bluuebunny/crossref_metadata_2025 huggingface dataset:
Some variables affecting reproducibility in capillary electrophoresis
Authors: Shihabi Zakariya K. , Hinsdale Mark E.
Published: May 2005

Several variables that can affect reproducibility of both the peak height and the migration time in capillary electrophoresis (CE) were investigated here. A great part of the imprecision in CE for migration time is related to the interaction of the analyte with the capillary wall. Within a run, acidic compounds with long migration times have a higher relative standard deviation. In general, conditions that decreased the migration time, such as a short capillary length, tended to enhance the reproducibility of the migration time. Sample size also affected the reproducibility of peak height. Too small or too large an injection volume diminished the reproducibility. Sample matrix, such as high levels of proteins or salts in the sample, decreased the reproducibility. Furthermore, improvement in reproducibility can be achieved by using internal standards (especially for migration time), thorough washing of the capillary, or removing the excess proteins by acetonitrile deproteinization. Acetonitrile has an added effect of producing sample stacking. Depending on the migration time and analyte concentration, the peak height and area yielded different reproducibility results.
Identification of proteins that are abnormally regulated in differentiated cultured human keratinocytes
Authors: Olsen Eydfinnur , Rasmussen Hanne Holm , Celis Julio E.
Published: May 2005

Comparison of the protein expression patterns of proliferating normal primary human keratinocytes plated in serum‐free medium (SFKM), supplemented with epidermal growth factor (EGF) and bovine pituitary extract (BPE), and similar cultures induced to differentiate by the addition of Dulbecco's modified Eagle medium (DMEM), containing 10% fetal calf serum (FCS), revealed several known and unknown polypeptides that are abnormally regulated in the differentiated cells. Upregulated proteins included keratins (keratins 6, 10/11, 14 and 16), members of the S100 protein family psoriasin, MRP8, MRP14 and S100c), actin‐binding proteins (gelsolin and tropomyosin 9220), annexins (annexins IV and VIII), hsp28, the fatty acid binding protein 5 (FABP5), the squamous cell carcinoma (SCC) antigen, members of the 14‐3‐3 family, involucrin, E‐cadherin, cystatin A, desmoglein and integrins α2 and β1, as well as several proteins of as yet unknown identity. The highest upregulated proteins corresponded to psoriasin (124.0 times), MRP8 (42.4 times), MRP14 (14.9 times), tropomyosin 9220 (11.5 times), involucrin (11.1 times), and FABP5 (9.1 times). FABP5, hsp28, and tropomyosin 9220 were also highly upregulated in quiescent keratinocytes indicating that their increased levels in the differentiated cell may be due to loss of proliferative activity. Highly downregulated proteins included PAI‐2, tropomyosins 9213, 9121 and 9122, keratin 5, calnexin, 14‐3‐3 beta and eta, nucleoside diphosphate kinase A, Rho GDIs, hsp60, hnRNPs H and C2, α‐enolase, eIF‐4D, thioredoxin, annexins III and V, moesin, nucleolar protein B23, GST π and PCNA/cyclin. Both the high expression of keratin 6 and 16 – which are markers for an alternative pathway of keratinocyte differentiation – as well as the extremely high upregulation of some members of the S100 protein family indicate that the cells have differentiated via an abnormal pathway.
Gaining insight into a complex organelle, the phagosome, using two‐dimensional gel electrophoresis
Authors: Burkhardt Janis , Huber Lukas A. , Dieplinger Hans , Blocker Ariel , Griffiths Gareth , Desjardins Michel
Published: May 2005

Phagosomes are the organelles formed de novo in a variety of cells by the internalization of large particulate materials, including a wide range of pathogenic microorganisms. We present here a systematic approach that can be used to study the polypeptide composition of phagosomes/phagolysosomes and to yield analytical information on the characteristics of their proteins. A density shift approach was used to isolate pure preparations of phagosomes filled with low density latex beads from mouse J774 and human U937 macrophages. High resolution two‐dimensional (2‐D) gel electrophoresis was performed to generate a map of the overall [35S]methionine‐labeled protein profile of the isolated phagosomes. The resulting map showed the minimal presence of over 200 polypeptides, indicating the complexity of this organelle. Comigration experiments showed that several phagosome polypeptides, among them several known proteins, are shared by the two species. Extraction with Triton X‐114 and sodium carbonate was performed to distinguish between membrane and soluble proteins, and sensitivity to a panel of proteases was measured to identify proteins exposed on the cytoplasmic face of the phagosome membrane. The general value of the 2‐D gel approach in the mapping of organelle proteins is discussed.
Accessibility to tissue‐specific genes from methylation profiles of mouse brain genomic DNA
Authors: Watanabe Sachihiko , Kawai Jun , Hirotsune Shinji , Suzuki Harukazu , Hirose Kenji , Taga Chiharu , Ozawa Naoki , Fushiki Shinji , Hayashizaki Yoshihide
Published: May 2005

