bluuebunny/crossref_metadata_2025 huggingface dataset:
Screening for urinary methadone by capillary electrophoretic immunoassays and confirmation by capillary electrophoresis‐mass spectrometry
Authors: Thormann Wolfgang , Lanz Matthias , Caslavska Jitka , Siegenthaler Peter , Portmann Rudolf
Published: May 2005

This paper characterizes competitive binding, electrokinetic capillary‐based immunoassays for urinary methadone using reagents which were commercialized for a fluorescence polarization immunoassay. After incubation of 25 μL urine with the reactants, a small aliquot of the mixture is applied onto a fused‐silica capillary and the unbound fluorescein‐labeled methadone tracer is monitored by capillary electrophoresis with on‐column laser‐induced fluorescence detection. Configurations in presence and absence of micelles were investigated, found to be capable of recognizing urinary methadone concentrations ⩾ 10 ng/mL, and shown to be suitable for rapid methadone screening of patient urines. Based upon shorter run times and a much better separation of free tracer and antibody‐tracer complex, conditions without micelles are preferred. For confirmation analysis of urinary methadone and its major metabolite, 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP), capillary electrophoresis in a pH 4.6 ammonium acetate‐acetic acid buffer was interphased to an atmospheric pressure ionization triple quadrupole mass spectrometry system. Using positive ion electrospray ionization and the tandem mass spectrometry mode with collision‐induced dissociation in the collision cell, fragmentation of the two substances was determined. For confirmation via direct urine injection or application of a urinary extract, in‐source fragmentation was employed and the first quadrupole was operated in the selected ion monitoring mode by switching between the masses of relevant precursor/product ion sets for methadone (m/z = 310, 265) and EDDP (m/z = 278, 249, 234). This capillary electrophoresis – mass spectrometry approach is shown to permit the confirmation of methadone and EDDP in patient urines that tested positive for methadone using electrokinetic capillary‐based immunoassays, a fluorescence polarization immunoassay, and capillary electrophoresis with UV absorption detection.
Capillary sample introduction of polymerase chain reaction (PCR) products separated in ultrathin slab gels
Authors: Bullard Katherine M. , Hietpas Paula Beyer , Ewing Andrew G.
Published: May 2005

Polymerase chain reaction (PCR) amplified short tandem repeat (STR) samples from the HUMVWF locus have been analyzed using a unique sample introduction and separation technique. A single capillary is used to transfer samples onto an ultrathin slab gel (57 μm thin). This ultrathin nondenaturing polyacrylamide gel is used to separate the amplified fragments, and laser‐induced fluorescence with ethidium bromide is used for detection. The feasibility of performing STR analysis using this system has been investigated by examining the reproducibility for repeated samples. Reproducibility is examined by comparing the migration of the 14 and 17 HUMVWF alleles on three consecutive separations on the ultrathin slab gel. Using one locus, separations match in migration time with the two alleles 42s apart for each of the three consecutive separations. This technique shows potential to increase sample throughput in STR techniques although separation resolution still needs to be improved.
Sequencing using capillary electrophoresis of short tandem repeat alleles separated and purified by high performance liquid chromatography
Authors: Marino Michael A. , Devaney Joseph M. , Smith Jonathan K. , Girard James E.
Published: May 2005

