immobiline gels due to low‐molecular mass ion adsorption and condition for salt front formation during electrophoretic desorption
Authors: Stoyanov Alexander V. , Righetti Pier Giorgio
Published: May 2005

A simple theoretical model of low molecular mass ion adsorption on a weak exchanger is proposed. For a system containing immobilized charges in uniform concentration the pH change values connected with neutral salt addition and followed by further washing and voltage application are evaluated. The question of stability of the moving boundary, arising in the area near the electrodes, is considered for the constant current stabilization at different values of Immobiline concentration, pK values of Immoboline and salt concentration. The threshold values of ion mobility capable of providing a sharp salt front formation are also evaluated for conditions of strong adsorption. We found that, with extremely acidic Immobilines, a sharp salt front does not arise under the latter condition, even in the case of very high Immobiline concentrations.
High sensitivity identification of proteins by electrospray ionization tandem mass spectrometry: Initial com‐ parison between an ion trap mass spectrometer and a triple quadrupole mass spectrometer
Authors: Figeys Daniel , Aebersold Ruedi
Published: May 2005

Recently, we have shown that a solid‐phase‐microextraction/capillary electrophoresis device coupled to an electrospray ionization triple quadrupole mass spectrometer through a microelectrospray interface represents a powerful analytical system for the rapid, conclusive and sensitive identification of proteins separated by gel electrophoresis. Here we report on the successful coupling of the same device to an electrospray ionization ion trap mass spectrometer and on the comparative evaluation of the performance of the triple quadrupole and ion‐trap‐based systems. In the ion trap mass spectrometer‐based system, using a tryptic digest of a calibrated bovine serum albumin sample, we achieved limits of detection in the single mass spectrometry (MS) and tandem MS mode, respectively, of 400 amol (if 20 μL of solution at a concentration of 20 amol/μL was applied). The system was also successfully used to identify six yeast proteins isolated from a single analytical two‐dimensional polyacrylamide gel. For the detection of unfragmented peptide ions both systems showed comparable sensitivity, whereas the ion‐trap‐based system showed superior performance with fragment ion spectra.
Comparison of in‐gel and on‐membrane digestion methods at low to sub‐pmol level for subsequent peptide and fragment‐ion mass analysis using matrix‐assisted laser‐desorption/ionization mass spectrometry
Authors: Courchesne Paul L. , Luethy Roland , Patterson Scott D.
Published: May 2005

The success of the mass spectrometric‐based approaches for the identification of gel‐separated proteins relies upon recovery of peptides, without high levels of ionization‐suppressing contaminants, in solvents compatible with the mass spectrometer being employed. We sought to determine whether in‐gel or on‐membrane digestion provided a significant advantage when low to sub‐pmol quantities of gel‐separated proteins were analyzed by matrix‐assisted laser‐desorption/ionization mass spectrometry (MALDI‐MS) with respect to the number and size of released peptides. Serial dilutions of five standard proteins of Mr 17 000 to 97 000 (from 16 pmol to 125 fmol) were electrophoresed and subjected to in‐gel digestion (using a microcolumn clean‐up protocol, Courchesne, P.L. and Patterson, S. D., BioTechniques, 1997, in press) or on‐membrane digestion following blotting to the PVDF‐based membranes, Immobilon‐P and Immobilon‐CD. Peptide maps were able to be obtained for all proteins at the detection limit of each method (Immobilon‐P and Immobilon‐CD, 0.5 pmol; and in‐gel, 125 fmol), and searches of Swiss‐Prot or a non‐redundant database (>193 000 entries) successfully identified all of the proteins, except β‐casein. Fragment‐ion spectra using a curved‐filed reflector MALDI‐MS were obtained from more than one peptide per protein at loads down to 250 fmol (except β‐casein). Using the uninterpreted data, a search of the nonredundant database and a six‐way translation of GenBank dbEST (> 2 208 000 entries total) was able to identify myoglobin, carbonic anhydrase II, and phosphorylase b.
Rapid mass spectrometric identification of proteins from two‐dimensional polyacrylamide gels after in gel proteolytic digestion
Authors: Li Guang , Waltham Mark , Anderson N. Leigh , Unsworth Edward , Treston Anthony , Weinstein John N.
Published: May 2005

