bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence patterns produced by incomplete enzymatic digestion or one‐step Edman degradation of peptide mixtures as probes for protein database searches
Authors: Jensen Ole N. , Vorm Ole , Mann Matthias
Published: May 2005

Mass spectrometric peptide mapping of proteins isolated by polyacrylamide gel electrophoresis is a rapid method for identifying proteins in sequence databases. A majority of tryptic peptide maps were found to contain pairs of peptide ion peaks separated by the molecular weight of the lysyl or arginyl residue. These peaks originate from amino acid sequence patterns such as Lys‐Lys where trypsin has cleaved C‐terminals to either one of the lysines. The peptide mass and the pattern define an N‐ or C‐terminal sequence tag. Searching sequence databases by such a sequence tag results in only a moderate number of matches and significantly reduces the number of database matches when used in combination with a peptide mass map. Two N‐ or C‐terminal sequence tags alone unambiguously identify a protein in most cases. The technique discussed here is simple, does not require additional measurments, and increases the percentage of protein samples that can be identified by their mass maps alone. N‐Terminal peptide sequence tags for database searching can also be generated by manual one‐step Edman degradation of the unseparated peptide mixture.
Real‐time velocity of DNA bands during field‐inversion gel electrophoresis
Authors: Sabanayagam Chandran R. , Holzwarth George
Published: May 2005

The velocity v of bands of double‐stranded, linear DNAs containing 48.5–5700 kbp was determined with 0.3 s resolution during field‐inversion agarose gel electrophoresis (FIGE) for a broad range of the forward pulse period T+, keeping the duration of the backward pulse T− = T+/3. Within 0.6 s or less after the field changed sign from − to +, the velocity showed a sharp positive peak; a similar spike, but with negative velocity, occurred immediately after the field changed from + to −. For long pulses, the magnitude of this spike increased with M0.36, reaching ten times the steady‐state velocity for M = 5.7 kbp. After this spike, the velocity dipped to 55–75% of its value in a steady field, then increased to a small secondary peak before reaching a steady‐state plateau. The duration of the velocity trough, and the time of the small peak, increased as M1. For standard FIGE conditions (ratio of forward:reverse pulse duration, T+:T− = 3:1; equal forward and reverse field amplitudes, E+ = E−), the mobility μ = ∫ vdt over a complete cycle was a minimum when E+ terminated at the end of the velocity trough. The minimum occurred because the velocity during E+ sampled primarily the trough, and because the backward velocity during E− was exceptionally large; the negative velocity spike was maximized when T+ terminated at the end of the velocity trough. Computer simulations of FIGE by Zimm (J. Chem. Phys. 1991, 94, 2187–2206) and by Duke and Viovy (J. Chem. Phys. 1992, 96, 8552–8563) generate real‐time velocities that are in excellent agreement with our experimental data.
Conformational dynamics of DNA during biased sinusoidal field gel electrophoresis
Authors: Masubuchi Yuichi , Oana Hidehiro , Matsumoto Mitsuhiro , Doi Masao , Yoshikawa Kenichi
Published: May 2005

DNA motion during biased sinusoidal field gel electrophoresis around the antiresonance condition was investigated by direct observation using fluorescent microscopy and Brownian dynamics simulation. Time development of the center of mass velocity, vx, and the principal value of the gyration tensor, Rl, was measured at this antiresonance condition. The typical stretch‐contract motion, which is observed in steady field and at high frequency field, is severely suppressed, and there are two or more dominant kinks that compete with each other. Analysis of the kink motion from the simulation results supports this picture.
Fluorescence resonance energy transfer visualization of DNA aggregates formed during electrophoretic separations in ultradilute hydroxyethylcellulose
Authors: Schwinefus Jeffrey James , Wang Shau‐Chun , Hammond Richard W. , Morris Michael D.
Published: May 2005

Fluoresence resonance energy transfer (FRET) microscopy is used to observe structures of DNA aggregates formed during electrophoresis in ultradilute hydroxyethylcellulose (HEC). FRET gives evidence of aggregates with a tightly packed core and random entanglement of DNA in the outer portions. HEC concentrations also appear to play a role in aggregate formation. DNA aggregates take longer to assemble in higher HEC concentration solutions and assume a less compact form than those formed at lower HEC concentrations.
Protein separation by electrophoresis in a nonsieving amphoteric medium
Authors: Blanco Stéphane , Clifton Michael J. , Joly Jean‐Louis , Peltre Gabriel
Published: May 2005

A numerical model has been used to study the separation of protein mixtures by zone electrophoresis in a nonsieving amphoteric medium. An amphoteric buffer fixes the pH of the solution close to its isoelectric point, where the buffer molecules are uncharged: they thus contribute very little to the conductivity of the solution. This means that high field strengths can be used for rapid separation without sacrificing resolution. The numerical study shows that in this process the band spreading that can reduce resolution is essentially due to differences in migration rate between protein‐rich and low‐protein zones: both differences in solution conductivity and in protein mobility are involved. Rules for judging the buffer capacity of amphoteric molecules are presented and it is shown how, for a given protein, the effectiveness of this technique varies with the range of pH in which it is applied.
Purification of thermamylase in multicompartment electrolyzers with isoelectric membranes: The problem of protein solubility
Authors: Esteve‐Romero Josep S. , Bossi Alessandra , Righetti Pier Giorgio
Published: May 2005