Polymerase chain reaction (PCR) amplified alleles need to be isolated and purified before carrying out additional analysis to confirm sequence, number of repeats and microvariants within a short tandem repeat (STR) locus. Also, PCR amplification of tetranucleotide repeat loci, used in DNA typing assays, often result in heteroduplex formation, adding to the complexity of analysis. Sequencing reactions require single specific target DNA for reliable sequencing analysis. Alkylated poly(styrene‐divinylbenzene) columns at elevated temperature and gradient elution conditions increase the efficiency of separation to allow for the purification of PCR products. Using the separation technique of ion‐paring reverse‐phase (IPRP) high performance liquid chromatography (HPLC), molecular biologists can separate and purify DNA fragments without alteration to the double‐stranded DNA sequencing properties. In this study, the IP‐RP chromatography technique has been demonstrated by separation of alleles of the short tandem repeat loci of TH01, vWA31, F13A01 and FES/FPS. Alleles differing in size range of 12 to 4 base pairs were separated by IPRP/HPLC and individual alleles were peak‐captured, then cycle‐sequenced. These HPLC fractions required no additional steps prior to cycle sequencing. Capillary electrophoresis (CE) was used to sequence the alleles. Furthermore, CE offers advantages over traditional slab methods via automation and higher applied voltages. Interestingly, unlike traditional gel electrophoresis, samples were introduced into the sieving matrix by electrokinetic injection, which allows for multiple injections from a single sample, a key feature for method development. Applied voltage was 320 V per centimeter using a nonderivatized fused silica capillary with an interior diameter of 50 μm and a total length of 47 centimeters. The total analysis time including capillary filling and pre‐electrophoresis was less than 30 min for a 220‐bp fragment. A sequencing rate of 530 bp/h was achieved using these conditions. By combining the techniques of HPLC separation and CE sequencing, the results confirmed the sequence and number of nucleotide repeats for each STR loci. An average sequencing efficiency of 97% was achieved. Additionally, this method defined the absence of a 9.3 microvariant for a TH01 heterozygous individual previously typed as a 9,9.3/10 using slab gel electrophoresis. The techniques described can be applied to other DNA purification and isolation problems.
Myoglobin intron variation in the Gouldian Finch Erythrura gouldiae assessed by temperature gradient gel electrophoresis
Authors: Heslewood Margaret M. , Elphinstone Martin S. , Tidemann Sonia C. , Baverstock Peter R.
Published: May 2005

In this study we show how the use of exon‐primed, intron‐crossing (EPIC) polymerase chain reaction (PCR) of a diploid intronic region, in conjunction with temperature gradient gel electrophoresis (TGGE), can be used to detect and rapidly assess allelic variation at the nucleotide level. We developed passerine‐specific primers to amplify and sequence a 762 bp region including the second intron of the myoglobin gene in the Gouldian Finch, Erythrura gouldiae. A POLAND plot based on this sequence indicated that TGGE in combination with heteroduplex analysis (TGGE/HA) should reveal nucleotide variation in the 160 bp low‐melting domain. Sequencing of the entire fragment from 19 Er. gouldiae revealed five nucleotide substitution differences within the low‐melt domain, all of which could be detected and differentiated by TGGE/HA, and an additional substitution in a section of the high‐melt domain which characterised another allele. A total of 181 individuals from four populations were screened for these six alleles.
Development of standard reference materials for diagnosis of p53 mutations: Analysis by slab gel single strand conformation polymorphism
Authors: O'Connell Catherine D. , Tian Jing , Juhasz Agnes , Wenz H.‐Michael , Atha Donald H.
Published: May 2005

We have amplified by polymerase chain reaction (PCR) a 2.0 kbp region of the p53 gene containing exons 5–9 from seven cell lines reported in the literature to contain the majority of mutations reported for this gene. Sequence analysis of these products show that all seven cell lines contain mutations within the mutational hot spots of the p53 gene. Six of the seven clones have single base substitutions and the seventh has a single base delection. We have analyzed the seven p53 single point mutations by single strand conformation polymorphism (SSCP) analysis using fluorescence slab gel electrophoresis (SG‐SSCP). Fluorescent‐labeled PCR primers were used for amplification of specific exons for mutation detection. SG‐SSCP was conducted using Model 373 and Model 377 DNA sequencers with GeneScan Software (Perkin Elmer, Applied Biosystem Division). Nine different gel systems were first tested for their ability to resolve the p53 mutations using the Model 373 instrument. Two gel systems were capable of resolving all of the mutations that were screened. Optimal results were obtained with 12% w/v acrylamide 50:1 plus 10% v/v glycerol. This gel system was used to evaluate the effect of temperature on the ability to resolve the mutations. The separation with respect to wild type varied for each mutation examined. Subambient temperature (20°C) was preferable overall for discrimination of these mutations as a group. We intend to use this system to examine a much larger panel of p53 mutation standards that are now under development.
Elution of glycoproteins from replicas of sodium dodecyl sulfate‐polyacrylamide gel electrophoresis gels
Authors: Szewczyk Boguslaw , Pilat Zbigniew , Bienkowska‐Szewczyk Krystyna , Summers Donald F.
Published: May 2005

A method for the elution of glycoproteins from sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) replicas of gels on polyvinylidene difluoride (PVDF) membranes is described. Ten model glycoproteins were resolved by SDS‐PAGE and then electrotransferred onto PVDF membranes. After reversible staining, glycoprotein bands were eluted with a mixture of SDS/Triton X‐100 at pH 9 or with a mixture of guanidinium hydrochloride/lysophosphatidylcholine at neutral pH. For both types of eluents, the final recoveries ranged from over 30% to about 80%. Good recoveries and mild conditions of elution render the method applicable for the structural elucidation of glycan chains.
Forensic validation of the short tandem repeat HUMACTBP2 using capillary electrophoresis
Authors: Dimo‐Simonin Noelle , Grange Fabienne , Kratzer Adelgunde , Brandt‐Casadevall Conxita , Mangin Patrice
Published: May 2005