We report a rapid method for identifying proteins resolved by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) using matrix‐assisted laser desorption ionization‐mass spectrometry (MALDI‐MS). In‐gel digestion was performed in a way such that the volume ratio of trypsin solution to gel plug was quantitatively controlled to promote reproducible digestion and to maximize the digestion yield. To make the digestion samples more compatible with MALDI‐MS, the volatile salt ammonium bicarbonate in the digestion buffer was largely removed prior to peptide extraction. Samples of mixed tryptic peptides from in‐gel digestion were used without purification to obtain molecular weights by MALDI‐MS with α‐cyano, 4‐hydroxy‐cinnamic acid as the matrix. Modifications of MALDI sample loading procedures improved the detetion sensitivity by one half to one order of magnitude. The peptide mass peaks in MALDI‐MS spectra were distinguished from those of impurities by using several types of controls, and masses were corrected by using trypsin autodigestion fragments as internal calibration standards. Two different peptidematching computer programs were used to interrogate sequence databases and identify proteins. Identification was enhanced by generation of orthogonal data sets (by using different proteases) and by including experimental values of isoelectric point (pI) and molecular weight to exclude false entries in the candidate lists. Approximately 1% of the material from a spot was used in each sample loading, and nine protein spots from rat liver 2‐D PAGE gels were identified correctly, as judged by comparison with identification results previously obtained from Edman sequencing. A previously identified low‐abundance spot was not identified by MALDI‐MS, presumably because there was insufficient material in a single gel. The sample handling procedure reported here should permit us to identify many 2‐D PAGE protein spots of medium abundance.
Immobilized pH gradient two‐dimensional gel electrophoresis and mass spectrometric identification of cytokine‐regulated proteins in ME‐180 cervical carcinoma cells
Authors: Matsui Neil M. , Smith Diana M. , Clauser Karl R. , Fichmann Jenny , Andrews Lori E. , Sullivan Christine M. , Burlingame Alma L. , Epstein Lois B.
Published: May 2005

Two‐dimensional (2‐D) polyacrylamide gel electrophoresis combined with mass spectrometry is a powerful combination of technologies that allows high resolution separation of proteins and their rapid identification. Immobilized pH gradient (IPG) first‐dimensional gels have several advantages over carrier ampholyte isoelectric focusing, including a high degree of reproducibility, good protein spot resolution, and a selection of pH range. Here we demonstrate the utility and efficacy of combining IPG 2‐D gel electrophoresis with mass spectrometry to identify interferon‐γ‐ (IFN) and tumor necrosis factor (TNF)‐regulated proteins in ME‐180 cervical carcinoma cells. Three cytokine‐regulated proteins have been identified, using imidazole‐zinc‐stained preparative IPG 2‐D gels and in‐gel tryptic digestion followed by matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometry for determination of peptide masses and sequences: (1) triosephosphate isomerase, a glycolytic pathway enzyme, (2) proteasome subunit C3, which is important in protein degradation, and (3) Ran, a GTP‐binding protein important in cell cycle regulation, protein import into the nucleus, and RNA export from the nucleus.
The Dictyostelium discoideum proteome – the SWISS‐2DPAGE database of the multicellular aggregate (slug)
Authors: Yan Jun X. , Tonella Luisa , Sanchez Jean‐Charles , Wilkins Marc R. , Packer Nicolle H. , Gooley Andrew A. , Hochstrasser Denis F. , Williams Keith L.
Published: May 2005

The cellular slime mold Dictyostelium discoideum is a eukaryotic microorganism which has developmental life stages attractive to the cell and molecular biologist. By displaying the two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) protein map of different developmental stages, the key molecules can be identified and characterised, allowing a detailed understanding of the D. discoideum proteome. Here we describe the preparation of reference gel of the D. discoideum multicellular aggregate, the slug. Proteins were separated by 2‐D PAGE with immobilised pH gradients (pH 3.5–10) in the first dimension and sodium dodecyl sulfate (SDS)‐PAGE in the second dimension. Micropreparative gels were electroblotted onto polyvinylidene difluoride (PVDF) membranes and 150 spots were visualised by amido black staining. Protein spots were excised and 31 were putatively identified by matching their amino acid composition, estimated isoelectric point (pI) and molecular weight (Mr) against the SWISS‐PROT database with the ExPASy AAcompID tool (http://expasy.hcuge.ch/ch2d/aacompi.html). A total of 25 proteins were identified by matching against database entries for D. discoideum, and another six by crossspecies matching against database entries for Saccharomyces cerevisiae proteins. This map will be available in the SWISS‐2DPAGE database.
Large‐scale protein modelling and integration with the SWISS‐PROT and SWISS‐2DPAGE databases: The example of Escherichia coli
Authors: Peitsch Manuel C. , Wilkins Marc R. , Tonella Luisa , Sanchez Jean‐Charles , Appel Ron D. , Hochstrasser Denis F.
Published: May 2005