The main α‐amylase from Bacillus licheniformis (called thermamylase because of its resistance to high temperatures, 90°C) has been subjected to purification by isoelectric focusing in multicompartment electrolyzers with isoelectric membranes. The enzyme tended to precipitate, producing severe smears in proximity of its pI value (7.18). Solubility could not be ameliorated by any of the known means typically adopted in isoelectric focusing and compatible with enzyme activity, such as addition of neutral and zwitterionic surfactants (e.g., Nonidet, 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propane‐sulfonate, up to 2%), mixed hydro‐organic solvents (glycerol, ethylene glycol, propylene glycol) and addition of zwitterions unable to form micelles, such as taurine. However, addition of sugars, notably saccharose, sorbitol, and, to a lesser extent, sorbose, greatly improved protein solubility in the pI proximity. The improvement was dramatic if these sugars were admixed with 0.2 M taurine. Additionally, the increment of solubility (which occurred when reaching a level of 40% of the different sugars) was accompanied by a large pI shift, typically reducing the pI value by as much as 0.4 pH units (e.g., from a pI of 7.18 in the absence of additives to a pI of 6.80 in presence of a mixture of 40% sucrose and 0.2 M taurine, the best solubilizer in all the series investigated). This apparent pI shift was not due to a change of pH gradient caused by the presence of additives, since pH measurements in the absence as well as presence of additives gave identical results. The results are explained by the theory of Timasheff and Arakawa on stabilization of protein structure by solvents: sugars (at ca. 1 M concentration) and zwitterions such as taurine belong to class I stabilizers, characterized by negative binding to proteins and by increasing the surface tension of water. As a result, the protein is in a state of “superhydration”, which might prevent binding to Immobilines in the gel matrix and might alter some pKs on the protein surface. In solutions of 40% saccharose and 0.2 M taurine, thermamylase could be successfully purified to a single isoelectric and isoionic band in the multicompartment electrolyzer.
Validation of highly discriminating multiplex short tandem repeat amplification systems for individual identification
Authors: Kimpton Colin P. , Oldroyd Nicola J. , Watson Stephanie K. , Frazier Rachael R. E. , Johnson Peter E. , Millican Emma S. , Urquhart Andrew , Sparkes Becky L. , Gill Peter
Published: May 2005

Short tandem repeat (STR) loci are routinely employed for individual identification. We have examined the performance and reproductibility of a highly informative co‐amplification system containing the tetranucleotide STR loci: HUMVWFA31/A, HUMTH01, D20S85, D8S1179, HUMFIBRA, D21S11, and D18S51, in conjunction with the amelogenin sex test, in addition to a modified system omitting the locus D20S85. Polymerase chain reaction (PCR) products were fluorescently detected on an automated sequencer and automatically sized against an internal size standard by Genescan software. Both systems were routinely able to type 500 pg of undegraded DNA. At DNA concentrations between 50–500 pg, partial profiles were produced, but no allelic drop‐out was observed. Balanced amplification of all loci occurred over a wide range of DNA concentrations from 50 pg to 10 ng. Alteration of reagent concentrations and cycling parameters from optimal resulted in variation in the efficiency of individual locus amplification relative to the other loci within the system. This was also observed at high ionic strength or extreme pH. However, at all reagent concentrations and conditions, allelic drop‐out was not observed. These multiplex systems have potential in both routine forensic and intelligence database applications.
Comparison of different electrophoretic methods for digital typing of the MS32 (D1S8) locus
Authors: Rodriguez‐Calvo María Sol , Bellas Susana , Soto Jose Luis , Barros Francisco , Carracedo Angel
Published: May 2005

Minisatellite variant repeat mapping by polymerase chain reaction (MVR‐PCR) is a new and powerful approach for individual identification from human DNA. This method has been successfully applied to the hypervariable human minisatellite MS32 (locus D1S8). In this work, we further investigate this locus using different electrophoretic techniques which include: agarose gels and nonradioactive hybridization, miniaturized polyacrylamide gels, followed by silver staining, and automatic detection of fluorescent labeled alleles. The advantages and disadvantages of each method are described. Also, MVR codes from a Galician population (NW Spain) are provided.
An exactly solvable Ogston model of gel electrophoresis II. Sieving through periodic gels
Authors: Slater Gary W. , Guo Hong L.
Published: May 2005

Recently, we developed a lattice model to study the dynamics of particles being electrophoresed in gels (G. W. Slater, H. L. Guo, Electrophoresis 1995, 16, 11–15). In Part I of this series (G. W. Slater, H. L. Guo, Electrophoresis 1996, 17, 977–988), we showed how to calculate the exact electrophoretic mobility of one‐site particles in the limit where the electric field intensity E is vanishingly small. Since we can solve the model for arbitrary gel structures in two or more dimensions, we compared our results with those of the Ogston‐Morris‐Rodbard‐Chrambach model (OMRCM) of gel electrophoresis, which assumes that the mobility (μ) of charged particles is directly proportional to the fractional gel volume (f) that is available to them. Our results and theoretical analysis indicated that the OMRCM is a mean‐field approximation that can be useful as a rough guide; however, it generally misses the subtle sieving effects related to the correlations between the position of the obstacles in a given gel structure. In this paper (Part II) we study, for two‐dimensional periodic gels, the exact relationships between the zero‐field mobility μ and the gel concentration C for larger particle sizes. The fact that μ is a strong function of the particle size suggests that we can separate large particles using two‐dimensional periodic gels (similar to those fabricated by W. D. Volkmuth and R. H. Austin, Nature 1992, 358, 600–602). We analyze our data using Ferguson‐like plots and we show that one can indeed use a generalized retardation coefficient, K, to estimate the effective pore size, aK and effective fiber size rK for these model gels. We conclude that the retardation coefficient is a useful concept to characterize a sieving structure even though it does not permit the inference of the exact gel structure.
Electrophoresis of DNA in novel thermoreversible matrices
Authors: Sassi Alexander P. , Barron Annelise , Alonso‐Amigo M. Goretty , Hion Daniel Y. , Yu Jessina S. , Soane David S. , Hooper Herbert H.
Published: May 2005