The DNA methylation status of a large number of genomic loci is visualized simultaneously and quantitatively as two‐dimensional gel spots in the newly developed restriction landmark genomic scanning with a methylation‐sensitive restriction enzyme (RLGS‐M). Here, we demonstrate that RLGS‐M using NotI as a methylation‐sensitive enzyme could also scan gene loci of mammalian genomes, since almost all of the NotI loci corresponding to randomly chosen RLGS‐M spots were located near or in transcriptional units (6 out of 7 NotI‐linking clones) when mouse brain genomic DNA was used. This supports the previous prediction that most NotI sites are located in CpG islands (Lindsay and Bird, Nature 1987, 327, 336–338). Furthermore, beginning with RLGS‐M spots we examined how to approach their corresponding RNA messages, whose expression may be associated with methylation. We compared RLGS‐M patterns among various developmental stages of the mouse brain from embryonic day 9.5 to postnatal 8 weeks or among in vitro cell lines, and detected alterations of RLGS‐M spots which were due to methylation of NotI sites. Two experiments using NotI‐linking clones or polymerase chain reaction (PCR) were carried out to approach to their corresponding RNA messages. Consequently, we isolated two PCR‐amplified clones (# 15 and # 91) which corresponded to methylatable loci and gave positive signals to mRNA from the adult brain. Furthermore, we identified two NotI‐linking clones (C211 and C198) whose corresponding NotI loci localized near or at transcriptional units and were methylated in cell lines. Thus, RLGS‐M is expected to be widely applicable for the isolation of tissue‐specific genes whose genomic loci are associated with DNA methylation.
Quantitative and qualitative genetic variation in two‐dimensional DNA gels of human lymphocytoid cell lines
Authors: Asakawa Jun‐Ichi , Kuick Rork , Neel James V. , Kodaira Mieko , Satoh Chiyoko , Hanash Samir M.
Published: May 2005

There is a continuing need for more efficient methods to examine human (and other) populations for altered germinal and somatic cell mutation rates. To this end, we have explored the potential usefulness of two‐dimensional (2‐D) electrophoresis of human DNA fragments obtained from restriction‐enzyme‐digested genomic DNA, using samples from father/mother/child trios. On a single 2‐D DNA preparation, approximately 2000 DNA fragments varying in size from 1.0 to 5.0 kbp in the first dimension and 0.3 to 2.0 kbp in the second dimension are visualized. To enter into a genetic analysis of quantitative variation, these fragments must exhibit positional and quantitative stability. With respect to the latter, if spots that are the product of two homologous DNA fragments are to be distinguished with the requisite accuracy from spots that are the product of only one fragment, the coefficient of variation of spot intensity should be approximately ⩽ 0.12. At present, 482 of the spots in our preparations meet these standards. In an examination of preparations based on three Japanese mother/father/child trios, 43 of these 482 spots were found to exhibit variations that segregated within families according to Mendelian principles. Additionally, of the 2000 spots, 1114 (of which the aforementioned 482 are a subset) were deemed appropriate for the study of qualitative variation. A total of 142 variable spots were identified; the heterozygosity index for these DNA fragments was 4.4%. The genetic nature of the additional variants was again established by their segregation according to Mendelian principles. We have established the feasibility of cloning fragments from such gels and determining their nucleotide sequence. This technology should be highly efficient in monitoring for mutation resulting in loss/gain/rearrangement events in DNA fragments distributed throughout the genome.
High resolution microscopic mapping of DNA using multicolor fluorescent hybridization
Authors: Windle Bradford , Silvas Emilio , Parra Irma
Published: May 2005

We describe a procedure for microscopically mapping the relative positions of DNA probes along extended strands of DNA. The procedure referred to as direct visual hybridization (DIRVISH) DNA mapping involves the simultaneous hybridization of multiple probes and the fluorescent colors, red, green and blue to produce images that convey high‐resolution mapping information. The images appear as long strings of fluorescent signals positioned as they are in the genome. A visual multi‐color map is generated within 2 days. Cosmid probes span a distance of 10 μm or more and have been observed to contain patterns within the strings of signals. We have developed computer imaging programs to scan through the strings of signals and plot the intensities. Scans through multiple signal strings for one cosmid probe revealed consistent patterns. We have interpreted the patterns as the result of suppression of repetitive DNA sequence hybridization. These patterns may prove useful as fingerprints for regions of DNA.
Molecular epidemiology of respiratory syncytial virus: A review of the use of reverse transcription‐polymerase chain reaction in the analysis of genetic variability
Authors: Cane Patricia A. , Pringle Craig R.
Published: May 2005

Respiratory syncytial virus (RSV) is the major viral cause of lower respiratory tract disease (bronchiolitis and pneumonia) in babies and infants. Infections with the virus occur as annual winter epidemics in temperate climates, placing considerable pressure on the provision of hospital beds. The virus is unusual in that it can reinfect individuals and it can infect babies despite the presence of maternal antibody. RSV has a negative sense nonsegmented RNA genome and as such is liable to high levels of mutation. This paper describes methods developed to determine the degree of genetic variability of the virus both during individual epidemics and worldwide. It is necessary for these methods to be quick, easy and cheap so that large numbers of samples can be analysed readily. They are based on extraction of viral RNA directly from clinical samples or from viral cultures, reverse transcription of the viral RNA, and then amplification of selected regions of the genome by the polymerase chain reaction (PCR). PCR products are then analysed by restriction mapping, or, if necessary, direct nucleotide sequencing. In this way isolates of RSV have been shown to fall into a number of genotypes, with epidemics being made up of cocirculating genotypes whose relative proportions vary with each epidemic. An understanding of the molecular epidemiology of this important human pathogen will be of significance in the search for an effective vaccine.
An improvement of restriction analysis of bacteriophage DNA using capillary electrophoresis in agarose solution
Authors: Klepárník Karel , Malá Zdeňka , Doškař Jiři , Rosypal Stanislav , Boček Petr
Published: May 2005