Experiments were performed to evaluate the forensic identification of the short tandem repeat (STR) HUMACTBP2 (human beta‐actin‐related pseudogene) using automated fluorescence‐based capillary electrophoresis. The HUMACTBP2 is a complex tetranucleotide STR locus with more than 32 alleles in the range of 202‐323 bp. The reproducibility of genetic typing using a fluorescent labeled allelic ladder was determined by comparison of the calculated fragment size after consecutive (within‐day) and nonconsecutive (day to day) injection. The maximum variation in size (window) observed for any allele was 0.23 bp for the within‐day and 0.8 bp for the day‐to‐day precision. Furthermore, it is possible to achieve a 1 bp resolution, the precision of the reproducibility assays being about 99.95%. Sixty blood samples and twenty stains were typed with both automated fluorescent sequencer ABI 373A and ABI 310. Identical genotypes were obtained with both techniques and the ABI 310 seemed to be more sensitive than the ABI 373A. A population sample of 197 unrelated individuals from southwest Switzerland was analyzed and the genotype frequencies observed were similar to those reported by others. Thirty‐one alleles and 126 genotypes were found. The observed heterozygosity was 0.934. Mixtures from two different blood samples varying in their ratio were typed and the minor fraction was detectable to about 1:10. The practical usefulness of the HUMACTBP2 is illustrated by analyzing casework samples. This validation study proves the usefulness of the HUMACTBP2 locus in forensics and the detection efficiency using fluorescent capillary electrophoresis.
Plasma protein adsorption patterns on emulsions for parenteral administration: Establishment of a protocol for two‐dimensional polyacrylamide electrophoresis
Authors: Harnisch Stephan , Müller Rainer H.
Published: May 2005

The two‐dimensional polyacrylamide electrophoresis (2‐D PAGE) of the plasma protein adsorption pattern previously established for polymeric nanoparticles was modified and transferred to oil in water emulsions for intravenous administration. The emulsions were incubated with citrated plasma, and separation from excess plasma was performed by centrifugation under optimized conditions: 15 000 g and three washing steps with 0.05 M phosphate buffer, pH 7.4. With this sample preparation, coalescence of droplets could be avoided and an unchanged surface area maintained, in addition the phosphate buffer minimized artificial IgG adsorption. Critical factors affecting sensitivity were contamination of the sample by oil residues and the use of thiourea in the immobilized pH gradients. Changes in the protein adsorption pattern caused by altered surface properties of the emulsion (i. e. adsorbed Poloxamer 407) were detectable when applying the optimized protocol. Knowledge of the protein adsorption patterns and their correlation to in vivo behavior opens the perspective for the development of intravenous emulsions for controlled drug delivery.
Affinity mode of capillary isoelectric focusing for the characterization of the biotin‐binding protein actinavidin
Authors: Okun Vadim M.
Published: May 2005

Different modes of capillary isoelectric focusing (CIEF) with salt mobilization and zwitterionic additive to cathodic mobilizer were applied to characterize the biotin‐binding protein actinavidin and its affinity properties. The analysis is performed in a neutral coated capillary with completely eliminated electroosmotic flow. Synthetic pI standards with absorption in the visible region were used in all runs and pI determinations were based on a fitted nonlinear calibration graph. CIEF of highly purified actinavidin in native conditions was revealed in about 12 peaks in pI range 6.2–7.5, with three major forms having pI 6.80, 6.86, 6.90. CIEF of a mixture of actinavidin and increasing concentrations of two affinity ligands (biotin and biotinylated oligonucleotide) demonstrated drastic changes in the number of protein isoforms. In the latter case it resulted in only one peak (pI 5.05) when the ligand was in excess. This method, which can be named affinity CIEF, was found to be well‐suited for studying structural changes, occurring in receptor proteins upon binding with a ligand. CIEF of the protein, performed under denaturing conditions (6 M urea in a solution of carrier ampholytes) is also reported. It was revealed in three isoforms with a pI more acidic than that of native actinavidin. It is demonstrated that careful selection of denaturing conditions was necessary for the reproducible results.
Separation of the enantiomers of ibuprofen and its major phase I metabolites in urine using capillary electrophoresis
Authors: Bjørnsdottir Inga , Kepp Dorte Rode , Tjørnelund Jette , Hansen Steen Honoré
Published: May 2005

