bluuebunny/crossref_metadata_2025 huggingface dataset:
Multimodal application of lectin affinity electrophoresis of α‐fetoprotein
Authors: Taketa Kazuhisa
Published: May 2005

Multimodal application of lectin affinity electrophoresis of α‐fetoprotein (AFP) glycoforms is reviewed. Crossed affinity immunoelectrophoresis developed by Bøg‐Hansen and others was extended to tandem‐lectin affinity electrophoresis by tandem lining of two different lectin gels and to mixed‐lectin affinity electrophoresis. By introducing an antibody‐affinity blotting technique for detection of separated glycoforms of AFP, several two‐dimensional combinations of lectin affinity electrophoresis became possible: two different lectins for the first and second dimension electrophoresis, lectin‐gradient affinity electrophoresis, and electrophoretic separation of lectin isoforms in the first‐dimension electrophoresis, followed by affinity electrophoresis against the separated lectin isoforms. Usefulness of the different modalities of lectin affinity electrophoresis for several analytical purposes has been described.
Two‐dimensional gel electrophoretic analysis of the changes after immortalization of human cells: Decrease of intracellular α‐2‐macroglobulin fragment
Authors: Kondo Tadashi , Sakaguchi Masakiyo , Yamada Hidenori , Namba Masayoshi
Published: May 2005

To study the mechanisms of immortalization of human cells, an early step in cancer development, we compared the cellular proteins of normal and immortalized human fibroblasts. Two‐dimensional gel electrophoresis showed that one spot with a molecular mass 20 kDa and an isoelectric point of 6.0, became significantly smaller after immortalization of human cells. Further, the spot was rarely observed in four human liver cancer cell lines. Investigation of the N‐terminal amino acids revealed that the spot was a fragment of α‐2‐macroglobulin. Although the 20 kDa fragment contains methionine, the spot was not labeled with [35S]methionine. Thus we concluded that the spot might be derived from the culture medium. These results indicated that intracellular metabolism of α‐2‐macroglobulin, which is a multifunctional protease inhibitor, changed after the cells were transformed.
Structural determination of N‐linked carbohydrates by matrix‐assisted laser desorption/ionization‐mass spectrometry following enzymatic release within sodium dodecyl sulphate‐polyacrylamide electrophoresis gels: Application to species‐specific glycosylation of α1‐acid glycoprotein
Authors: Küster Bernhard , Hunter Ann P. , Wheeler Susan F. , Dwek Raymond A. , Harvey David J.
Published: May 2005

This paper describes a sensitive method for analysis of N‐linked carbohydrates released enzymatically from within the gel following separation of glycoproteins (50–100 pmols) by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The separated bands containing the glycoproteins were cut from the gel, destained, reduced and alkylated. N‐linked glycans were then released by in‐gel incubation with peptide N‐glycosidase‐F (PNGase‐F) and extracted with water and acetonitrile. Sialic acid‐containing glycans were converted into methyl esters by reaction with methyl iodide, salts and reagents were removed by passage through a mixed‐bed column of ion‐exchange resins and the glycans were examined by matrix‐assisted laser desorption/ionization (MALDI)‐mass spectrometry. Structural determination of the released glycans was performed by exoglycosidase digestion. Following glycan release and extraction, the protein could be digested within the gel with trypsin, and the masses of the tryptic peptides could be compared with those generated from a sequence database for protein identification. The method is applied to the analysis of N‐linked glycans from α1‐acid glycoprotein from man, cow, sheep and dog. Major species‐specific differences in glycosylation were found. Thus, although all four species used N‐acetyl‐neuraminic acid, only cow and sheep additionally used N‐glycolyl‐neuraminic acid. Biantennary glycans were the predominant carbohydrates in cow, sheep and dog but man produced more triantennary glycans and a substantial amount of tetraantennary sugars. Fucosylation was only found in glycans from man and cow and both cow and sheep glycans were found to have β1‐3‐ and well as β1‐4‐linked galactose residues in the antennae.
'98 Escherichia coli SWISS‐2DPAGE database update
Authors: Tonella Luisa , Walsh Brad J. , Sanchez Jean‐Charles , Ou Keli , Wilkins Marc R. , Tyler Margaret , Frutiger Séverine , Gooley Andrew A. , Pescaru Ioana , Appel Ron D. , Yan Jun X. , Bairoch Amos , Hoogland Christine , Morch Fabienne S. , Hughes Graham J. , Williams Keith L. , Hochstrasser Denis F.
Published: May 2005

The combination of two‐dimensional polyacrylamide gel electrophoresis (2‐DPAGE), computer image analysis and several protein identification techniques allowed the Escherichia coli SWISS‐2DPAGE database to be established. This is part of the ExPASy molecular biology server accessible through the WWW at the URL address http://www.expasy.ch/ch2d/ch2d‐top.html. Here we report recent progress in the development of the E. coli SWISS‐2DPAGE database. Proteins were separated with immobilized pH gradients in the first dimension and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the second dimension. To increase the resolution of the separation and thus the number of identified proteins, a variety of wide and narrow range immobilized pH gradients were used in the first dimension. Micropreparative gels were electroblotted onto polyvinylidene difluoride membranes and spots were visualized by amido black staining. Protein identification techniques such as amino acid composition analysis, gel comparison and microsequencing were used, as well as a recently described Edman “sequence tag” approach. Some of the above identification techniques were coupled with database searching tools. Currently 231 polypeptides are identified on the E. coli SWISS‐2DPAGE map: 64 have been identified by N‐terminal microsequencing, 39 by amino acid composition, and 82 by sequence tag. Of 153 proteins putatively identified by gel comparison, 65 have been confirmed. Many proteins have been identified using more than one technique. Faster progress in the E. coli proteome project will now be possible with advances in biochemical methodology and with the completion of the entire E. coli genome.
Mining the human proteome: Experience with the human lymphoid protein database
Authors: Hanash Samir M. , Teichroew Daniel
Published: May 2005