Seven representatives of the serogroup B Staphylococcus aureus bacteriophages, 29, 53, 55, 83A, 85, ϕ11 and 80α, were examined by capillary electrophoresis (CE) for genomic homology using DNA restriction analysis. Genomic DNA of individual bacteriophages was cleaved by HindIII restriction endonuclease, and the resulting restriction fragments were separated by standard horizontal agarose slab gel electrophoresis (SGE) as well as by CE in low‐melting‐point agarose solutions. The number and size of restriction fragments identified by both methods were compared. The high separation power of CE makes it possible to extend the restriction fragment patterns. In most of the restriction patterns, some additional restriction fragments as small as 150 bp, not identified by SGE, were detected. With respect to speed, high separation efficiency, low sample consumption and automation, CE offers a simple procedure for processing of multiple samples cost‐effectively in a reasonable time. The comparison of the complemented restriction patterns of the different phage strains and the subsequent identification of their common fragments leads to a deeper understanding of their phylogenetic relationships. The genome homologies expressed for individual phage pairs in terms of coefficient F values ranged from 15 to 69%. These values are in good accordance with the degree of DNA homology of these phages as determined by DNA hybridization studies and thermal denaturation analysis of DNA by other authors. The total size of each phage genome was estimated by adding the sizes of individual restriction fragments.
Hydrolytically stable cellulose‐derivative coatings for capillary electrophoresis of peptides, proteins and glycoconjugates
Authors: Huang Mingxian , Plocek Jiri , Novotny Milos V.
Published: May 2005

A method for the preparation of versatile, hydrolytically stable coatings of fused silica capillaries is described. First, the HNO3‐pretreated silica surface is reacted with 7‐oct‐1‐enyltrimethoxysilane to form a stable self‐assembled alkylsilane monolayer. Secondly, the copolymer of hydroxypropylcellulose and 2‐hydroxyethyl methacrylate is statically coated onto a hydrophobized wall. This allows the formation of a highly crosslinked copolymer layer which is tightly bound to the wall surface. Hydrolytical stability in the pH range of 2–10 for several weeks has been demonstrated. The coating generates a low electroosmotic flow at alkaline pH, which can be easily masked by using special buffers. The versatility of this column technology is documented by separations of peptides, glycoproteins and derivatized oligosaccharides.
Electrophoretic analysis of bile proteins from patients with and without gallstones
Authors: Ota Yukiko , Ido Kenichi , Kimura Ken , Fukui Emiko , Ikemoto Shigenori
Published: May 2005

Bile proteins from 82 cases with various types of gallstones (pure cholesterol stones, cholesterol stones with a small amount of other substances, mixed stones and pigment stones) and 9 controls without gallstones were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). A unique bile protein with a molecular mass of 32 kDa was identified. The 32 kDa protein could be stained on immunoblotting membranes using immunoperoxidase with concanavalin A. The 32 kDa protein was found in all controls but only in a portion (22.2–36.4%) of the patients with gallstones of various types. Following electrophoretic elution from the SDS‐PAGE gels, the 32 kDa protein was analyzed by reversed phase – high‐performance liquid chromatography, yielding three peaks for controls and only two peaks for patients with gallstones.
High resolution, standard format two‐dimensional protein electrophoresis using disposable glass micropipettes and non‐dedicated equipment
Authors: Sarmiento Marion
Published: May 2005

A method for high resolution, two‐dimensional polyacrylamide gel electrophoresis (PAGE) of proteins on a standard format that utilizes nondedicated electrophoretic equipment is described. This method combines key features of various previous protocols into an improved approach which can readily be applied by persons experienced only in sodium dodecyl sulfate (SDS)‐PAGE. The method yields high‐quality two‐dimensional protein separations that are reliably reproducible and uniform between different analyses. High protein resolution is attained and preserved primarily by facilitating the execution of the first isoelectric focusing (IEF) dimension and streamlining the initiation of the second (SDS‐PAGE) dimension. Important features of this method include the use of piperazine diacrylate cross‐linker, disposable glass micropipettes, and conical sample chambers in the IEF dimension. In addition, the equilibration of the first‐dimensional gels is accomplished at the same time the gels are loaded onto the second dimension. Loss of protein resolution due to diffusion after the end of the first dimension is reduced by minimizing the time required to load the SDS‐PAGE dimension. The method is versatile and adaptable to the analysis of one or many samples. The high degree of resolution, reproducibility and uniformity attained by this method are sufficient to abrogate the need for dedicated equipment in most applications.
Cross‐species identification of proteins separated by two‐dimensional gel electrophoresis using matrix‐assisted laser desorption ionisation/time‐of‐flight mass spectrometry and amino acid composition
Authors: Cordwell Stuart J. , Wilkins Marc R. , Cerpa‐Poljak Anne , Gooley Andrew A. , Duncan Mark , Williams Keith L. , Humphery‐Smith Ian
Published: May 2005