A capillary electrophoresis method for determination of the enantiomers of ibuprofen and its major phase I metabolites: 2′‐hydroxyibuprofen and 2′‐carboxyibuprofen in urine samples have been developed. Cyclodextrins and linear dextrins have been investigated as chiral selectors. Simultaneous chiral separation of the enantiomers of ibuprofen, 2′‐hydroxyibuprofen and 2′‐carboxyibuprofen was obtained using a mixture of dextrin 10 and heptakis (2,3,6‐tri‐O‐methyl)‐β‐cyclodextrin in a 2‐[N‐morpholino]ethanesulphonic acid buffer, pH 5.26. The electroosmotic flow was reversed using hexadimethrine bromide as a buffer additive. The method can be used for the determination of the free enantiomers of ibuprofen, 2′‐hydroxyibuprofen and 2′‐carboxyibuprofen as well as for the indirect determination of their glucuronic acid conjugates in urine samples.
Microevolution during epidemic spread of Neisseria meningitidis (minireview)
Authors: Achtman Mark
Published: May 2005

Similar to many other naturally transformable bacteria, Neisseria meningitidis has yielded many examples where horizontal genetic exchange has resulted in genetic variation of individual strains. Epidemic strains are purified of genetic variants due to bottlenecks during the spread from country to country, resulting in clonal descent. Occasionally, clonal replacement also occurs during epidemic spread. These processes occur rapidly in serogroup A meningococci; such that after their descent from a common ancestor, clonally related bacteria have diversified at numerous loci within the last decades.
Mosaic genes and their role in penicillin‐resistant Streptococcus pneumoniae (minireview)
Authors: Hakenbeck Regine
Published: May 2005

Penicillin resistance in clinical isolates of Streptococcus pneumoniae is mediated by mosaic genes encoding altered penicillin binding proteins. Mosaic sequence blocks are the result of a genetic exchange between related streptococcal species. It is likely that resistance has emerged in commensal streptococci before being transferred into the pneumococcus. Closely related mosaic genes are found in different pneumococcal clones and in different streptococcal species isolated worldwide since the first reports on such strains in the late 70s, demonstrating the importance of commensal streptococci for the spread of selectable markers in naturally transforming pathogens.
Activity staining of pectinesterase on polyacrylamide gels after acidic or sodium dodecyl sulfate electrophoresis
Authors: Hou Wen‐Chi , Lin Yaw‐Huei
Published: May 2005

Pectinesterase (PE), from commercial orange peels or ammonium sulfate fractionation (50–80% saturation) of pea pods, was detected on polyacrylamide gels after native acidic polyacrylamide gel electrophoresis (PAGE) or sodium dodecyl sulfate (SDS)‐PAGE by using the synthetic substrate β‐naphthyl acetate (β‐NA). The release of β‐naphthol (at 322 nm) from β‐NA was proportional to PE activity. The PE activity bands on polyacrylamide gels after native acidic PAGE or SDS‐PAGE were stained with a combination of tetrazotized o‐dianisidine and β‐NA. This fast and sensitive method can be used for enzyme purification and characterization.
Use of thiourea to increase the solubility of membrane proteins in two‐dimensional electrophoresis
Authors: Rabilloud Thierry
Published: May 2005

The separation of membrane proteins by high‐resolution two‐dimensional electrophoresis was carried out. At high loads, these proteins are prone to precipitation, resulting in poor resolution. It is shown here that the use of thiourea, previously described for focusing in immobilized pH gradients, can be extended to conventional isoelectric focusing. As thiourea inhibits acrylamide polymerization, a modified photopolymerization system must be used. These modifications result in higher solubility of proteins during IEF, thereby increasing the resolution and capacity of the two‐dimensional gels.
A two‐dimensional electrophoretic study of serum amyloid A and C‐reactive protein in infants and children
Authors: Bruun Cathrine Foyn , Sanchez Jean‐Charles , Hochstrasser Denis F. , Marhaug Gudmund , Husby Gunnar
Published: May 2005