We demonstrate the feasibility of temperature‐sensitive polymers as novel matrices for capillary and slab electrophoresis of DNA. These matrices combine the ease‐of‐use of agarose with resolution properties of polyacrylamide. Two classes of matrices are used: (i) aqueous suspensions of gel microspheres and (ii) solutions containing uncross‐linked temperature‐sensitive polymers. When heated, the viscosity of these solutions drops dramatically because of the phase transition behavior of these polymers, and therefore these formulations are easy to pour or load. Results are presented for separation of double‐stranded DNA fragments (< 2000 bp) in capillary, tube, and slab electrophoresis. Preliminary results are also presented for separation of single‐stranded sequencing fragments by capillary electrophoresis. In the tube format, good resolution was obtained for ΦX174/HaeIII fragments. In the slab format, excellent resolution of double‐stranded DNA was obtained, including simulaneous separation of a 10 bp ladder up to 150 base pairs. In the capillary format, pBR322/MspI fragments were completely resolved, and single‐base resolution of sequencing fragments was obtained up to 150 bases. We believer that temperature‐sensitive polymers represent a new and promising class of electrophoretic media.
Comment on mobility‐shift computations featuring cage effects
Authors: Cann John R.
Published: May 2005

Theoretical mobility‐shift patterns are computed by solution of conservation equations for electrophoresis coupled with chemical reaction. The chemical reaction term is often formulated in terms of dissociation of the protein‐DNA complex in a gel cage. This formulation assumes that once the dissociated protein escapes the cage, it goes down a sink and is totally lost. This implies that the concentration of the escaped protein is too low to affect sign ficantly the rates of protein‐DNA association along its migration pathway.
Three novel mutations of the NF1 gene detected by temperature gradient gel electrophoresis of exons 5 and 8
Authors: Horn Denise , Robinson Peter N. , Böddrich Annett , Buske Annegret , Tinschert Sigrid , Nürnberg Peter
Published: May 2005

We screened a total of 100 unrelated patients with neurofibromatosis type 1 (NF1) for mutations in exons 5 and 8 of the NF1 gene using temperature gradient gel electrophoresis (TGGE). Careful interpretation of exon 5 TGGE patterns was necessary due to interference by an exonic polymorphism. Three novel mutations were identified: a stop mutation in exon 5 (Q239X) caused by a C→T transition at cDNA nucleotide position 715, a transition at the invariant G of the splice accceptor site in intron 4c (G655‐1A), and a transversion at the invariant G of the splice donor site in intron 8 (G1185+1T). Analysis of mRNA revealed the predicted abnormal splice products. While skipping of exon 5 causes a shift in the reading frame with a premature stop codon downstream in the middle of exon 6, skipping of exon 8 leads to an in‐frame deletion with the predicted protein product being shortened by 41 amino acids.
Separation mechanism of pullulan solution‐filled capillary electrophoresis of sodium dodecyl sulfateproteins
Authors: Nakatani Manabu , Shibukawa Akimasa , Nakagawa Terumichi
Published: May 2005

The separation mechanism of capillary electrophoresis of sodium dodecyl sulfate (SDS)‐proteins using pullulan with a molecular mass range of 50 000–100 000 as a separation matrix was investigated. The pullulan solution was filled into fused‐silica capillaries whose inner walls were coated with linear polyacrylamide through chemically stable Si‐C linkages. Baseline separations of SDS proteins were achieved at concentrations ranging from 3–10% w/v of pullulan. The entanglement threshold of pullulan solution was found to be around 0.5% w/v, indiacating migration of SDS‐proteins through an entangled pullulan network. Ferguson plots exhibited a linear relationship between log mobility and pullulan concentration. Linear relationships were also obtained for double logarithmic plots of the electrophoretic mobility and protein molecular mass. These results show that the separation is based on mass discrimination in accordance with the Ogston theory.
Two‐dimensional electrophoretic studies on down‐regulated intracellular transferrin in human fibroblasts immortalized by treatment with either 4‐nitroquinoline 1‐oxide or 60Co gamma rays
Authors: Kondo Tadashi , Mihara Koichiro , Inoue Yusuke , Namba Masayosi
Published: May 2005

The immortalization of human cells is an important early step of carcinogenesis. To investigate the mechanisms of immortalization, we established three immortalized human fibroblast cell lines by treatment with either 4‐nitroquinoline 1‐oxide (4NQO) or 60Co gama rays. Using two‐dimensional gel electrophoresis (2‐D PAGE), we identified proteins which are down‐regulated in these immortalized cell lines. Their isoelectric points ranged between 5.5 and 6.3, and their molecular masses were approximately 80 kDa. The proteins were also decreased in another human fibroblast cell line immortalized with simian virus 40 (SV40) and two human cervical cell lines. The proteins were present in a cytoskeletal fraction, reacted with anti‐transferrin antibody and some of them were phosphorylated. The proteins were identified as transferrin. Although the 2‐D PAGE patterns of our proteins and serum transferrin of different origin were distinct from each other, their isoelectric points and molecular weights were similar. We implicate some functions of intracellular transferrin, other than an iron transporter, in immortalization of human cells and discuss a new aspect of transferrin metabolism in the cells.
Multiple capillary DNA sequencer that uses fiber‐optic illumination and detection
Authors: Quesada Mark A. , Zhang Shiping
Published: May 2005