Amino acid analysis and matrix‐assisted laser desorption/ionisation time‐of‐flight (MALDI‐TOF) mass spectrometry were used to identify nine of twelve proteins originally separated by two‐dimensional electrophoresis and derived from an organism poorly defined at the molecular level (Spiroplasma melliferum). Two of three unidentified proteins appeared to be novel. The percentage amino acid composition and the molecular mass of peptide fragments generated by tryptic digestion were used to search the PIR/SWISS‐PROT and MOWSE databases respectively. Lists of candidate proteins were independently generated and ranked from data obtained by both methods. A putative identification was allocated when a single candidate protein appeared in both lists of computer‐generated rankings. Results were verified using N‐terminal protein microsequencing. The combined use of amino acid composition and MALDI‐TOF mass spectrometry allowed a high degree of confidence to be placed in such identifications because they were based upon homologous data sets of at least 20 parameters (16 amino acids and 4–10 tryptic digest fragments). A further two parameters, estimated Mr and, to a lesser extent, pI, were also used to reinforce this measure of confidence. Ranking of candidate proteins by one method alone could lead to false identification. Both techniques can process large numbers of samples rapidly. In light of the increasing number of entries in both gene and protein databases, this approach is likely to become an essential first step for the characterisation of proteins, particularly across species boundaries.
Microchip electrophoresis with sample stacking
Authors: Jacobson Stephen C. , Ramsey J. Michael
Published: May 2005

A fused quartz microchip with a serpentine column geometry is fabricated to perform rapid microchip electrophoresis of dansylated amino acids. A 67 mm separation column is constructed in a 7 × 10 mm area on a quartz substrate using standard photolithographic, etching and deposition techniques. Buffer and sample flows within the channel manifold are precisely controlled through potentials applied to the reservoirs. To enhance the detection limits, a stacking injection technique is used to concentrate the sample at the inlet of the separation column. The stacked injections exhibit high reproducibility (2.1% relative standard deviation in peak area). Using a separation length of 67 mm and a separation field strength of 1100 V/cm, separations are performed in ⩽ 15 s generating ≈︁ 40 000 theoretical plates.
Solid‐phase fluorescent labeling reaction of picomole amounts of insulin in very dilute solutions and their analysis by capillary electrophoresis
Authors: Pinto Devanand M. , Arriaga Edgar A. , Sia Samuel , Li Zhong , Dovichi Norman J.
Published: May 2005

The fluorescent labeling of peptides at concentrations as low as 10−8 M can be achieved by using a solid‐phase reactor. Using oxidized insulin chain B as a test peptide, we demonstrate the use of an Immobilon CD membrane to capture and preconcentrate peptides. Insulin chain B can then be labeled with a fluorogenic reagent, 3‐(2‐furoyl)quinoline‐2‐carboxaldehyde, while it is still attached to the membrane. Unwanted fluorescent products (attributed to secondary reactions) can be washed away with methanol without significant removal of the labeled insulin chain B, which then can be extracted with a low pH buffer. The analysis by micellar electrokinetic capillary chromatography with post‐column laser‐induced fluorescence detection (mass limit of detection of 2.4 × 10−21 moles insulin chain B) results in electropherograms that show great improvement in terms of unwanted peaks and high number of theoretical plates (up to 20 million). The use of the solid‐phase reactor allows easy handling of as little as 5 picomoles of insulin chain B.
Capillary electrophoretic resolution of phosphorylated peptide isomers using micellar solutions and coated capillaries
Authors: Tadey Tanya , Purdy William C.
Published: May 2005

The addition of sodium dodecyl sulfate (SDS) micelles in the running buffer can be used to resolve mono‐ and diphosphorylated isomers of the insulin receptor peptide by capillary electrophoresis. The effect of SDS on peptide resolution is very dependent on pH. Complete resolution of three monophosphorylated isomers is achieved in uncoated capillaries filled with phosphate buffer containing 25 mM SDS and buffered at pH 6.1. Resolution of the diphosphorylated isomers can be significantly improved by using polyacryla‐mide coated capillaries. In coated capillaries electroosmotic flow is negligible and the migration order of the isomers is reversed. This allows for a longer period of interaction between the diphosphorylated isomers and the micelle and therefore selectivity is improved. Efficiency of all peptide isomers was also improved in coated capillaries due to reduced adsorption to the capillary wall.
Fused‐silica capillaries with surface‐bound dextran layer crosslinked with diepoxypolyethylene glycol for capillary electrophoresis of biological substances at reduced electroosmotic flow
Authors: Mechref Yehia , Rassi Ziad El
Published: May 2005

This report is concerned with the introduction of novel surface modification involving the covalent attachment of branched, high molecular weight dextrans and subsequent crosslinking with polyether chains on the inner surface of fused silica capillaries with the aim of producing hydrophilic capillaries with reduced electroosmotic flow (EOF). Three different molecular mass dextrans, namely 45, 71 or 150 kDa were covalently attached to the capillary surface, and subsequently crosslinked with diepoxypolyethylene glycol (PEG). This chemistry produced stable coating over a wide range of conditions including high and low pH aqueous solutions. Moreover, the various dextran‐PEG‐coated capillaries exhibited reduced electroosmotic flow. These features of the dextran‐PEG coatings were useful for the separations of basic and acidic proteins, and provided high resolution separation for closely related acidic monosaccharides and sialooligosaccharides

Presented as a poster at Electrophoresis Forum '94, Munich, Germany, October 24–26, 1994

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Affinity electrophoretic determination of oligosaccharide specificity of Butea monosperma agglutinin
Authors: Wongkham Sopit , Taketa Kazuhisa , Liu Miao , Taga Hiroko
Published: May 2005

