bluuebunny/crossref_metadata_2025 huggingface dataset:
Separation of cis/trans conformers of human and salmon calcitonin by low temperature capillary electrophoresis
Authors: Thunecke Frank , Fischer Gunter
Published: May 2005

Conformer‐specific recognition of peptides and proteins has often been proved with the aid of indirect methods. Here we provide an analytical approach for a direct investigation of separated isomers. Cis/trans conformers of the peptide hormones human (hCT) and salmon (sCT) calcitonin exhibit different migration properties in capillary zone electrophoresis at subambient temperatures. Calcitonin consists of 32 amino acids with two proline residues incorporated. It is the longest unstructured peptide for which a conformer separation by capillary electrophoresis has yet been achieved. Lowering the temperature yielded a splitting into two and three peaks for sCT and hCT, respectively, in acidic buffer. The peak ratios of 66:34 for sCT and 71:23 for hCT are in good agreement with the conformer distribution previously reported from nuclear magnetic resonance (NMR) studies. The addition of different organic modifiers (5–20% v/v) to the running buffer does not improve the separation. The observed merging of conformer peaks in buffer containing 20% v/v 2,2,2‐trifluoroethanol (TFE) is attributed to structure formation.
Sodium alkyl ether sulfate preparative electrophoresis for the preparation of reaction centers without H‐subunit from Rhodopseudomonas viridis
Authors: Miyake Jun , Hara Masayuki , Asada Yasuo , Morimoto Yasuhiro , Shirai Makoto
Published: May 2005

Sodium alkyl ether sulfate (AES), an analog of sodium dodecyl sulfate (SDS) was used in polyacrylamide gel electrophoresis (PAGE) for the partial decomposition of the photosynthetic reaction center (RC) of Rhodopseudomonas viridis. Unlike SDS, AES did not completely dissociate RC into its subunits but selectively detached H‐subunit from RC to give RC(‐H) without losing the spectroscopic nature of RC. For the denaturation of RC(‐H), AES was found to be as mild as 3‐[3‐cholamidopropyl dimethylammonio]‐1‐propanesulfonate (CHAPS).
A heparin‐binding peptide from human serum amyloid P component characterized by affinity capillary electrophoresis
Authors: Heegaard Niels H. H.
Published: May 2005

Affinity capillary electrophoresis (CE) was used for a detailed characterization of the binding between heparin and a peptide isolated from the heparin‐binding serum protein amyloid P component (SAP). The peptide corresponds to a tryptic fragment (T3) comprising amino acids 14–38 of SAP. By including ligands in the electrophoresis buffer various glycosaminoglycans could be screened for binding of T3 using one sample aliquot. The binding was found to be highly specific for heparin and heparin fragments down to tetramers and appeared strongest at a slightly alkaline pH while no binding could be demonstrated with heparan sulfate, chondroitin sulfate, desulfated heparin, mannose 6‐phosphate and phosphotyrosine. The T3‐heparin complexes were sufficiently stable to perform quantitative measurements of the binding using preequilibration of samples prior to a CE‐mediated separation of bound and free T3‐peptide. Plots based on quantitation of analyte peaks corresponding to free and complexed T3 yielded a dissociation constant of 1.5 μm for the interaction with heparin. The results indicate that a specific subfraction of the heparin molecules is active in binding interactions with the peptide. The affinity CE approach proved to be useful for these studies because of its sensitivity to complex formation involving charged ligands and the possibility of achieving separations under native conditions. Also advantageous is the low sample consumption and the ability to analyze unlabeled reactants in solution.
Using capillary electrophoresis/frontal analysis to screen drugs interacting with human serum proteins
Authors: McDonnell Patricia A. , Caldwell Gary W. , Masucci John A.
Published: May 2005

We have used capillary electrophoresis in the frontal analysis mode (CE/FA) to determine the binding capacity of β‐adrenoceptor blocking drugs to individual serum proteins, serum protein mixtures and human serum. The free drug concentration was directly measured from the height of the frontal peak and used to calculate the bound drug concentration. From the bound drug concentration, the percentage of drug bound to the serum proteins α1‐acid glycoprotein (AGP) and human serum albumin (HSA) was then determined. In addition to determining the percent of a drug bound to a protein, the drug‐protein association constant (Ka) was determined for AGP binding to β‐blockers. The data‐estimated association constants were consistent with literature values. The CE/FA studies on the β‐adrenoceptor blocking drugs and the serum proteins indicated that HSA, AGP, high density lipoprotein (HDL), and low density lipoprotein (LDL) were the main contributors to serum binding for this series of compounds. The serum‐drug binding data sorted the β‐adrenoceptor blocking drugs into high and low binding categories. The protein mixture (AGP + HSA + HDL + LDL) resulted in dividing the β‐blockers into the same high/low rankings. The protein mixture (AGP + HSA + HDL + LDL) was amenable to automation, did not autoaggregate, and had constant concentrations for the proteins.
Smith/Birnstiel mapping of genome rearrangements in Pseudomonas aeruginosa
Authors: Heuer Thomas , Bürger Christiane , Tümmler Burkhard
Published: May 2005