Knowledge‐based molecular modelling of proteins has proven useful in many instances, including the rational design of mutagenesis experiments, but it has generally been limited by the availability of expensive computer hardware and software. To overcome these limitations, we developed the SWISS‐MODEL sever for automated knowledge‐based protein modelling. The SWISS‐MODEL server uses the Brookhaven Protein Data Bank as a source of structural information and automatically generates protein models for sequences which share significant similarities with at least one protein of known three‐dimensional structure. We have now used the software framework of the server to generate large collections of protein models, and established the SWISS‐MODEL Repository, a new database for automatically generated and theoretical protein models. This repository is directly integrated with the SWISS‐PROT and SWISS‐2DPAGE databases through the ExPASy World Wide Web server (URL is http://expasy.hcuge.ch). Here we present an illustration of this process by an application to the Escherichia coli sequences.
Characterization of target antigens from anti‐neutrophil cytoplasmic antibodies in autoimmune hepatitis type‐I
Authors: Kellner Roland , Orth Thomas , Mayet Werner‐Johannes
Published: May 2005

The occurrence of anti‐neutrophil cytoplasmic antibodies (ANCA) has been described in sera of patients with autoimmune hepatitis (AIH). The significance of this finding remains uncertain and the nature of the target antigen(s) has not yet been defined. We studied 32 sera from patients with AIH type‐I and prepared extracts of human neutrophils to identify the target antigen(s). A 43 kDa dominant immunoreactive protein was found and identified as the cytoskeletal component actin. Initial studies to define the antigenic determinants identified three different actin domains.
Computer‐supported analysis (MegaBlot) of allopurinol‐induced changes in the autoantibody repertoires of rats suffering from experimental lens‐induced uveitis
Authors: Grus Franz‐H. , Augustin Albert J. , Zimmermann Christoph W.
Published: May 2005

Using a new quantitative immunoblot technique (MegaBlot and artificial neural network) sera of rats with lens‐induced uveitis (LIU) were tested against Western blots (WB) of sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS‐PAGE) separations of protein fractions from rat lenses. The blots were scanned using digital image analysis and evaluated by multivariate analysis of discriminance and an artificial neural network. Five groups of LIU rats (male Wistar rats, n = 28) were investigated: no treatment, single doses of methylprednisolone (MPR; 7.5 mg/kg body weight, intravenous), allopurinol (AL; 50 mg/kg body weight, intravenous), a combination of both drugs (AL and MPR), and repeated application of AL (ALFR; 50 mg/kg body weight intravenous every 2 weeks during the immunization period and a daily dose of approximately 25 mg/kg body weight, orally). ALFR, MPR, and MPRAL show statistically significant immunological effects in LIU treatment Classification of WBs using both discriminance analysis or neural network resulted in 100% correct assignment; and 82% (discriminance analysis) and 86% (neural network) of WBs were correctly assigned using only unknown blots for classification (not studied before). The MegaBlot technique is able to detect therapeutically induced changes in autoantibody repertoires allowing therapeutic control and a prognostic view of treatment.
Characterization of Chlamydia trachomatis L2‐induced tyrosine‐phosphorylated HeLa cell proteins by two‐dimensional gel electrophoresis
Authors: Birkelund Svend , Bini Luca , Pallini Vitaliano , Sanchez‐Campillo Maria , Liberatori Sabrina , Clausen Johannes D. , Østergaard Søren , Holm Arne , Christiansen Gunna
Published: May 2005

Chlamydia trachomatis is an obligate intracellular bacteria, inducing its own uptake in nonprofessional phagocytes either by phagocytosis or pinocytosis. We have previously shown that C. trachomatis L2 induces tyrosine phosphorylation of eukaryotic proteins upon their entry by phagocytosis. In this paper we characterize the tyrosine‐phosphorylated proteins by two‐dimensional gel electrophoresis. In immunoblotting with anti‐phosphotyrosine antibodies of C. trachomatis L2‐infected HeLa cells, but not with uninfected cells, two rows of spots were observed with a molecular mass of 69 and 71 kDa and pI from 5.0 to 5.2. In addition, a single spot of 100 kDa and pI 6.2 was observed.
A two‐dimensional electrophoresis database of human breast epithelial cell proteins
Authors: Giometti Carol S. , Williams Katherine , Tollaksen Sandra L.
Published: May 2005