We have undertaken an effort in the past five years aimed at developing a database of lymphoid proteins detectable by two‐dimensional (2‐D) polyacrylamide gel electrophoresis. The database contains 2‐D patterns and derived information pertaining to: (i) polypeptide constituents of unstimulated and stimulated mature T cells and immature thymocytes; (ii) cultured T cells and cell lines that have been manipulated by transfection with a variety of constructs or by treatment with specific agents; (iii) single cell‐derived T and B cell clones; (iv) cells obtained from patients with lymphoproliferative disorders and leukemia; and (v) a variety of other relevant cell populations. The database has experienced a substantial expansion in 2‐D patterns it contains, numbering currently 9167 individual 2‐D patterns. This number represents a fraction of the 30682 2‐D patterns maintained in our databases. The capacity to design and undertake experiments, produce high‐quality 2‐D patterns, and to undertake simple or rudimentary analyses of 2‐D patterns to meet the basic needs of the experiments for which the 2‐D gels were produced has exceeded the capacity to fully and uniformly integrate information generated from any gel image or experiment, across all images and experiments. While only a fraction of the information in the 2‐D patterns contained in the lymphoid database has been mined, novel findings derived from querying the database point to the merits of this protein based approach. Additional resources have recently been put into place to mine more effectively data pertaining to protein expression in lymphoid cells.
Proteomic changes associated with degeneration of myelin‐forming cells in the central nervous system of c‐myc transgenic mice
Authors: Jensen Niels A. , Celis Julio E.
Published: May 2005

Myelin is necessary for the conduction of high frequency and high velocity nerve impulses in the central nervous system of mammals, and severe neurological disturbances develop as a result of myelin loss. In this report, we have characterized changes in the brain proteomic profile of transgenic mice that develop a c‐myc‐induced degenerative disorder of myelin. Marked differences were seen in the accumulation of cytoskeletal proteins associated with the pathological condition fibrous gliosis in the optic nerves of affected animals, including upregulation of glial fibrillary acid protein and vimentin. In addition, the expression of several major myelin proteins, including five isoforms of myelin basic protein, four isoforms of cyclic nucleotide 3′‐phosphodiesterase, and myelin‐associated glycoprotein, was markedly reduced in the brains of c‐myc transgenic mice as revealed by immunocytochemistry and by two‐dimensional immunoblots. A number of novel proteomic disease marker candidates were revealed, which displayed pronounced changes in their expression profile. Sequence determination of these proteins and molecular cloning of their mRNAs will provide an opportunity to further evaluate their roles in the disease process.
Protein changes observed in pacing‐induced heart failure using two‐dimensional electrophoresis
Authors: Heinke Monique Y. , Wheeler Colin H. , Chang Dennis , Einstein Rosemarie , Drake‐Holland Angela , Dunn Michael J. , Remedios Cristobal G. Dos
Published: May 2005

Rapid ventricular pacing in dogs results in a low output cardiomyopathic state which is similar to idiopathic dilated cardiomyopathy in man. However, the pathophysiological mechanisms which cause this failure following pacing are unknown. Five dogs underwent rapid ventricular pacing. Hearts were stimulated at 245 beats per min (bpm) for four weeks and then reduced to 190 bpm to stabilize the failure. Six unoperated dogs were used as controls. This paper compares the two‐dimensional gel electrophoresis (2‐DE) protein patterns of left ventricular samples from the paced myocardium with the control dogs. Changes in protein expression were analyzed qualitatively and semi‐quantitatively. In the paced dog samples 69 protein spots were significantly altered of which 42 were decreased and 27 were elevated. One qualitative change was observed: elongation factor Tu was present only the control hearts. Of these proteins, 20 have been identified by a combination ofN‐terminal protein microsequencing, peptide mass profiling by mass spectrometry, amino acid compositional analysis, and by comparison with databases of canine and human ventricular proteins. Ten of these are associated with mitochondria and energy production, including: pyruvate dehydrogenase E1 component, isocitrate dehydrogenase subunit α, HSP60 and HSP70, creatine kinase M and fatty acid binding protein. The cytoskeletal protein desmin was detected in reduced quantities and a spot corresponding to a fragment of desmin was increased. These results indicate that the development of heart failure in the paced dog involves alterations in mitochondrial energy production, the cytoskeleton and calcium activation.
Method development in pharmaceutical analysis employing capillary electrochromatography
Authors: Angus Peter D. A. , Stobaugh John F. , Victorino Estelita , Payne Kent M. , Demarest Charles W. , Catalano Tom
Published: May 2005