The oligosaccharide specificity of newly isolated Butea monosperma agglutinin (BMA) was determined by two‐dimensional lectin affinity electrophoresis of α‐fetoprotein (AFP) with concanavalin A, lentil lectin, erythroagglutinating phytohemagglutinin and Allomyrina dichotoma lectin, of which the specificities to known AFP oligosaccharides had been established. Effects of neuraminidase treatment on the reactivities of AFP to the lectins were also studied. The results indicated that BMA had the highest affinity for the exposed Gal residues of nonreducing termini of biantennary complex‐type oligosaccharides, and that the affinity was reduced to zero in the following order by the presence of monosialyl residue of the Man α→3 arm, monosialyl residue of the Man α1→6 arm, monosialyl residue of the Man α1→3 arm and bisecting GlcNAc, and disialyl residues. BMA did not recognize Neu5Ac α2→6 and Neu5Ac α2→3 substitutions of Gal. These characteristics of BMA were shown to be useful in identifying malignancy‐associated alteration of AFP sugar chains.
Mapping of Chlamydia trachomatis proteins by Immobiline‐polyacrylamide two‐dimensional electrophoresis: Spot identification by N‐terminal sequencing and immunoblotting
Authors: Bini Luca , Sanchez‐Campillo Maria , Santucci Annalisa , Magi Barbara , Marzocchi Barbara , Comanducci Maurizio , Christiansen Gunna , Birkelund Sevend , Cevenini Roberto , Vretou Evangelia , Ratti Giulio , Pallini Vitaliano
Published: May 2005

Proteins from purified elementary bodies of Chlamydia trachomatis were separated by two‐dimensional gel electrophoresis on nonlinear wide‐range immobilized pH gradients in the first dimension and polyacrylamide gradient gels in the second dimension. The maps obtained with this system are highly reproducible and resolve ca. 600 spots. By using immunoblot analysis with specific antibodies and/or N‐terminal amino acid sequencing, we established the positions of a number of described chlamydial proteins, such as the major outer membrane protein (MOMP) the 60 kDa cystein‐rich outer membrane protein (OMP2), the DnaK‐like, GroEL‐like, and macrophage infectivity potentiator (MIP)‐like proteins, the plasmid‐encoded pgp3 protein, two ribosomal proteins (S1 and L7/L12), and the protein‐elongation factor EF‐Tu. Other proteins, for which gene assignment was not possible, have been identified by three parameters (Mr, pI and N‐terminal sequence). This work provides a preliminary basis for a future and progressive compilation of a genome‐linked database of chlamydial proteins.
Use of internally controlled reverse transcriptase‐polymerase chain reaction for absolute quantitation of individual multidrug resistant gene transcripts in tissue samples
Authors: Zhang Fang , Riley Joan , Gant Timothy W.
Published: May 2005

Research into the relative importance of P‐glycoprotein (pgp) overexpression, in comparison to other mechanisms of multidrug resistance (mdr) phenotype development, has been hampered by difficulties of measurement in clinical samples. The small size, heterogeneity and often poor quality of clinical specimens renders the quantitation of mdr mRNA species by Northern analysis or RNAse protection difficult. The reverse transcriptase‐polymerase chain reaction (RT‐PCR) assay has both the sensitivity and specificity to make it suitable for the analysis of mdr mRNA levels in clinical samples. It also has a significant speed advantage over Northern and RNAse protection analysis. Unfortunately the variable nature of the reactions involved have made it difficult to obtain accurate and reproducible quantitative data. To overcome this difficulty we constructed internal RNA standards to each human and rat mdr mRNA species. When included in the RT‐PCR reaction these internal standards allow both comparative and absolute quantitation of individual mdr family mRNA levels.
Detection of glycosaminoglycans as a copper (II) complex in capillary electrophoresis
Authors: Toida Toshihiko , Linhardt Robert J.
Published: May 2005

Glycosaminoglycans including heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate and hyaluronic acid were analyzed by reversed polarity capillary electrophoresis. Detection was achieved at 240 nm based on the formation of a copper (II) complex in copper sulfate solution at low pH. Glycosaminoglycans having a high ratio of iduronic acid to glucuronic acid, as well as ones having lower molecular weight, gave the highest detection sensitivity using this method. Detection of the copper (II)‐heparin complex is extremely sensitive, permitting the analysis of as little as 10−9 g. This method was also successfully applied to the analysis of heparin oligosaccharides that lacked a chromophore prepared from heparin using controlled, low pH nitrous acid depolymerization.
Acid‐catalyzed reductive amination of aldoses with 8‐aminopyrene‐1,3,6‐trisulfonate
Authors: Evangelista Ramon A. , Guttman András , Chen Fu‐Tai A.
Published: May 2005

The reductive amination of monosaccharides with 8‐aminopyrene‐1,3,6‐trisulfonate (APTS) in seven different organic acids including the commonly used acetic acid was investigated by capillary electrophoresis (CE) with laser‐induced fluorescence (LIF) detection. The correlation between the yields of the saccharide‐APTS adducts and pKa of the organic acid catalyst is consistent with general acid catalysis of the rate‐determining step of the reductive amination reaction. Derivatization in the presence of organic acids of higher strength than acetic acid produced substantially higher yields of APTS‐sugar adducts, an effect which is more pronounced for N‐acetylamino sugars. Optimum yields were obtained using citric acid as a catalyst. Conversion of a few nanomoles of neutral saccharides to the APTS derivatives is achieved at 75°C in less than 60 min.
Determination of carbohydrates as their dansylhydrazine derivatives by capillary electrophoresis with laser‐induced fluorescence detection
Authors: Perez Sherryl A. , Colón Luis A.
Published: May 2005