As sequencing of the human genome progresses, attention is turning to when and where specific genes are being expressed and how that expression is regulated. The human breast, with the highly specific, but transient, function of milk production (lactation), exemplifies human gene regulation. The molecular mechanisms for the dramatic structural and functional changes involved in shifting from lactation‐capable to lactation‐incapable tissue are poorly understood, as are the mechanisms that result in deviation from normal breast cell growth into different types of breast neoplasms. We are using quantitative two‐dimensional electrophoresis (2‐DE) to determine which proteins are present in different types of human breast cells (milk‐producing and ‐nonproducing, estrogen‐receptor‐positive and ‐negative, normal and malignant) and which proteins change in abundance in response to stimuli that trigger cell differentiation, growth, or death. A composite map of proteins found in human breast cells is being generated and used as an index of human genes that are differentially expressed, both qualitatively and quantitatively. Proteins found in 15 different types of human breast cells, two from healthy tissue (from milk and reduction mammoplasty tissue) and 13 from tumor tissue, are now included in the composite map. Copies of the human breast epithelial cell protein map are available on the World Wide Web (URL: www.anl.gov/CMB/PMG/projects/index_hbreast.html) with links to quantitative data and identifications for proteins found to be differentially expressed in these epithelial cells. Links to the Swiss‐Prot and enzyme metabolic pathway databases are also provided. The World Wide Web presentation is designed to allow public access to the available 2‐DE data together with logical connections to databases providing genome‐related information.
Renal cell carcinoma and normal kidney protein expression
Authors: Sarto Cecilia , Marocchi Alessandro , Sanchez Jean‐Charles , Giannone Daniela , Frutiger Séverine , Golaz Olivier , Wilkins Marc R. , Doro Giancarlo , Cappellano Francesco , Hughes Graham , Hochstrasser Denis F. , Mocarelli Paolo
Published: May 2005

Renal cell carcinoma (RCC), a human kidney cancer from the proximal tubular epithelium, accounts for about 3% of adult malignancies. Molecular and cytogenetic analysis have highlighted deletions, translocations, or loss of heterozygosity in the 3p21–p26, a putative RCC locus, as well as in 6q, 8p, 9pq, and 14pq. Studies on phenotypic expression of human kidney tissue and on post‐translational modifications in RCC have not yet provided a marker for early renal cell carcinoma diagnosis. Current dignostic methods do not help to detect the tumor before advanced stages. We therefore used two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) to study normal and tumor kidney tissues in ten patients suffering from RCC. A human kidney protein map in the SWISS‐2DPAGE database accessible through the ExPASy WWW Molecular Biology Server was established. Of 2789 separated polypeptides, 43 were identified by gel comparison, amino acid analysis, N‐terminal sequencing, and/or immunodetection. The comparison between normal and tumor kidney tissues showed four polypeptides to be absent in RCC. One of them was identified as ubiquinol cytochrome c reductase (UQCR), whose locus has elsewhere been tentatively assigned to chromosome 19p12 or chromosome 22. A second polypeptide was identified as mitochondrial NADH‐ubiquinone oxido reductase complex I whose locus is located on chromosome 18p 11.2 and chromosome 19q 13.3. These result suggest that the lack of UQCR and of mitochondrial NADH‐ubiquinone oxidoreductase complex I expression in RCC may be caused by unknown deletions, or by changes in gene transcription or translation. It might indicate that mitochondrial disfunction plays a major role in RCC genesis or evolution.
A two‐dimensional gel database of human colon carcinoma proteins
Authors: Ji Hong , Reid Gavin E. , Moritz Robert L. , Eddes James S. , Burgess Antony W. , Simpson Richard J.
Published: May 2005