An 8‐capillary prototype electrophoresis system for DNA sequencing has been construted. The sequence of 400–450 bases can be obtained from each capillary in less than an hour form sequencing reactions generated with four‐color fluorescent terminators. Illumination of each capillary and collection of fluorescence is through individual optical fibers. Resolution of the DNA ladder is through a replaceable sieving matrix of linear polyacrylamide in reusable coated capillaries. Light from an argon ion laser is introduced into a fused biconically tapered fiber‐optic splitter, and individual fibers deliver approximately 10 mW of 514 nm light to each of the eight electrophoresis capillaries. Illumination and collection are by fibers normal to the surface of the electrophoresis capillary and at right angle to each other. Illumination by a fiber with low numerical aperture and collection by a fiber with high numerical aperture provides good sensitivity and signal‐to‐noise ratios without the need for microlenses (limit of detection: 1.5 × 10−11 M fluorescein analog dye with a signal‐to‐noise ratio of 2). The eight collection fibers are passed in parallel through holographic filters for Rayleigh rejection and into an imaging spectrograph, which simultaneously displays the full fluorsecence spectrum (475–648 nm) from the eight capillaries in parallel on the surface of an intensified chargecoupled device (CCD). The CCD is read out at a rate of 3.4 complete images per second.
The use of elevated column temperature to extend DNA sequencing read lengths in capillary electrophoresis with replaceable polymer matrices
Authors: Klepárnik Karel , Foret František , Berka Jan , Goetzinger Wolfgang , Miller Arthur W. , Karger Barry L.
Published: May 2005

Capillary electrophoresis with a replaceable linear polyacrylamide matrix operated at elevated column temperatures of 55° and 60°C was used to exted the separation of DNA sequencing fragments to lengths greater than 800 bases. A solid‐state heater was employed to provide stable, uniform temperature control over a significant portion of the capillary. The polymer matrix, 3% w/v linear polyacrylamide in a denaturing buffer, was replaced in the capillary after each run. Using dye‐labeled primers and Sequenase chemistry on an M13mp18 single‐stranded template, four‐color separations for the sequencing products were obtained, with read lengths in excess of 800 bases. This paper also briefly discusses the effects of buffer denaturants and capillary temperature on separation speed, resolution, and gel compression.
Fast capillary electrophoresis‐laser induced fluorescence analysis of ligase chain reaction products: Human mitochondrial DNA point mutations causing Leber's hereditary optic neuropathy
Authors: Muth Jochen , Williams P. Mickey , Williams Stephen J. , Brown Michael D. , Wallace Douglas C. , Karger Barry L.
Published: May 2005

High speed capillary electrophoresis‐laser‐induced fluorescence (CE‐LIF) has been used to separate and detect point mutations using the ligase chain reaction (LCR). The method utilizes short capillary columns (7.5 cm effective length) and fields of 400 V/cm to analyze DNA‐ethidium bromide complexes using an He/Ne laser. The method was first demonstrated with a commercially available kit for LCR based on a lacI gene fragment inserted in a Blusescript® II phagemid. LCR‐CE‐LIF was then applied to detect point mutations in human mitochondrial DNA. resulting in Leber's hereditary optic neuropathy (LHON). Three severe mutations were analyzed in which the original base is substituted by a thymidine base at positions 3460, 11778 and 14459. Appropriate primers were designed with polyT tails for length discrimination of pooled samples. Successful detection of mutated samples was achieved, with appropriate correction for small amounts of nonspecific ligated product. The method is rapid, easy to implement, and automatale.
Rh D/d genotyping by quantitative polymerase chain reaction and capillary zone electrophoresis
Authors: Cossu Gianfranco , Angius Antonia , Gelfi Cecilia , Righetti Pier Giorgio
Published: May 2005

A safe and reliable method for determining RhD type (positive or negative) and zygosity (D/D or D/d) could have applications, for instance, in the prediction of the D genotype of a father in couples where there is an RhD‐negative woman at risk of fetal alloimmunization. Capillary zone electrophoresis (CZE) is proposed as a novel, reliable and powerful method for quantitative evaluation of polymerase chain reaction (PCR) products. RhD is determined by amplifying a 136 bp region common to the RhCcEe and RhD genes and a 186 bp region specific of the RhD gene. RhD positive and negative samples are identified by polyacrylamide gel slab electrophoresis, followed by silver staining of the DNA bands, and by CZE in sieving liquid polymers, with direct on‐line peak densitometric evaluation by exploiting the intrinsic UV absorbance of the DNA fragments at 254 nm. The CZE method allows not only a reliable assessment of the RhD type (the presence of both 136 bp and 186 bp fragments indicating an RhD‐positive type, the presence of only the 136 bp fragment indicating an RhD‐negative type), but also a rapid determination of the zygosity based on the quantitative expression ratio of the 136 bp/186 bp pair. Thus, a 2:1 peak ratio clearly indicates a D/D homozygous individual, whereas a 3:1 peak ratio gives evidence of a D/d heterozygous individual.
Stereoselective interaction of drug enantiomers with human serum transferrin in capillary zone electrophoresis
Authors: Kilár Ferenc
Published: May 2005