Capillary electrochromatography (CEC) has been employed to explore method development for a series of structurally related polar neutral compounds of pharmaceutical relevance. Capillaries with dimensions of 75 μm ID × 25 cm length (34.5 cm total) were packed with Spherisorb ODS‐1, Hypersil phenyl, and Hypersil MOS (all 3 μm particles) and were compared in the reversed‐phase mode in order to determine which phase provided the best initial performance and thus serve as the phase of choice for additional method development experiments. The various separation parameters examined for their effect on efficiency, k, resolution, and linear velocity included percent and type of organic modifier, buffer concentration, voltage, and temperature. All separations were conducted with an acidic mobile phase (aqueous mobile phase component, pH 3.0). The separation efficiencies obtained were on the order of 200000–260000 plates/m, which equates to reduced plate heights of 1.22 for columns packed with Spherisorb ODS‐1. Repeatable column‐to‐column separation performance was demonstrated.
Application of heparin to chiral separations of antihistamines by capillary electrophoresis
Authors: Jin Yingkun , Stalcup Apryll M.
Published: May 2005

A study of the chiral separations of antihistamines, including pheniramine, chlorpheniramine, brompheniramine, carbinoxamine and doxylamine in capillary electrophoresis (CE) was accomplished using heparin as a chiral additive (CA) and phosphate buffer as the background electrolyte. Several factors were shown to affect both the selectivity and the migration time, including concentration of heparin, concentration of buffer, and the pH. A dual mechanism involving both inclusion complexation and ionic interactions with heparin is thought to be responsible for the chiral recognition. In the pH range of 2.6–3.5 and reversed polarity, baseline resolutions were obtained using a wide range of buffer and heparin concentrations. Typically, chiral resolution was obtained within 50 min.
The effect of blob size and network dynamics on the size‐based separation of polystyrenesulfonates by capillary electrophoresis in the presence of entangled polymer solutions
Authors: Cottet Hervé , Gareil Pierre , Viovy J.‐L.
Published: May 2005

This work focuses on the separation of standard polystyrenesulfonates (PSS), with molecular masses (Mr) between 16 and 990 × 103 in capillaries filled with semidilute (entangled) linear hydrophilic polymers. Contrary to cross‐linked chemical gels, which produce permanent networks, solutions of linear polymers lead to dynamic networks. The analytical performances and migration mechanisms are discussed on the basis of experiments performed in solutions of linear polyethyleneoxides and derivatized celluloses of various molecular masses. The influence of the mesh size and of the lifetime of the obstacles of the separating network has been investigated in detail. The mesh size is assimilated to the blob size of the separating polymer and is a decreasing function of its concentration. The lifetime of the obstacles of the network, identified with the reptation time of the polymer chain, characterizes its dynamics. This characteristic time increases with both the molecular weight of the separating polymer and its concentration. Its impact was first examined at fixed blob size. Then, the influence of the blob size was studied while keeping the reptation time of the network constant. By doing so, the existence of interactions between the solute and the separating polymer or between the solute and capillary wall can be more safely assessed. It appears that the reptation time of the mesh has a large influence on the electrophoretic mobility of the PSSs under a threshold value, which is of the order of magnitude of the time taken by the PSS to migrate on the blob size. Also shown are separations using networks made up with mixtures of polyethyleneoxides of the same nature and same mass concentration, but of very different molecular masses. This latter approach allows one to adapt the viscosity of the solution and the dynamics of the network, keeping the blob size constant.
Analysis of antisense oligonucleotides by on‐capillary isotachophoresis and capillary polymer sieving electrophoresis
Authors: Khan Kamran , Van Schepdael Ann , Hoogmartens Jos , Saison‐Behmoaras Tula , Van Aerschot Arthur
Published: May 2005

An attempt was made to evaluate the stability of an antisense oligonucleotide against nucleases present in HBL 100ras cells. To detect nanomolar concentrations of the oligonucleotide, a sensitive detection system was required. A combination of capillary electrophoresis/laser‐induced fluorescence (CE‐LIF) with fluorescence derivatization did not improve the sensitivity significantly and also resulted in loss of separation of the derivatized sample. On‐column isotachophoresis for the preconcentration of oligonucleotide samples in DB‐17 coated capillaries filled with hydroxyethyl cellulose solution could be an alternative. The isotachophoresis (ITP) step allows injection of up to 40% of the capillary volume without loss in peak resolution and peak efficiency. Using ITP‐capillary polymer sieving electrophoresis (CPSE), the limit of quantitation at a signal‐to‐noise ratio of 10 was 73 ng/mL for a 12‐mer oligonucleotide. Using these conditions, the gain in sensitivity was 125.
Subnanomolar detection limit for sodium dodecyl sulfate — capillary gel electrophoresis using a fluorogenic, noncovalent dye
Authors: Harvey Michael D. , Bandilla Dirk , Banks Peter R.
Published: May 2005

Picomolar limits of detection are obtained using the noncovalent, fluorogenic dye, Sypro Red. The size separation of four commonly used sodium dodecyl sulfate ‐ capillary gel electrophoresis (SDS‐CGE) molecular weight markers with 8% linear polyacrylamide (PAA) as the sieving matrix is used to construct a calibration curve for molecular weight determinations. SDS‐CGE purity and molecular weight determination of purified chorismate mutase‐prephenate dehydrogenase (CMPD) from Escherichia coli is shown to be comparable in accuracy with slab gel SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE). A migration time precision study indicates excellent reproducibility. Sypro red labeling of SDS‐bovine serum albumin (SDS‐BSA) complexes at nanomolar protein concentrations suggests assay detection limits surpassing those of silver staining. This detectability exceeds that achieved in previous SDS‐CGE laser‐induced fluorescence studies. This approach is expected to be easily adapted for use with commercial polymer formulations and automated instrumentation.
Sodium dodecyl sulfate‐capillary electrophoresis of proteins in a sieving matrix utilizing two‐spectral channel laser‐induced fluorescence detection
Authors: Craig Douglas B. , Polakowski Robert M. , Arriaga Edgar , Wong Jerome C. Y. , Ahmadzadeh Hossein , Stathakis Costas , Dovichi Norman J.
Published: May 2005