The master two‐dimensional gel database of human colon carcinoma cells currently lists cellular proteins from normal crypts and the colorectal cancer cell lines LIM 1863, LIM 1215 and LIM 1899 (Ward et al., Electrophoresis 1990, 11, 883–891; Ji et al., Electrophoresis 1994, 15, 391–405). Updated two‐dimensional electrophoretic (2‐DE) maps of cellular proteins from LIM 1215 cells, acquired under both nonreducing and reducing conditions, are presented. Fifteen cellular proteins are identified in the reducing 2‐DE gel map, and seven in the nonreducing gel map, along with a tabular listing of their Mr/pI loci and mode of identification. We also include our mass spectrometric based procedures for identifying 2‐DE resolved proteins. This procedure relies on a combination of capillary column (0.10–0.32 mm internal diameter) reversed‐phase HPLC peptide mapping of in‐gel digested proteins, peptide mass fingerprinting, sequence analysis by either collision‐induced dissociation or post‐source‐decay fragmentation, and protein identification using available database search algorithms. These data, and descriptions of the micro‐techniques employed in this laboratory for identifying 2‐DE resolved proteins can be accessed via the internet URL: http://www.ludwig.edu.au.
Electrophoretic analysis of the “cross‐class” interaction between novel inhibitory serpin, squamous cell carcinoma antigen‐1 and cysteine proteinases
Authors: Nawata Shugo , Nakamura Kazuyuki , Tanaka Tatehiko , Numa Fumitaka , Suminami Yoshinori , Tsunaga Nagato , Kakegawa Hisao , Katunuma Nobuhiko , Kato Hiroshi
Published: May 2005

We investigated the “cross‐class” interaction between cysteine proteinases and a novel inhibitory serpin, recombinant squamous cell carcinoma (rSCC) antigen‐1, which inhibits a serine proteinase, chymotrypsin. rSCC antigen‐1 inhibited the cysteine proteinases, papain, papaya proteinase IV and cathepsin L. Interestingly, although rSCC antigen‐1 formed sodium dodecyl sulfate (SDS)‐ and heat‐stable complexes with chymotrypsin, rSCC antigen‐1 gave the 40 kDa fragment and small molecular mass peptide by incubation with papain without forming an SDS‐ and heat‐stable complex. The cleavage was observed between the Gly353‐Ser354 bond, indicating that rSCC antigen‐1 interacts with cysteine proteinases not at the predicted reactive site P1‐P1′ portion (Ser354‐Ser355), but at the Gly353‐Ser354 of the P2‐P1 portion. These findings promote understanding of the “suicide inhibition” mechanism of SCC antigen‐1 against cysteine proteinases.
Separation of enantiomers of drugs by capillary electrophoresis. Part 4: Hydroxyprpyl‐gammacyclodextrin as chiral solvating agent
Authors: Koppenhoefer Bernhard , Epperlein Ulrich , Xiaofeng Zhu , Bingcheng Lin
Published: May 2005

In an extended chiral drug screening program, enantioseparation of 86 racemic drugs was tested with hydroxypropyl‐γ‐cyclodextrin as chiral solvating agent (CSA). A total of 30 drugs out of 86 could be resolved in this straightforward approach. The number of experiments performed under identical conditions allows a statistical treatment of the data. The enantioseparation of the analytes is correlated with their interaction strength with the CSA. Hence, the concentration of the CSA is a crucial parameter for optimization of the enantioseparation, as shown by a subset of 23 examples.
Chiral interactions in capillary zone electrophoresis: Computer simulation and comparison with experiment
Authors: Ingelse Benno A. , Sarmini Karim , Reijenga Jetse C. , Kenndler Ernst , Everaerts Frans M.
Published: May 2005

Chiral interaction in capillary electrophoresis can be modeled using pK values, mobilities of analytes, and their formation constants with the chiral selector. An existing steady‐state simulation program for CE (HPCESIM) was recently extended with a chiral submenu involving the chiral parameters listed above. These were experimentally determined in both our laboratories for mandelic acid and terbutaline using hydroxypropylated β‐cyclodextrin as chiral selector. A comparison was made between both sets of parameters and between experimental electropherograms and those obtained from simulation. Error analysis of the results indicate the sensitivity of the obtained results.
Determination of the enantiomeric purity of N‐propionyl‐6,7‐dimethoxy‐2‐aminotetralin by cyclodextrin‐modified micellar electrokinetic chromatography
Authors: Castelnovo Pierfrancesco , Albanesi Carlo
Published: May 2005

N‐Propionyl‐6,7‐dimethoxy‐2‐aminotetralin (Z12231A), a useful intermediate in the synthesis of dopaminergic agonists, was resolved into its enantiomers by high‐performance capillary electrophoresis. Cyclodextrin‐modified micellar electrokinetic chromatography was employed with a combination of chiral selectors both in the aqueous phase (a hydroxyalkyl‐β‐cyclodextrin) and in the micellar phase (sodium taurodeoxycholate). The absolute amount of the two chiral selectors as well as their ratio were found to be the most critical parameters in order to optimize separation. A resolution factor >3.5 was obtained, allowing a high amount of solute to be loaded in order to improve the detection limit. The reproducibility of the assay was also evaluated.