Stereoselective interaction of drugs with human serum transferrin in capillary zone electrophoresis is described. The substances passed a pseudo‐stationary protein zone applied in a coated capillary and the possible chiral separation of the optical isomers was followed. Drugs with different structures were screened and the enantiomers of bupivacaine, propranolol and promethazine as well as the diastereomers of labetalol were resolved. Racemic mixtures of atenolol and pindolol enantiomers could not be resolved under these conditions.
Application of capillary zone electrophoresis for analysis of thyreostatics
Authors: Křivánková Ludmilla , Krásenský Simon , Boček Petr
Published: May 2005

Capillary zone electrophoresis was optimized for the separation of thiouracil, methylthiouracil and propylthiouracil. Methylthiouracil could be determined in various types of urine (human, bovine, horse), either without any pretreatment or in ethyl acetate extracts, within 15 min. For identification, the simultaneous detection at three UV wavelengths (216, 245 and 278 nm) was advantageously used while for quantification the wavelength of the absorbance maximum at 278 nm was preferred. Under optimized conditions a linear response of the detector in the concentration range 0.1–100 ppm was obtained. On analysis of untreated urine, a detection limit of 0.5 ppm was found; for urine extracts the detection limit was 0.1 ppm. Univocal peak identification, based on absorption at three wavelength, was only possible above 2 ppm. Relative standard deviation for standard solutions of methylthiouracil, diluted in the background electrolyte, was 1%; for methylthiouracil in extracts dissolved in the background electrolyte it was 4.5%, and for methylthiouracil in untreated urine, 12.7%.
Data‐adaptive algorithms for calling alleles in repeat polymorphisms
Authors: Stoughton Roland , Bumgarner Roger , Frederick William J. , McIndoe Richard A.
Published: May 2005

Data‐adaptive algorithms are presented for separating overlapping signatures of heterozygotic allele pairs in electrophoresis data. Application is demonstrated for human microsatellite CA‐repeat polymorphisms in LiCor 4000 and ABI 373 data. The algorithms allow overlapping alleles to be called correctly in almost every case where a trained observer could do so, and provide a fast automated objective alternative to human reading of the gels. The algorithm also supplies an indication of confidence level which can be used to flag marginal cases for verification by eye, or as input to later stages of statistical analysis.
Screening for renal carcinoma associated mutations in the von Hippel‐Lindau tumor suppressor gene by temperature gradient gel electrophoresis
Authors: Wenzel Michael , Enczmann Jürgen , Uhrberg Markus , Hernández Ana , Wiese Ulrich , Ackermann Rolf , Schmitz‐Draeger Bernd , Ebert Thomas , Wernet Peter
Published: May 2005

Renal cell carcinoma is the most common neoplastic disease of the adult kidney and occurs in its sporadic and hereditary form. Approximately 57% of all renal carcinomas of the clear cell type analyzed revealed a mutation in the von Hippel‐Lindau disease (VHL) gene. In the present work, temperature gradient gel electrophoresis (TGGE) is presented as a rapid and precise polymerase chain reaction (PCR)‐employing methodology for the detection of mutations in the VHL gene. The theoretical efficiency of TGGE to detect mutations in every base pair of the gene was calculated. According to computer analysis, at least 92% of all known mutations in the VHL gene are detectable. This calculated figure appears to be in agreement with the experimental results. Primary difficulties in analyzing exon 1 of the VHL gene were overcome by the employment of psoralen‐cross‐linked PCR fragments. In addition, TGGE analysis was used in screening for possible mutations in thirteen renal carcinoma samples. With this protocol TGGE is successfully added to the array of methods for the screening of VHL mutations.
Translationally controlled tumor protein: A protein identified in several nontumoral cells including erythrocytes
Authors: Sanchez Jean‐Charles , Schaller Dominique , Ravier Florence , Golaz Olivier , Jaccoud Sylviane , Belet Monique , Wilkins Marc R. , James Richard , Deshusses Jacques , Hochstrasser Denis
Published: May 2005

The translationally controlled tumor protein (TCTP) is a growth‐related protein which is regulated at the translational level. It is present in mammals, higher plants and Saccharomyces cerevisiae. This study was undertaken to localize and further characterize the TCTP in human cell lysates using two‐dimensional gel electrophoresis, monoclonal antibodies, and 45Ca‐gel overlay. TCTP was found in several healthy and tumoral cells including erythrocytes, hepatocytes, macrophages, platelets, keratinocytes, erythroleukemia cells, gliomas, melanomas, hepatoblastomas, and lymphomas. It could not be detected in kidney and renal cell carcinoma (RCC). A monoclonal antibody raised against TCTP detected three isoforms likely due to post‐translational modifications. A calcium binding property was found as well as heat stability and cytoplasmic localization. The high degree of homology from plants to man and its expression in many tissues suggests that TCTP most likely has a cell housekeeping function.
Capillary electrophoresis of organotin compounds with indirect UV detection
Authors: Whang Kuo‐Shu , Whang Chen‐Wen
Published: May 2005