We report a method for protein labeling, separation by capillary electrophoresis in a polymer sieving matrix, and detection by laser‐induced fluorescence. Different dyes are used to label standard and sample proteins. A two‐spectral channel detector resolves fluorescence from the sample and standards. Comparison of the migration time of the sample and standards permits the precise determination of molecular weight, irrespective of variations in run‐to‐run migration times.
Enhanced sensitivity for sequence determination of major histocompatibility complex class I peptides by membrane preconcentration ‐ capillary electrophoresis ‐microspray ‐ tandem mass spectrometry
Authors: Naylor Stephen , Ji Qinchung , Johnson Kenneth L. , Tomlinson Andy J. , Kieper William C. , Jameson Stephen C.
Published: May 2005

Sequence analysis of antigenic major histocompatibility complex (MHC) class I peptides requires minimizing sample loss and enhancing mass spectrometric sensitivity. In order to facilitate such analyses, we have coupled on‐line membrane preconcentration‐capillary electrophoresis (mPC‐CE) with microspray mass spectrometry (mPC‐CE‐μMS) and tandem mass spectrometry (mPC‐CE‐μMS/MS). Specifically, cell lysate from ∼ 109 EG‐7 mouse tumor cells was immunoprecipitated and the released MHC class I peptides were subjected to reverse‐phase HPLC. An HPLC fraction containing antigenic peptide(s) shown to induce T‐cell stimulation was subjected to mPC‐CE‐μMS. Approximately 10 μL (from 100 μL) of the fraction was pressure‐injected and concentrated on a styrenedivinylbenzene (SDB) impregnated membrane. The peptides were eluted from the membrane with ∼100 nL of 80% methanol, sandwiched between a leading stcking buffer (LSB, also serving as CE separation medium) of ∼110 nL of 0.1% acetic acid in 10% methanol, and a trailing stacking buffer (TSB) of ∼ 110 nL of 0.1% NH4OH. On application of the CE voltage the peptides are subjected to moving boundary transient isotachophoresis and focused. The peptides were separated in a Polybrene‐coated capillary with application of −20 kV in reverse polarity mode and subsequently sprayed via an emitter coupled to the CE capillary by a liquid junction containing a platinum wire. An ion at m/z 482.3 was detected and subjected to mPC‐CE‐μMS/MS and determined to be SIINFEKL, a peptide (OVA) known to be antigenic in the mouse model system. Sensitivity enhancement over conventional mPC‐CE‐MS and MS/MS was ∼100‐fold.
Fluorescence imaging of light absorption for axial‐beam geometry in capillary electrophoresis
Authors: Johansson Jonas , Johansson Thomas , Nilsson Staffan
Published: May 2005

A new method for investigation of axial‐beam absorption detection for improved detection limits in microcolumn separations is reported. The method is based on fluorescence imaging of light absorption along a separation capillary. The probing UV light is introduced at one end of the capillary and shows an exponential fall‐off along the capillary. As the UV light propagates through the sample peaks, an additional loss in intensity will be observed. In order to view the absorption profile along the capillary, a background fluorophore is added to the buffer. A charge‐coupled device (CCD) detector and imaging optics are placed beside the capillary to view the capillary in a direction perpendicular to the capillary. Signal integration is employed for consecutive exposures as well as for neighboring detector pixels in order to increase the signal‐to‐noise ratio. Measurements for stilbene 3 with sulforhodamine B as a background fluorophore are presented. The characteristics of the detection method and potential improvements are discussed.
A computer‐controlled variable light attenuator for protection and autoranging of a laser‐induced fluorescence detector for capillary zone electrophoresis
Authors: Merz Jerry M. , Mort Andrew J.
Published: May 2005

Photodetectors have a limited range over which they can measure light intensities for any particular setting. The intensity of light reaching the detector can be kept within this range by using a liquid crystal variable light attenuator controlled by a computer that continuously checks the amount of light reaching the detector and adjusts the attenuation to an appropriate level. Using such a system we have constructed an intensified charge‐coupled device (ICCD) camera‐based detector with a dynamic range of over six orders of magnitude which is never exposed to damaging or saturating light levels.
Dual UV‐absorbing background electrolytes for simultaneous separation and detection of small cations and anions by capillary zone electrophoresis
Authors: Xiong Xiang , Li Sam F. Y.
Published: May 2005