A mixture of mono‐ and disaccharides was derivatized with dansylhydrazine in a relatively fast chemical reaction (15 min) which is selective toward aldehydes and ketones. The products of the derivatization reaction were then separated by capillary electrophoresis (CE) using laser‐induced fluorescence (LIF) as the detection mode. The smallest amount of carbohydrate derivatized and determined by CE/LIF was 10 pmol contained in 100 μL of the final reaction mixture. The CE/LIF method provided a linear response for the carbohydrates tested (over 2–3 orders of magnitude), with a limit of detection of 100 amol. Separation efficiencies over 250 000 theoretical plates were obtained. The procedure was used to analyze a tear fluid sample to demonstrate the applicability of the method for the glucose determination in small volumes of biological samples.
Analysis of Stevia glycosides by capillary electrophoresis
Authors: Mauri Pierluigi , Catalano Giovanna , Gardana Claudio , Pietta Piergiorgio
Published: May 2005

The determination of diterpene glycosides from Stevia rebaudiana leaves using capillary electrophoresis is described. Analyses were performed on fused silica capillaries with 20 mM sodium tetraborate buffer, pH 8.3, and 30 mM sodium dodecyl sulfate. The effect of the organic solvent injected with the sample solution on the electrophoretic solution has been confirmed, and an absolute amount of 1.6 nL per injected sample was optimal. Rebaudioside A and steviolbioside were isolated by semipreparative high performance liquid chromatography (HPLC), and their structure was assessed by mass spectrometry.
Detection and differentiation of pectic enzyme activity in vitro and in vivo by capillary electrophoresis of products from fluorescent‐labeled substrate
Authors: Zhang Zhiquan , Pierce Margaret L. , Mort Andrew J.
Published: May 2005

A sensitive assay is described for the detection of pectate‐depolymerizing enzymes using capillary electrophoresis of a fluorescent end‐labeled pectate oligomer. The labeled oligomer is allowed to react with the enzyme either in vitro or in vivo, such as inside the intercellular spaces of a cotton cotyledon, and after an appropriate incubation time the products are analyzed by capillary electrophoresis. The site and mode of action of the pectate‐depolymerizing activity can be inferred from the products. Both endo‐ and exopolygalacturonase activity, and lyase activity, were distinguished. Since only the fluorescent oligomer and products from its labeled reducing end are detected, there is no interference from other compounds; only pectic enzyme activity is detected. By this type of analysis we can show that there is considerable endo‐ and exopolygalacturonase activity in the intercellular spaces of cotton cotyledons.
Immobilized metal ion affinity gel electrophoresis: Quantification of protein affinity to transition metal chelates
Authors: Baek Won‐Ok , Haupt Karsten , Colin Carole , Vijayalakshmi Mookambeswaran A.
Published: May 2005

This paper describes some recent advances in the methodology of immobilized metal ion affinity gel electrophoresis. Four different ways to incorporate metal chelate ligands in agarose and polyacrylamide‐based electrophoresis gels are evaluated, a new polymerizable metal chelating ligand, allyl–2‐hydroxy‐3‐(N,N‐dicarboxymethyl)amino‐propyl ether, is introduced, and the determination of affinity constants described. The affinities of model proteins (ribonucleases A and B and cytochromes c from different species) for the transition metal chelate iminodiacetic acid‐Cu(II) were studied. The results were found to be in agreement with literature data on immobilized metal ion affinity chromatography, and the polymer nature and the different chemistries used influenced the affinity only quantitatively, keeping the basic mechanisms of interaction unchanged.
Federated two‐dimensional electrophoresis database: A simple means of publishing two‐dimensional electrophoresis data
Authors: Appel Ron D. , Barioch Amos , Sanchez Jean‐Charles , Vargas J. Reynaldo , Golaz Olivier , Pasquali Christian , Hochstrasser Denis F.
Published: May 2005

While a two‐dimensional electrophoresis (2‐DE) database is a relatively old concept, in recent years it generated renewed interest within the 2‐DE community due to two main factors: (i) The high reproducibility of the current 2‐DE method allows 2‐DE images to be exchanged and compared between laboratories. (ii) The recent development of faster and more powerful techniques for protein identification such as microsequencing, matrix‐assisted laser desorption ionization‐mass spectrometry (MALDI‐MS) and amino acid composition makes the production of reference protein maps and 2‐DE databases cost‐ and time‐effective. Additionally, the Internet network's current increase in popularity, combined with the rapid growth of Internet‐connected laboratories, provides a straightforward means of publishing and sharing 2‐DE data. While a small number of laboratories have already successfully published their data over the net, the increasing number of 2‐DE database servers that are currently being set up will sooner or later require some kind of standardization. Unfortunately, standardization can be a long and cumbersome process inevitably leading to undesirable compromises. A federated database offers a simple and efficient way to publish and share 2‐DE data without the need for standardization. Taking advantage of Internet protocols such as World Wide Web, they allow each laboratory to maintain their own database and to interconnect it with other similar databases through the use of active cross‐references. This paper first presents guidelines for building a federated 2‐DE database that may easily be followed by most laboratories. It then briefly reviews the state‐of‐the‐art in networked 2‐DE databases, and finally describes the SWISS‐2DPAGE database which fully implements the concept of a federated 2‐DE database.
The yeast SWISS‐2DPAGE database
Authors: Sanchez Jean‐Charles , Golaz Olivier , Frutiger Séverine , Schaller Dominique , Appel Ron D. , Bairoch Amos , Hughes Graham J. , Hochstrasser Denis F.
Published: May 2005