A capillary electrophoresis (CE)‐indirect UV absorbance detection method for organotin compounds is described. The five triorganotin cations, viz., trimethyltin (TMT), triethyltin (TET), tripropyltin (TPT), tributyltin (TBT) and triphenyltin (TPhT), were efficiently separated by CE in 10 min with a 5 mM acetate buffer (pH 4.5) containing 3 mM 4‐aminopyridine (4‐AP). 4‐AP also functioned as a UV‐absorbing coion for the indirect detection of these UV‐transparent organotin species. Simultaneous separation of di‐ and triorganotins was achieved with the addition of α‐cyclodextrin (α‐CD) as a modifier in the electrophoresis buffer. Linearity (r≥ 0.995) of about two orders of magnitude was generally obtained. The concentration limits of detection for the organotin compounds studied were between 2 and 20 μM (as tin). The relative standard deviation (RSD) values were in the range 1.3–7.1%. The applicability of the method for the analysis of marine sediment was examined.
Identification of mouse liver proteins on two‐dimensional electrophoresis gels by matrix‐assisted laser desorption/ionization mass spectrometry ofin situenzymatic digests
Authors: O'Connell Kathy L. , Stults John T.
Published: May 2005

A number of proteins from a silver‐stained two‐dimensional (2‐D) electrophoresis gel of mouse liver whole‐cell lysate were identified by peptide mass mapping and sequene database searching. The excised protein spots were processed byin situreduction and alkylation, followed by Lys‐C digestion. The masses of the resulting peptide mixtures were measured with a matrix‐assisted laser desorption/ionization (MALDI) reflectron‐time‐of‐flight mass spectrometer. These masses were used successfully to search a protein sequence database. Optimized silver staining and digestion protocols allowed proteins to be identified routinely at the low picomole level. The high mass accuracy and resolution provided by delayed extraction were important for high specificity in the database search. Fragment ion data obtained by MALDI post‐source decay (PSD) measurements not only provided confirmation of peptide identification, but could be used to identify the protein from a single peptide without spectral interpretation.
Convergence of amino acid compositions of certain groups of proteins aids in their identification on two‐dimensional electrophoresis gels
Authors: Galat Andrzej , Rioux Vincent
Published: May 2005

The amino acid composition (AAC) versus the protein identity (PI) method was used for establishment of the identities of proteins from bovine brain and kidneys which were prefractionated on a CM52 cation exchanger and by preparative flat‐bed isoelectric focusing. Established identities of proteins whose AACs converge with those of other members of their proper superfamily are reliable. Groups of convergent AACs can be extracted from protein databases using the standard root‐mean‐square rule (Rmsd) with measures the difference between the AAC of chosen protein versus those in the database. Convergence of AACs of proteins is dependent on several factors such as the upper limit of Rmsd, the limits of variations of molecular mass (m) and isoelectric point (pI), the number of proteins with similar AACs present in protein databases, and the domain structure of proteins. AACs of many proteins remain unique if the Rmsd is maintained with 1.5–1.0 with m ± 3kDA and pI ± 4. Certain groups of multidomain proteins have quasi‐unique AACs only if the Rmsd is restrained to a value within 1.0 and 0.7. Convergence of AACs of certain groups of proteins may indicate that a common biological function exists for some members of each group. The AAC‐PI method may become an additional search tool for protein functions.
An on‐line two‐dimensional polyacrylamide gel electrophoresis protein database of adult Drosophila melanogaster
Authors: Ericsson Christer , Pethö Zoltàn , Mehlin Hans
Published: May 2005

An annotated two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) protein database of adult Drosophila melanogaster has been constructed, based on the protein patterns of heads, thoraces and abdomens of adult male and female Drosophila melanogaster. About 1200 major protein spots are catalogued. Common proteins, found in all body parts, as well as bodypart‐ and sex‐specifically expressed proteins are reported. Of the major proteins, 91, or 7.5%, are differentially expressed in the two sexes or in different body parts, at least in part reflecting specific functional requirements. At the present time 43 proteins, or about 3.5% of the detected proteins, have been identified. These data can be accessed interactively from our World Wide Web (WWW) server through clickable inline gel images and hypertext links. Identified protein spots are cross‐referenced, through hypertext links, to the SWISS‐PROT annotated database of protein primary sequences and the Fly‐Base database of Drosophila genomic data. Our reference gels can be used to gain immediate access to protein spot identify and to the pattern of differentially expressed proteins in Drosophila melanogaster. The work presented in this article ties together information from protein 2‐D PAGE, molecular biology and geneties and offers a uniform way to access this large volume of data.
“Active” two‐dimensional electrophoresis of rat liver DNA‐polymerase α
Authors: Naryzhny Stanislav N.
Published: May 2005

The approach of so‐called active gel analysis was used to determine the position and appearance of the catalytic subunit of rat liver DNA polymerase α on a two‐dimensional (2‐D) electrophoretic map. In this case a polyacrylamide gel containing DNA was used for the second dimension. DNA presence does not change the 2‐D protein pattern but makes it possible to conduct a polymerase reaction directly in the gel after separation. A crude extract of rat liver nuclei was used for analysis. The extract is quickly, isolated and contains mainly DNA polymerase α activity. It was shown that this enzyme restores its activity after 2‐D electrophoresis and sodium dodecyl sulfate (SDS) elution. After polymerase reaction with labeled dNTPs and autoradiography, the catalytic polypeptide or, rather, polypeptide cluster is revealed as chains of spots (possibly because of the presence of different hydrolyzed and phosphorylated forms). These spots are located on the 2‐D electrophoretic map in the region corresponding to molecular masses of 160, 140, and 130 kDa and pI 5.5–6.2.
The different expression of proteins recognized by monoclonal anti‐heat shock protein 70 (hsp70) antibody in human colonic diseases
Authors: Stul Jiřri , Bureš Jan , Jandík Pavel , Langr František , Kovářová Hana , Macela Aleš
Published: May 2005