The simultaneous separation and detection of small cations and anions by capillary zone electrophoresis (CZE) with indirect ultraviolet (UV) detection was successfully demonstrated in a background electrolyte (BGE) containing two UV‐absorbing components. Benzylamine, imidazole, benzenesulfonic acid, sulfosalicylic acid, and pyromellitic acid were tested as the components of the BGE. The success of the simultaneous separation of the cations and anions is dependent upon the proper selection of the electrolyte components and control of the migration of the ions towards the detector. High pH is beneficial to the detection of anionic analytes but not to the separation of cationic analytes because of large electroosmotic flow produced under this condition. The upper pH limit of the working pH range is confined by the pKa value of the cationic component of the BGE. The influence of pH and total electrolyte concentration on the electroosmatic flow (EOF) counteracted each other. This counteraction effect imposes an upper limit on the change of total electrolyte concentration at certain pH. It was found that the EOF should be larger by at least 10 × 10−5 cm2V−1V1 than the electrophoretic mobilities of the anions so that the anions could be detected on the cathodic side within reasonable times and with good peak shapes. In the imidazole‐sulfosalicylic acid BGE, the detection limits (signal to noise, S/N = 3) for the cations and anions ranged from 100 to 900 ppb. In the benzylamine‐pyromellitic acid BGE, K+, Na+, Li+, CH3 COO−, HPO42−, F−, C1O3−, C1O4−, NO3−, NO2−, Cl− and SO42− were separated within twelve minutes. The strategies for selection of the electrolyte components of the binary BGE were also discussed.
Capillary electrophoresis/laser‐induced fluorescence detection of fluorescein as a groundwater migration tracer
Authors: Ferguson Patrick L. , Grange Andrew H. , Brumley William C. , Donnelly Joseph R. , Farley John W.
Published: May 2005

Capillary electrophoresis (CE) has been applied to the determination of the groundwater migration tracer dye fluorescein based on laser‐induced fluorescence (LIF) detection and compared to determinations obtained with traditional spectrofluorimetry. Detection limits of injected dye in the low parts per trillion (ppt) ranges have been accomplished with both CE/LIF based on the Ar ion laser and with a spectrofluorimeter. This approach was used for a real‐world problem in determining groundwater migration between adjacent Resource Conservation and Recovery Act (RCRA) and Superfund sites by the Environmental Sciences Division in response to regional needs and as application of new analytical tools under development. Fluorescent dye was injected into source wells and then was determined in monitoring wells by extracting pads that adsorbed the dye or by directly determining the dye in the water using solid‐phase extraction (SPE), a preconcentration technique. The approaches based on CE/LIF exhibits increased specificity over existing approaches due to the separation and unique migration time of the dye. Additional studies were aimed at achieving sub‐ppt levels in the water using solid‐phase extraction and field‐amplified injection techniques.
Steady‐state electrolysis of an ampholyte solution and possibility of violation of the “law of pH monotony”
Authors: Stoyanov Alexander V. , Righetti Pier Giorgio
Published: May 2005

The problem of stationary electrolysis of a single‐component ampholyte solution is analyzed. The effect of nonconstant relative mobility is taken into account. It is demonstrated that an incorrect dissociation model (i.e., expressing the resulting ampholyte flux by the arithmetic sum of the fluxes for “cationic” and “anionic” species) may lead to the violation of the so‐called “law of pH monotony”.
Recent developments in capillary isoelectric focusing with whole‐column imaging detection
Authors: Fang Xiaohong , Tragas Charalambos , Mao Qinglu , Pawliszyn Janusz , Wu Jiaqi
Published: May 2005

Capillary isoelectric focusing (CIEF) is a high resolution technique for protein separation. The on‐column single point detector requires a mobilization step which lengthens the analysis time and causes an uneven resolution along the separation column. The real time and whole column imaging detection has been developed for performing CIEF without mobilization. Three types of imaging detection systems have been developed: optical absorption, refractive index gradient, and laser induced fluorescence. This technique provides a fast analysis speed (about 6 min) and a good resolution of 0.03 pH unit level. Using the absorption imaging detector, ampholyte‐free IEF in tapered capillary is being demonstrated, which eliminates the interference of the expensive carrier ampholytes for protein detection in UV region. Recent advancements in this imaged CIEF technique as well as its applications are reviewed.
Optimization of solid phase microextraction ‐ capillary zone electrophoresis ‐ mass spectrometry for high sensitivity protein identification
Authors: Figeys Daniel , Zhang Yanni , Aebersold Ruedi
Published: May 2005

We have previously described the use of a solid phase extraction (SPE) ‐ capillary zone electrophoresis (CZE) ‐ tandem mass spectrometry (MS/MS) system for protein analysis at the low femtomole to subfemtomole level. Here we describe the systematic optimization of a number of parameters which facilitate the use of the SPE‐CZE‐MS/MS system and further enhance its performance. Specifically, we describe a robust SPE cartridge design which can be assembled without the use of glue, the evaluation of procedures to chemically modify the inner wall of the fused‐silica capillaries used in the system to improve separation and reproducibility, and the comparison of different reverse‐phase (RP) resins used for the SPE cartridge. We also explored the effects of transient isotachophoresis with respect to system performance and compatibility with different fused‐silica surface coatings, the RP resins used, and MS/MS. The enhanced performance of the optimized system is demonstrated by the analysis of calibrated tryptic digests of bovine serum albumin (BSA).
Analysis of sequence homogenisation in rDNA arrays of Haemonchus contortus by denaturing gradient gel electrophoresis
Authors: Gasser Robin B. , Zhu Xingquan , Chilton Neil B. , Newton Lisa A. , Nedergaard Trine , Guldberg Per
Published: May 2005