The systematic sequencing of the yeast genome will soon be completed. A new challenge has been launched by the EUROFAN (European Functional Analysis) project whose goal is to elucidate the physiological and biochemical function of newly discovered open reading frames (ORF) from yeast. One of the approaches is to use protein‐based technologies such as two‐dimensional gel eletrophoresis and protein identification in order to establish a yeast reference map. Modified protein patterns can be compared to the reference map which hopefully will help identify changes related, for example, to growth processes or developmental events. This paper describes the yeast SWISS‐2DPAGE database in which charge separation was obtained using immobilized pH gradient (IPG). Proteins identified by gel comparison, amino acid composition analysis and/or microsequencing are recorded and described in an accessible uniform format. We have identified more than one hundred polypeptides, several of which were newly mapped. In addition, the yeast SWISS‐2DPAGE database can be freely accessed through the World Wide Web (WWW) network on the ExPASy molecular biology server.
Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and centrifuge blotting: Preparation of polypeptides for amino‐terminal sequence analysis
Authors: Hermansen Leonila F. , Juul Jessie , Sletten Knut
Published: May 2005

The applicability and reproducibility of a previously described (L. F. Hermansen et al., Electrophoresis 1993, 14, 1302–1306) centrifuge‐blotting procedure for capturing subnanomolar amounts of protein on polyvinylidene difluoride membranes for direct Edman degradation was further investigated. Proteins with different molecular masses were centrifuge‐blotted onto Immobilon CD membranes. Simultaneous blotting and desalting was achieved with an overall yield of 15–56% after 2 h centrifugation for proteins with a molecular mass of 12–30 kDa. Centrifugation of myoglobin for 6 h resulted in an overall yield of 72%. The subnanomolar amounts obtained were also sufficient to conduct cyanogen bromide cleavage in situ on proteins with blocked NH2‐terminus and to generate sequence information.
Capillary zone electrophoresis in agarose gels using absorption imaging detection
Authors: Palm Anders , Lindh Curt , Hjertén Stellan , Pawliszyn Janusz
Published: May 2005

A simple home‐built electrophoretic unit and a commercially available chargecoupled device (CCD) camera with image acquisition and analysis software were used to study the separation process in zone electrophoresis experiments in 4 cm long, round capillaries (inside diameter 0.2 mm). Several capillaries could be investigated simultaneously. The absorption imaging system was used not only to follow the course of the separation but also to study the interaction between biologically active substances (proteins, detergents, enzymes and substrate). Since the system allows visual on‐line observation of the separation one can rapidly decide when the analysis is finished, which often shortens the analysis time. The electrophoresis method presented is suitable also for preparative runs, since direct visualizations of a solute zone allows it to be excised and then used for further studies.
Alpha‐2‐HS‐glycoprotein (AHSG) polymorphism in semen and saliva
Authors: Yasuda Toshihiro , Takeshita Haruo , Tsubota Etsuko , Sawazaki Kazumi , Iida Reiko , Nadano Daita , Kishi Koichiro
Published: May 2005

Polymorphism of alpha‐2‐HS‐glycoprotein (AHSG) was demonstrated in human semen and whole saliva samples by thin‐layer polyacrylamide gel isoelectric focusing (IEF) and immunoblotting. Although the seminal AHSG IEF patterns were found to differ from those of plasma AHSG from the corresponding donors, incorporation of Nonidet P–40 into the IEF gel (pH 4.2–4.9) enabled us to phenotype seminal AHSG correctly. Salivary AHSG, however, exhibited IEF patterns similar to those of the corresponding plasma AHSG. By treating the samples with neuraminidase, it was possible to determine the AHSG types using 2–5 μL semen and 50–100 μL whole saliva samples. The AHSG types determined separately in 47 sets of semen, whole saliva, urine and plasma samples from the same donors correlated perfectly with each other. AHSG typing could, therefore, provide an additional discriminant characteristic in the forensic examination of semen and saliva samples.
Identification of stress proteins in endothelial cells
Authors: Portig Irene , Pankuweit Sabine , Lottspeich Friedrich , Maisch Bernhard
Published: May 2005

Endothelial cells have been shown to play a major role in the pathophysiology of various diseases including ischemic heart disease and viral infection leading to myocarditis or dilated cardiomyopathy, conditions in which stress proteins (heat shock protein – hsp; glucose‐related protein – grp) are likely to be involved. For further characterization of stress proteins and their possible role in these diseases, the major stress proteins in human endothelial cells were separated by two‐dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimension and identified by immunoblotting and either N‐terminal or internal amino acid sequencing, respectively. Ubiquitin, hsp27, hsp60, hsp70, heat shock cognate protein 70, grp78 and grp75 were found to be constitutively expressed; hsp72 was found in stressed cells, exclusively, in line with results obtained in other human cell lines. Three additional proteins with molecular masses between 34 and 40 were regulary detected in stressed cells that were found to have identical amino acid sequences with those of members of the hsp70 familiy.
S‐Pyridylethylation of intact polyacrylamide gels and in situ digestion of electrophoretically separated proteins: A rapid mass spectrometric method for identifying cysteine‐containing peptides
Authors: Moritz Robert L. , Eddes James S. , Reid Gavin E. , Simpson Richard J.
Published: May 2005