Analysis of the expression of hsp70 (70 kDa heat‐shock proteins) in normal and pathological tissues might prove their potential diagnostic and prognostic values. In the present study, we combined high‐resolution two‐dimensional polyacrylamide gel electrophoresis with immunoblotting to study hsp70 expression in normal, preneoplastic and neoplastic colonic mucosa. By monoclonal anti‐hsp70 antibody, recognizing both the constitutive and inducible forms of hsp70, we have detected six charge isoforms localized in the pH 5.1–5.3 range and in the molecular mass range of 68–69 kDa in normal colonic mucosa. Immunostaining of hsp70 in polypous and malignant tissues revealed qualitative as well as quantitative changes in the expression of more acidic isoforms of hsp70 in comparison with normal tissue. Furthermore, the different basic isoforms of hsp70 were detected in chronically inflamed colonic mucosa from patients suffering from ulcerative colitis (UC) or Crohn's disease (CD). Besides the standard hsp70 protein pattern, additional proteins with molecular masses of about 39, 40, 74 and 75 kDa were variably immunostained in normal and pathological specimens. These observations suggest that hsp70 expression may be closely linked to disease etiology and/or pathophysiology.
Structural transitions of human serum albumin: An investigation using electrophoretic techniques
Authors: Gianazza Elisabetta , Galliano Monica , Miller Ingrid
Published: May 2005

The influence of pH and urea concentration on the electrophoretic mobility of native and reduced human serum albumin was evaluated by zonal electrophoresis across transverse urea gradients as well as by migration across transverse pH gradients in gels containing varying concentrations of urea. Exposure to urea results in a change of both pI and hydrodynamic volume of the albumin molecule. At acidic pH, the former effect is brought about by lower urea concentrations than the latter, as made evident by a biphasic denaturation curve; in alkaline buffers, all structural transitions occur at once, and a typical sigmoidal curve is observed. Below pH 6, the lower the pH, the lower the urea concentration causing albumin denaturation. For instance, in the presence of 3 M urea, below pH 5 > 95% of the protein is present in its denatured state, above pH 8 > 90% is in its native form, whereas in the 6.5–7.5 pH range the two components have similar abundance. Also, the reversibility of the transition between folding and unfolding depends upon pH, and is complete only above pH 6. After inclusion of β‐mercaptoethanol in the albumin sample the urea concentration required to bring about protein unfolding increases between pH 4 and 6 and decreases thereafter.
Prothymosin α: Efficient sequence determination by experimental and theoretical capillary electrophoretic support
Authors: Weiler Angelika , Stoeva Stanka , Tsitsilonis Ourania E. , Voelter Wolfgang
Published: May 2005

A rapid and efficient primary structure elucidation of prothymosin (ProTα) and its enzymatic fragments was performed by a combination of matrix‐assisted laser desorption ionization mass spectrometry (MALDI), automatic N‐terminal Edman degradation, and the available theoretical predictions of electrophoretic mobility in capillary electrophoresis (CE) as a basis suggesting active sites of ProTα in the different bioassays

Presented at the “Elektrophorese Forum ”︁96” meeting of the German Electrophoresis Society, held at the Technical University Munich, October 23–25; 1996

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Computer analysis of two‐dimensional electrophoresis gels: A new segmentation and modeling algorithm
Authors: Bettens Eva , Scheunders Paul , Van Dyck Dirk , Moens Luc , Van Osta Peter
Published: May 2005

The complexity of the spot patterns of two‐dimensional electrophoresis gels made it necessary to use image processing techniques to analyze the gels. An important issue in the analysis is the detection and quantification of the protein spots. In this paper we describe a new technique to segment and model the different spots. For the segmentation of a gel into its different spot regions we apply a watershed technique, which is robust and efficient. For the quantification of the spots, a new spot model is constructed, based on diffusion principles. Besides the advantage of having a physical interpretation, the model is demonstrated to be superior to the commonly used Gaussian models.
Comparison of natural and recombinant isoforms of grass pollen allergens
Authors: Petersen Arnd , Grobe Kay , Lindner Buko , Schlaak Max , Becker Wolf‐Meinhard
Published: May 2005

More than 95% of grass pollen allergic patients possess IgE antibodies against grass group I, a heterogeneous group of glycoproteins found in all temperate grasses. We studied the structural variability of the group I allergens in single species and among different grasses. By 2‐DE blotting using patients' IgE and monoclonal antibodies, we detected IgE‐reactive isoforms with molecular masses between 32 and 37 kDa and focusing in a wide pI ranging from 4.7 to 7.6. While the group I allergens of timothy grass (Phl p 1) were composed of 37 and 35 kDa components, only single isoforms were found for ryegrass (Lol p 1) and velvet grass (Hol 1 1): 32 and 34 kDa, respectively. By N‐terminal microsequencing we determined single amino acid substitutions in different‐sized group I allergens. The post‐translational modifications (one N‐glycosylation site, two hydroxylated proline residues and seven cysteine residues for potential disulfide formations), which contribute to IgE reactivity, were identical in all. From the cDNA sequences we deduced protein sequence homologies > 90%, a result which might explain the high IgE cross‐reactivity among the grasses. In order to test whether recombinant group I grass allergens can act as substitutes for the natural forms, we expressed rPhl p 1 in E. coli and in P. pasteuris. 2‐DE immunoblotting again demonstrated a microheterogeneity in molecular mass and pI. While the E. coli products were free from post‐translational modifications, rPhl p 1 from Pichia is a heterogeneous glycoprotein fraction with a carbohydrate content of about 15%. This rPhl p 1 is hyperglycosylated compared to the nPhl p 1, which only has a 5% carbohydrate content