Testing different theories of concerted evolution experimentally has been hampered mainly due to the lack of appropriate model systems and technical limitations. In this study, we employed a denaturing gradient gel electrophoresis (DGGE) approach for the display and definition of nucleotide variations in the second internal transcribed spacer (ITS‐2) of ribosomal DNA (rDNA) of the parasitic nematode, Haemonchus contortus. The ITS‐2 was amplified from individual adult nematodes by PCR and subjected to DGGE. Of the 94 individuals (representing nine different populations) analysed, 13 different DGGE profiles were displayed. Eighteen bands representing those profiles were excised and sequenced. Sequencing defined 13 different types of ITS‐2 with 12 nucleotide variations (4 transitions, 5 transversions, 1 insertion and 2 deletions) which could be related to particular positions of the predicted secondary structure for the ITS‐2 pre‐rRNA. The results showed that individuals of interbreeding populations of H. contortus can have rDNA arrays that are partially or fully homogenised for different sequence variants (despite interindividual variation), suggesting that the homogenisation process is driven mainly by intrachromosomal exchange. The findings also demonstrated the capacity of the DGGE‐sequencing strategy to quantify the frequency of ITS‐2 sequence types within individual nematodes from different populations without the need for cloning or Southern blot procedures. This has important implications for studying the mechanisms of sequence homogenisation in rDNA and pre‐rRNA processing as well as for elucidating speciation events and population differentation at the molecular level.
Short tandem repeat (STR) locus HUMD8S306 in a large population sample from Germany
Authors: Benecke Mark , Knopf Manuela , Voll Wolfgang , Oesterreich Wolfgang , Jacobi Yvonne , Edelmann Jeanett
Published: May 2005

Applied DNA typing in medico‐legal investigations, in criminalistic practice, and in paternity cases often relies on high inclusion and exclusion probabilities. For that reason, the short autosomal tandem repeat locus D8D306 was validated for forensic use and incorporated into a nonoverlapping multiplex reaction with HUMDHFRP2 and HUMCD4. The allele frequencies of D8S306 in four different regions of Germany (n = 1220 alleles) were determined for use in a population database; the allele distributions did not significantly deviate from each other. The hererozygosity of D8S306 is 83%, expected exclusion chance in stain cases is 96% (paternity cases: 69%), the lowest amount of sucessfully amplified DNA was 30 pg. The alleles are in Hardy‐Weinberg equilibrium (exact test).
Rapid fluorescent monitoring of total protein patterns on sodium dodecyl sulfate‐polyacrylamide gels and Western blots before immunodetection and sequencing
Authors: Alba F. Javier , Daban Joan‐Ramon
Published: May 2005

The fluorogenic dye 2‐methoxy‐2,4‐diphenyl‐3(2H)‐furanone (MDPF) has been used for the detection of total protein patterns on polyvinylidene difluoride (PVDF) membranes. Fluorescent staining of protein bands on membranes with this covalent dye is completed in 20 min. Wet membranes are translucent, allowing protein visualization by transillumination with ultraviolet light. The resulting images can be recorded using Polaroid film or a charge‐coupled device camera. Electrophoretic bands containing 5–10 ng of protein can be detected on the MDPF‐stained Western blot. When proteins are directly transferred to the membrane using a slot blotting device, as little as 0.5 ng of protein can be detected. Previous visualization of protein bands on sodium dodecyl sulfate‐polyacrylamide gels with the noncovalent fluorescent dye Nile red (Alba et al., BioTechniques, 1996, 21, 625–626) does not interfere with further MDPF staining and fluorescent detection of these bands transferred to PVDF membranes. Thus, Nile red and MDPF staining can be performed sequentially, allowing the rapid monitoring of total protein patterns on both the electrophoretic gel and Western blot. Using the conditions described in this study, MDPF staining does not preclude further N‐terminal microsequencing and immunodetection of specific bands with polyclonal antibodies.
An electrophoretic study on interactions of albumins of different species with immobilized Cibacron Blue F3G A
Authors: Miller Ingrid , Gemeiner Manfred
Published: May 2005

Albumins of different species, varying in electrophoretic mobility, were compared in their interaction with the dye Cibacron Blue F3G A. Immobilized by coupling to a high molecular weight dextran (“blue dextran”), the dye was used as a ligand in affinity electrophoresis in different setups. One‐dimensional electrophoresis with blue dextran entrapped in an intermediate gel and two‐dimensional applications with transverse gradients (affinity titration curves, zonal electrophoresis in linear ligand gradients) were performed. Compared to the human homologue, animal albumins display more complex patterns and interaction profiles, depending on pH and ionic strength of the buffers. Results may differ considerably from those obtained by affinity chromatography, illustrating the additional screening potential of the electrophoretic methods. Comparison of different samples under the influence of ligand competition, reducing conditions, or denaturing agents gives supplementary information on conformational behavior of the proteins.
DNA preparation and efficient microsatellite analysis from insect hemolymph
Authors: Gerken Thomas , Kurtz Joachim , Sauer Klaus P. , Lubjuhn Thomas
Published: May 2005

A simple and time‐saving method for DNA preparation for efficient microsatellite analysis is described. The method is based on thermal treatment of only 1–5 μL of insect hemolymph in a Chelex 100‐suspension. Since hemolymph withdrawal does not harm the insects, analysis of mating systems, population structure and phylogenetic reconstruction can be conducted with minimal experimental influence.
Identification of animal species in ground meat mixtures by multivariate analysis of isoelectric focusing protein profiles
Authors: Skarpeid Hans‐Jacob , Kvaal Knut , Hildrum Kjell Ivar
Published: May 2005