In‐gel proteolytic digestion of acrylamide‐gel separated proteins is a method widely used for generating peptide fragments for the purpose of identifying proteins by Edman degratation, tandem mass spectrometry, and peptide‐mass fingerprinting. However, it is well recognised for disulfide‐bonded proteins electrophoresed under reducing conditions that if no precautions are taken to minimise disulfide bond formation during protein digestion or peptide isolation, complex peptide maps can result. Here, we describe an improved method for in‐gel protein digestion. It consists of first reducing and S‐pyridylethylating Coomassie Brilliant Blue R‐250‐stained proteins immobilised in the whole gel slab with dithiothreitol and 4‐vinylpyridine, excising the individual stained and alkylated proteins, and then digesting them in situ in the gel matrix with trypsin or Achromobacter lyticus protease I. Peptide fragments generated in this manner are extracted from the gel piece and purified to homogeneity by a rapid (≤12 min) reversed‐phase high performance liquid chromatography (HPLC) procedure, based upon conventional silica supports. Recoveries of peptides are increased by S‐pyridylethylation of acrylamide‐immbilised proteins prior to in‐gel digestion. Further, the levels of gel‐related contaminants, which otherwise result in suppression of sample signals during electrosprayionisation mass spectrometry, are greatly reduced by the reduction / alkylation step. Additionally, we demonstrate that S‐β‐(4‐pyridylethyl)‐cysteine containing peptides can be readily identified during reversed‐phase HPLC by absorance at 254 nm, and during electrospray ionisation tandem mass spectrometry by the appearance of a characteristic‐pyridylethyl fragment ion of 106 Da. The position of cysteine residues in a sequence can be determined as phenylthiohydantoin S‐β‐(4‐pyridylethyl)‐cysteine during Edman degradation, and by tandem mass spectrometry.
Ultraviolet matrix assisted laser desorption ionization‐mass spectrometry of electroblotted proteins
Authors: Schreiner Martin , Strupat Kerstin , Lottspeich Friedrich , Eckerskorn Christoph
Published: May 2005

Direct mass spectrometric analysis of proteins electroblotted onto polyvinylidene fluoride membranes after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis is demonstrated by matrix‐assisted laser desorption ionization‐mass spectrometry (MALDI‐MS) with a linear time‐of‐flight instrument, equipped with a nitrogen laser (337 nm). The blotted proteins were desorbed directly from the blotting membrane after incubation with sinapinic acid as matrix. Different commercially available membranes resulted in high quality protein signals for hydrophobic membranes exhibiting high specific surface areas (Immobilon PSQ or Trans‐Blot) or for charged membranes (Immobilon CD). Systematic investigations with standard proteins were performed to compare standard preparation procedures for ultraviolet (UV) MALDI‐MS on stainless steel sample stages and preparation of proteins immobilized onto membranes either by direct application from protein solutions (spotting) or by electrotransfer from gels (electroblotting). Aspects such as mass resolution, reproducibility from shot to shot and spot to spot, mass accuracy, and preservation of protein localization are addressed in this paper.
Statistical features in the lakes‐straits model and the influence of hernias
Authors: Disch Christian , Loomans Dirk , Sokolov Igor M. , Blumen Alexander
Published: May 2005

We evaluate numerically the mobility of DNA chains under field‐inversion gel electrophoresis (FIGE) conditions in the framework of the lakes‐straits model introduced by Zimm (Phys. Rev. Lett. 1988, 61, 2965–2968; J. Phys. Chem. 1991, 94, 2197–2206). We extend the model by allowing both simple and also multiple‐branched hernias; this is achieved by arranging the data structure used in the algorithm so that each fragment in a lake can be treated separately. We show that the existence of hernias allows the probe to migrate faster and that with hernias the mobility minimum in FIGE shifts to smaller field periods. These effects occur only if the electric field is strong enough. We also discuss the influence of the model's parameters on the mobility.
Automatic matrix determination in four dye fluorescence‐based DNA sequencing
Authors: Yin Zhongbin , Severin Jessica , Giddings Michael C. , Huang Wei‐an , Westphall Michael S. , Smith Lloyd M.
Published: May 2005

The four dye fluorescence detection strategy is a widely used approach to automated DNA sequence analysis. An important aspect of data processing in this approach is the multicomponent analysis to deduce the concentrations of four fluorophores from fluorescence emission intensities at four different wave‐lengths. This requires knowledge of the correct transformation matrix M. The matrix M is a function both of the fluorophores employed and the fluorescence detection system. M is typically determined either by a calibration process with individual dyes, or by choosing four well‐separated individual peaks corresponding to the four different dyes. Both are time‐consuming and complicated procedures for routine use. An automatic scheme for finding M directly from raw sequence data is presented here. This facilitates data analysis and the underlying algorithm may also find utility in other multispectral applications.