Presented at the “Elektrophorese Forum ”︁96” meeting of the German Electrophoresis Society, held at the Technical University Munich, October 23–25, 1996

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Capillary zone electrophoresis at subzero temperatures II: Chiral separation of biogenic amines
Authors: Ma Stacey , Horváth Csaba
Published: May 2005

The separation of enantiomer pairs of eight biogenic amines by capillary zone electrophoresis (CZE) was investigated with heptakis (2,6‐di‐O‐methyl)‐β‐cyclodextrin as the chiral selector at temperatures ranging from −20° to 40°C by using a commercial electrophoresis unit retrofitted with an external thermostated refrigerated circulating bath in order to assist the original cooling system. Sodium phosphate in both neat aqueous and methanolic media at pH 2.5, as measured by the glass pH electrode, were used with the fused silica capillary from 1.5° to 40°C and −20° to 40°C, respectively. The effect of temperature on enantioselectivity was found to depend on the number of phenolic hydroxyl groups in the molecule. Upon lowering the temperature from 40° to −20°C, the chiral selectivity of the system, as measured by the relative mobility difference, increased tenfold for the amines with two vicinal phenolic hydroxyls, whereas the increase was insignificant for those having no phenolic hydroxyl groups. The complex formation constants of three amines which have the same molecular structure but the number of phenolic hydroxyl groups were determined at different temperatures and the thermodynamic parameters as well as compensation temperatures for the process were evaluated. Whereas the compensation temperature was 690 K for the amine without phenolic hydroxyl group, it was < 400 K for the amines with one or two phenolic hydroxyl groups. The difference in the compensation temperatures indicates that the intrinsic mechanisms of their complexation with the chiral selector are not the same; this may account for the discrepancies observed in the temperature dependency of the chiral selectivity. The enthalpy change per phenolic hydroxyl group was 2.5 kcal mol−1, which compares favorably with the typical value for a single hydrogen bond. Therefore, when the amines have phenolic hydroxyl groups, the strong increase in chiral selectivity with decreasing temperature may be due to enhanced H‐bonding between the cyclodextrin and the phenolic hydroxyls under the conditions employed in this study.
Electrokinetic chromatography employing an anionic and a cationic β‐cyclodextrin derivative
Authors: Jakubetz Hansjörg , Juza Markus , Schurig Volker
Published: May 2005

β‐Cyclodextrin, 1, can act as a chiral buffer additive in capillary zone electrophoresis (CZE) owing to its ability to form diastereomeric complexes with the enantiomers of ionic compounds. Reaction of 1 with 2,3‐epoxy‐propyltrimethylammonium chloride gave preponderantly 6‐O‐(2‐hydroxy‐3‐trimethyl‐ammoniopropyl) derivatives (2) of varying degrees of substitution (d.s.). Analogous reaction of 1 with 1,3‐propanesultone yielded 6‐O‐(sulfo‐n‐propyl) derivatives (SPE‐β‐CD, 3) of varying d.s. The purity and the d.s. of 2 and 3 were determined by 1H NMR and electrospray ionization mass spectrometry (ESI‐MS). These charged β‐cyclodextrin derivatives were used for the chromatographic enantiomer separation of a series of neutral barbiturates, of chlorthalidone, terbutaline, warfarin, salbutamol and brompheniramine by cyclodextrin electrokinetic chromatography (CD‐EKC). During the separation of chlorthalidone with 3 as chiral additive, a reversible interconversion of the enantiomers (enantiomerization) was observed at temperatures below 20°C.
Enantioseparation of nonsteroidal anti‐inflammatory drugs by capillary electrophoresis using mixtures of anionic and uncharged β‐cyclodextrins as chiral additives
Authors: Fillet Marianne , Hubert Philippe , Crommen Jacques
Published: May 2005

Nine nonsteroidal anti‐inflammatory drugs (NSAIDs) were enantioseparated by capillary electrophoresis using an anionic cyclodextrin derivative (sulfobutyl ether β‐cyclodextrin or carboxymethyl‐β‐cyclodextrin) in combination with a neutral cyclodextrin as chiral additives to a pH 3 phosphoric acid‐triethanolamine buffer. In the presence of a negatively charged cyclodextrin, the analytes were given an appropriate mobility but relatively low enantioselectivities were generally obtained when such a cyclodextrin was the only selector added to the buffer. The addition of an uncharged cyclodextrin, such as the native β‐cyclodextrin or one of its derivatives (dimethyl‐, trimethyl‐ and hydroxypropyl‐β‐cyclodextrin), to this kind of buffer containing an anionic cyclodextrin, was found to give rise to considerable improvement in chiral resolution for all compounds studied. Resolution and analysis time were optimized by varying the nature and concentration of the two cyclodextrins. The best compromise was usually achieved by the simultaneous addition of sulfobutyl ether β‐cyclodextrin and trimethyl‐β‐cyclodextrin. Under optimum conditions, the enantiomers of all NSAIDs examined could be completely separated (most often with resolution values higher than 5) in short analysis times (generally lower than 15 min).