Two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) was used to analyze C‐reactive – (CRP) and serum amyloid A protein (SAA) in infants and children. Five SAA isotypes were identified. CRP showed vertical streaking, and its optical density values correlated with immunoturbidimetric measurements. As evaluated by densitometry, both proteins showed an age‐dependent variation. In more than 50% of the neonates, SAA was present in equal or higher amounts than CRP, and only SAA1α could be detected. In children, CRP was expressed in higher amounts than SAA, and both SAA1α and SAA2α were present. N‐terminally modified forms of both isotypes were present regardless of age, including in premature infants. These results suggest that the overall synthesis of the gene products SAA1α and SAA2α is developmentally regulated, but at the same time that their N‐terminal processing occurs independently of developmental factors. The presented data suggest that SAA has an important function in neonates, and that the role of SAA as an infection marker in this population should be investigated further.
Divergent and common groups of proteins in glands of venomous snakes
Authors: Rioux Vincent , Gerbod Marie‐Christine , Bouet Françoise , Ménez André , Galat Andrzej
Published: May 2005

Protein contents of venom‐producing glands from the sea‐snake Laticauda colubrina (LC) and terrestrial Vipera Russelli (VR) were studied using high‐resolution two‐dimensional gels: isoelectric focusing followed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (IEF/SDS‐PAGE) and nonequilibrium pH gradient electrophoresis (NEPHGE) followed by SDS‐PAGE. Tentative identities of numerous proteins were established using their amino acid compositions and in certain cases the identities were verified by microsequencing of their N‐terminals and internal fragments. As expected, we found several proteins known to be present in the venom of the respective snakes. These include numerous isoforms of phospholipase A2 (PLA2) in both snake glands, various neurotoxins in LC glands and factor IX/factor X‐binding protein, hemorrhagic factor and coagulation factor X activating enzyme in Russell's viper glands (VR). Not unexpectedly, we also found a number of cell housekeeping proteins, cytoskeletal proteins, proteins that are necessary for folding, such as heat‐shock proteins, protein disulfide‐isomerase and peptidylprolyl cis/trans isomerases. Unexpectedly, however, the glands of Laticauda colubrina and Russell's viper include a large quantity of antihemmorhagic factor and inhibitor of PLA2, respectively, that have been previously described in snake plasma. The possible reason associated with the presence of these components in venom glands is discussed.
Two‐dimensional gel electrophoresis separation and N‐terminal sequence analysis of proteins from Clostridium pasteurianum W5
Authors: Flengsrud Ragnar , Skjeldal Lars
Published: May 2005

Proteins from Clostridium pasteurianum were separated by two‐dimensional gel electrophoresis. Amino‐terminal sequence determination and sequence analysis allowed the identification of 20 proteins, while 11 protein sequences remained unidentified and one protein appeared to have a blocked amino terminus.
Two‐dimensional gel database of human breast carcinoma cell expressed proteins: An update
Authors: Rasmussen Richele K. , Ji Hong , Eddes James S. , Zugaro Lisa M. , Reid Gavin E. , Simpson Richard J. , Dorow Donna S.
Published: May 2005

Previously, we reported a two‐dimensional gel map and database with molecular weight/isoelectric point (Mr/pI) loci for 22 proteins expressed in the breast carcinoma cell line, MDA‐MB231 (Rasmussen et al., Electrophoresis 1997, 18, 588–598). Here we update this database with Mr/pI loci for a further nine cytoplasmic proteins and three Triton X‐114 solubilised membrane proteins from MDA‐MB231 cells. In addition, a novel protein, previously represented only in expressed sequence tag (EST) databases, has been identified as a Triton X‐114 soluble protein and assigned an Mr/pI locus. During the course of isolating proteins from the Triton X‐114 fraction, we compared recoveries of proteins in sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels after isoelectric focusing (IEF) using either immobilised pH gradients or carrier ampholytes. In these experiments, a significantly higher proportion of membrane proteins were visible in SDS‐polyacrylamide gels after the use of carrier ampholytes for the first dimension. We also report our mass spectrometric‐based procedure for identifying two‐dimensional electrophoresis (2‐DE) gel‐resolved proteins, combining in‐gel enzymatic digestion, 0.2 mm internal diameter (ID) capillary column reversed‐phase high‐performance liquid chromatography (RP‐HPLC) peptide mapping and electrospray ionisation — ion trap — mass spectrometry.
Characterization of rat brain stathmin isoforms by two‐dimensional gel electrophoresis‐matrix assisted laser desorption/ionization and electrospray ionizationion trap mass spectrometry
Authors: Zugaro Lisa M. , Reid Gavin E. , Ji Hong , Eddes James S. , Murphy Anne C. , Burgess Antony W. , Simpson Richard J.
Published: May 2005