The present work investigates the possibility of constructing a multivariate calibration model to predict the composition of ground meat with respect to animal species, based on intensity profiles from isoelectric focusing of water‐soluble proteins in mixtures of ground meat. Samples containing various amounts of beef, pork and turkey meat were analyzed by isoelectric focusing in immobilized pH‐gradients. The gels were photographed and the images transferred to a digital format. The digital images were processed in order to reduce background and optimize signal strength. Resulting gel profiles were analyzed by multivariate regression allowing the determination of sample composition with prediction errors close to 10%.
Separation of oligonucleotides and DNA fragments by capillary electrophoresis in dynamically and permanently coated capillaries, using a copolymer of acrylamide and β‐D‐glucopyranoside as a new low viscosity matrix with high sieving capacity
Authors: Chiari Marcella , Damin Francesco , Melis Alessandra , Consonni Roberto
Published: May 2005

New copolymers of acrylamide and β‐D‐glucopyranoside were synthesized and characterized. The different reactivity of the two monomers towards radical polymerization meant we could control the growth of the polymer chains whose length was inversely related to the number of glucose residues incorporated in the copolymers. The properties of these polymers were investigated in the separation of oligonucleotides and double‐stranded DNA by capillary electrophoresis (CE) in coated and uncoated capillaries. The new copolymers were a suitable matrix for CE due to their high‐resolving capacity and low viscosity. We also looked into the advantages of a new method of dynamic suppression of electroosmotic flow based on the addition of small amounts (0.03–0.05%) of dimethylacrylamide to the sieving and to the running buffer. A complete test was run on the reproducibility and efficiency of separations carried out in a permanently and dynamically coated capillary, and the advantages and disadvantages of the two methods were compared.
A simple method for the determination of isoelectric points of ampholytes with closely spaced pKa values using pressure‐mediated capillary electrophoresis
Authors: Glukhovskiy Pavel V. , Vigh Gyula
Published: May 2005

A simple method, based on a modified version of pressure‐mediated capillary electrophoresis (PreMCE) has been developed for the determination of the isoelectric points of ampholytes which have closely spaced pKa values. This new pI‐determination PreMCE method (i) can be easily executed on most commercial capillary electrophoresis instruments; (ii) it can use small, impure samples, (iii) unlike isoelectric focusing methods in natural pH gradients, it does not require a linear pH gradient, and (iv) it eliminates the pI errors that are due to chromatographic retention on the walls of the separation chamber.
Faster capillary electrophoresis separation of wheat proteins through modifications to buffer composition and sample handling
Authors: Bean Scott R. , Lookhart George L.
Published: May 2005

Studies were conducted to produce faster, simpler, more rugged protocols for separating wheat proteins by high performance capillary electrophoresis (HPCE). Three areas were targeted for improvement: initial capillary equilibration procedures, buffer composition, and post‐separation rinsing procedures. For the initial equilibration of capillaries, a brief rinse with a hydroxypropylmethylcellulose (HPMC) solution was the most critical factor for successful separation of wheat proteins. To reduce separation time and maintain resolution, β‐alanine and glycine were each used in place of sodium phosphate as buffer ions. Two isoelectric buffers, aspartic acid and iminodiacetic acid (IDA) were also tested. Each of these four buffer systems generated substantially lower currents, and provided faster separations, than sodium phosphate‐based buffers. Finally, post‐separation rinsing procedures were re‐examined with the goal of reducing the time necessary to rinse the capillary after each separation. A critical factor in achieving this goal was removal of albumins and globulins prior to separation. These proteins bind to the capillary wall and cause rising baselines and excessive peak tailing. Once these proteins were removed, capillaries could be rinsed with buffer for only 2 min between separations. Capillary equilibration procedures were shortened from 90 min to 30 min. Likewise, separation times were reduced by ∼ 40% (25 min to 15 min) by using glycine in place of sodium phosphate in the separation buffer. Finally, post‐separation times were reduced by 80% (10 min to 2 min). Overall, these factors resulted in a reduction in total separation time of 50% (35 to 17 min) and maintained high resolution separations and good run‐to‐run repeatability.
Protein patterns of human nasal and bronchoalveolar lavage fluids analyzed with two‐dimensional gel electrophoresis
Authors: Lindahl Mats , Ståhlbom Bengt , Svartz Jesper , Tagesson Christer
Published: May 2005

We have previously described the protein patterns of human nasal lavage fluid (NLF) and bronchoalveolar lavage fluid (BALF) after two‐dimensional gel electrophoresis (2‐DE). We now report the identification of a number of additional proteins in these 2‐DE patterns. Several plasma proteins (α2‐macroglobulin, haptoglobin α1‐chain, IgA S chain, ceruloplasmin, α1‐microglobulin, amyloid P and apolipoprotein A‐1) could be included both in the BALF and NLF spot pattern data bases by matching with a master plasma 2‐DE pattern (SWISS‐2DPAGE). Furthermore, lysozyme, lactoferrin and the antiinflammatory proteins lipocortin‐1 and Clara cell protein 16 (CC‐16) were identified by matching with reference proteins and Western immunoblots. Significant differences in the levels of some of the identified proteins were found between NLF and BALF, and between BALF from smokers and nonsmokers. Transferrin, hemopexin and haptoglobin α1 were lower in NLF than BALF, while IgA, lysozyme and lactoferrin were higher in NLF than BALF. One form of α1‐microglobulin was more abundant in NLF than in BALF, while the opposite was found for a second form of the same protein. Moreover, the levels of IgA, ceruloplasmin and the pro‐form of apolipoprotein A‐1 in BALF were lower in smokers than in nonsmokers. The possibility to describe and analyze differences in NLF and BALF 2‐DE patterns at the protein spot level may have wide clinical applications.
Fire hazards of organic materials—small scale assessment of flammability and smoke generation
Authors: Brown J. R. , Dunn P.
Published: May 2005