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

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

Owing to the complexity of higher eukaryotic cells, characterization of a complete proteome is likely to be difficult to achieve. However, advantage can be taken of the cell compartmentalization to build organelle proteomes, which can moreover be viewed as specialized tools to study specifically the biology and “physiology” of the target organelle. Within this frame, we report here the construction of the human mitochondrial proteome, using placenta as the source tissue. Protein identification was carried out mainly by peptide mass fingerprinting, but other methods were also used (N‐terminal microsequencing, blotting). The optimization steps in two‐dimensional (2‐D) electrophoresis needed for proteome research are discussed. However, the relative paucity of data concerning mitochondrial proteins is still the major limiting factor in building the corresponding proteome, which should be a useful tool for researchers working on human mitochondria and their deficiencies

This work can be found on the Internet at the following address: http://www‐dsv.cea.fr/MitoPick/Default.html

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Correlation of acidic and basic carrier ampholyte and immobilized pH gradient two‐dimensional gel electrophoresis patterns based on mass spectrometric protein identification
Authors: Nawrocki Arkadiusz , Larsen Martin R. , Podtelejnikov Alexandre V. , Jensen Ole N. , Mann Matthias , Roepstorff Peter , Görg Angelika , Fey Stephen J. , Larsen Peter Mose
Published: May 2005

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

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

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

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

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

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

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

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

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

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

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

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

The electrophoretic mobility of five single tandem repeats (STRs) and four amplified fragment length polymorphisms (AFLPs) in polyacrylamide gels was tested under denaturing and nondenaturing conditions. Relative anomalous mobility in nondenaturing conditions was found in one AT‐rich AFLP (3′ ApoB) and in two AT‐rich STRs (HUMACTBP2 and HUMF13A1). In these cases, highly anomalous electrophoretic behavior was found even when changes were made in the %T value alone. In such cases typing results were affected by the gel composition. The mobility of these systems was nevertheless normal under denaturing conditions. As a consequence of this study, we recommended that the typing of these systems should only be performed under denaturing conditions and we also recommend the further study of the electrophoretic behavior of repetitive DNA polymorphisms, especially AT‐rich systems, before they are used in forensic or genetic applications.
Synthesis of zwitterionic acrylic acid buffers for isoelectric focusing in immobilized pH gradients
Authors: Bellini Marco P. , Manchester Keith L.
Published: May 2005

A range of zwitterionic acrylic acid derivatives, buffering in the neutral and basic pH ranges, have been synthesized by the Mannich reaction of malonic acid, formaldehyde and a secondary amine. These compounds include 2‐(4‐morpholinomethyl)propenoic acid pK2 7.59 ± 0.03 (23°C), 2‐[bis(2‐hy‐droxyethyl)aminomethyl]propenoic acid pK2 ≈ 8.6 (20°C), 2‐[bis(2‐hydroxy‐propyl) aminomethyl]propenoic acid pK2 ≈ 8.7 (20°C), 2‐[N‐(2‐hydroxyethyl)‐N‐methylaminomethyl]propenoic acid pK2 9.22 ± 0.08 (22°C), 2‐[N‐ethyl‐N‐(2‐hydroxyethyl)aminomethyl]‐propenoic acid pK2 ≈ 9.6 (20°C), and 2‐[4‐(2‐carboxyprop‐2‐enyl)piperazinylmethyl]propenoic acid, which has a sigmoidal buffering profile over the pH range 3–10. These zwitterionic acrylic acid buffers were successfully copolymerized with acrylamide to prepare immobilized pH gradients (IPGs) in the neutral to alkaline portion of the pH range. Bovine erythrocyte carbonic anhydrase isozymes were resolved on a pH 5–8 IPG prepared using 2‐[4‐(2‐carboxyprop‐2‐enyl)piperazinylmethyl]propenoic acid as the immobilized buffer and horse heart myoglobin was focused on pH 7.1–8.1 and pH 7.5–7.7 IPGs, using 2‐(4‐morpholinomethyl)propenoic acid as the immobilized buffer. In both cases the pK 9.3 Immobiline compound was used as the strongly basic titrant. These new compounds, besides possessing more hydrophilic residues than the corresponding commercial basic acrylamido buffers (Immobilines), resist hydrolysis at alkaline pH values.