The flammability and smoke generation properties of a number of organic materials used in ships, including laminated materials, rigid and flexible cellular polymers, cable insulting materials and adhesives, have been evaluated. The relative flammabilities were determined by the limiting oxygen index method that gives convenient, reproducible, numerical ratings of materials. Smoke generation was assessed photometrically by measuring light attenuation under standard conditions and is reported in terms of the total amount of smoke generated, as well as the times for the smoke density to reach an arbitrary ‘critical’ level and the maximum value. Materials with low flammability and low smoke generation characteristics have been identified, as well as those that would be hazardous in the event of a fire.
A new approach to testing the flammability characteristics of surface coatings
Authors: Barnett Brian A.
Published: May 2005

This paper describes the background information which led to the development of a technique specifically designed to test the flammability performance of coatings applied to metal substrates. Design parameters for the new test are discussed and the prototype apparatus is described in detail. The mode of operation is explained and some results are tabulated for a range of typical coating materials. Conclusions based on the behaviour of the coatings tested are drawn in the text. These conclusions are, however, biased towards the Naval environment and application, and may not be universally applicable. The main implications of this work so far are that it is possible to improve the performance of existing fire retardant paint systems by: (1) ensuring that only the minimum thickness is applied to non‐flammable substrates; (2) that adequate time is allowed for solvent elimination before deliberate exposure to risk; (3) using a white coating if possible; (4) increasing the durability of the coating to reduce the need for cosmetic overcoating, (i.e. re‐coating purely to improve appearance), e.g. by using a thermosetting resinous binder in place of the conventionally used alkyd resin.
Fire behaviour of beds and bedding materials
Authors: Woolley W. D. , Ames S. A. , Pitt A. I. , Murrell J. V.
Published: May 2005

Full‐scale fire tests on domestic beds fully equipped with bedding materials have been carried out in an experimental compartment‐corridor facility at the Fire Research Station, Borehamwood, U. K. Mattresses made of hair, spring interior, foam rubber and polyurethane of various types, together with mattress covers of cotton, flame‐retarded cotton or proofed nylon were studied. The effectiveness of protective hair or glass fibre interlining was examined. The study has shown that a rapid development of fire in bed and bedding materials can take place with certain combinations of mattresses and their covers. The types of covers extremely important in overall fire development, particularly with polyurethane mattresses. A substantial improvement in the fire behaviour of many of the principal types of beds tested can be achieved by a careful selection of bedding materials, such as the type of mattress cover, and in certain cases by the use of protective interlinings.
Development of a stratified ceiling layer in the early stages of a closed‐room fire
Authors: Zukoski E. E.
Published: May 2005

A simple analytical model has been developed to determine the time required for a room to fill with products of combustion from a small fire. The room is assumed to be closed except for small openings at either the floor or ceiling level and the assumption is made that the leak is large enough to allow the transient pressure term in the energy equation to be neglected. Products of combustion are assumed to occupy a layer next to the ceiling and the model predicts the growth of the thickness and the mean density of this layer as a function of time. The analysis shows that times required to fill a typical room are small. For example, a typical bedroom fills with products from a 20 kW fire in several minutes. The time required to fill a room and the mean density of ceiling layer are determined in terms of fire size, room geometry, leak position, fire elevation and geometry.
Relative toxicity of pyrolysis gases from materials: Effects of chemical composition and test conditions
Authors: Hilado Carlos J. , Cumming Heather J.
Published: May 2005

Relative toxicity test data on 270 materials are presented, based on test procedures developed at the University of San Francisco. The effects of chemical composition, using data on 13 types of synthetic polymers and eight types of fabrics, are discussed. Selected materials were evaluated using nine test conditions with the USF method, and using methods developed at the FAA Civil Aeromedical Institute, Douglas Aircraft Company and San Jose State University.
A two zone model of flame propagation applied to H2 + air flames and HCL inhibited flames
Authors: Brown N. J. , Fristrom R. M.
Published: May 2005

This communication presents a two zone flame model which allows computation of flame propagation velocity and average properties in the radical generation region and fuel attack region of the flame. The approach is computationally simpler than formal flame theory calculations; however, it provides only limited information since detailed structural information is sacrificed for computational simplicity. The zonal approach to flame structure is described and justification is provided for the various approximations which characterize the model. The model is largely a chemical one; however, the balance maintained between the radical molecule attack reaction and diffusional transport of radicals into the reaction region controls the propagation rate. The model was tested on five H2 + air flames, and the computed propagation velocities compared favorably with experimental values. HCL inhibition of the stoichiometric H2 + air flame was investigated, and the zonal approach was found suitable for studying flame inhibition.