A novel application of the Smith/Birnstiel technique is presented for the analysis of intraspecies genomic diversity in small genomes. Rare‐cutter total/N frequent‐cutter partial restriction digestions are separated in N separate lanes by pulsed‐field gel electrophoresis, blotted and hybridized with rare‐cutter fragment ends as probes. The evaluation of the autoradiogram results in high‐resolution restriction maps of 10–200 kbp regions. The technique was applied to the analysis of genome rearrangements in Pseudomonas aeruginosa strains. Comparison of the region encoding the tryptophan biosynthesis genes in the PAO and the IATS serotype 5 strains revealed that shared sequence characterized by almost identical restriction fingerprints was interrupted in the serotype 5 strain by small islands displaying strain‐specific restriction site signatures. A multistep rearrangement in a hypervariable chromosome region downstream of the phn locus was detected in serial airway isolates from a patient with cystic fibrosis.
Bacterial phylogeny based on comparative sequence analysis (review)
Authors: Ludwig Wolfgang , Strunk Oliver , Klugbauer Sabine , Klugbauer Norbert , Weizenegger Michael , Neumaier Judith , Bachleitner Marianne , Schleifer Karl Heinz
Published: May 2005

Comparative sequence analysis of small subunit rRNA is currently one of the most important methods for the elucidation of bacterial phylogeny as well as bacterial identification. Phylogenetic investigations targeting alternative phylogenetic markers such as large subunit rRNA, elongation factors, and ATPases have shown that 16S rRNA‐based trees reflect the history of the corresponding organisms globally. However, in comparison with three to four billion years of evolution the phylogenetic information content of these markers is limited. Consequently, the limited resolution power of the marker molecules allows only a spot check of the evolutionary history of microorganisms. This is often indicated by locally different topologies of trees based on different markers, data sets or the application of different treeing approaches. Sequence peculiarities as well as methods and parameters for data analysis were studied with respect to their effects on the results of phylogenetic investigations. It is shown that only careful data analysis starting with a proper alignment, followed by the analysis of positional variability, rates and character of change, testing various data selections, applying alternative treeing methods and, finally, performing confidence tests, allows reasonable utilization of the limited phylogenetic information.
Mycobacterial genome structure (minireview)
Authors: Philipp Wolfgang J. , Schwartz David C. , Telenti Amalio , Cole Stewart T.
Published: May 2005

Genome maps have been constructed for the mycobacterial pathogens Mycobacterium leprae and Mycobacterium tuberculosis, as well as for the attenuated vaccine strain Mycobacterium bovis BCG Pasteur. While the chromosomes of M. tuberculosis and M. bovis BCG Pasteur show extensive conservation at the grosslevel, comparison with M. leprae revealed a high degree of diversification, with a mosaic‐like pattern apparent. The ordered libraries of M. tuberculosis and M. leprae produced during the course of these studies played a central role in the genome sequencing projects of these two bacilli, showing the utility of this approach for systematic sequencing of bacterial genomes.
Chromosome geometry and intraspecific genetic polymorphism in Gram‐positive bacteria revealed by pulsed‐field gel electrophoresis (minireview)
Authors: Leblond Pierre , Decaris Bernard
Published: May 2005

Pulsed‐field gel electrophoresis (PFGE) proved to be a powerful approach to study bacterial genomics. The genome structure and genetic polymorphism of Gram‐positive bacteria from the high G+C (Streptomyces) and low G+C (Streptococcus) groups have been studied. PFGE allowed the estimation of the size of their genome at about 8 Mbp and 1.8 Mbp, respectively, and to get an insight into their chromosome geometry. Thus, physical mapping of the genome of wild‐type Streptomyces ambofaciens strains revealed the linearity of the 8 Mbp chromosomal DNA and its typical invertron structure, while the 1.8 Mbp chromosome of Streptococcus thermophilus was shown to be circular. These findings disproved the long‐standing idea of universality of bacterial chromosome circularity. In addition, strains belonging to the species S. ambofaciens and S. thermophilus allowed us to characterize the genetic polymorphism at the intraspecific level. Within the S. thermophilus species, comparison of the physical maps showed a relative conservation of gene order as well as restriction sites along the chromosome. In contrast, variable loci were characterized that revealed localized genome rearrangements. The most spectacular of these corresponded to horizontal gene transfer events of sequences. In S. ambofaciens, the physical maps of three isolates pointed to the conservation of the genetic organization. However, a strong polymorphism was observed in the terminal regions of the linear chromosomal DNA. Previous PFGE studies in S. ambofaciens gave proof of a high structural instability of a limited region of the chromosome called unstable region (i.e., DNA rearrangements such as deletions and amplifications). These intraclonal rearrangements create an impressive intraspecific polymorphism of genome size and shape (linear or circular). In both organisms, the DNA rearrangements are restricted to particular regions of the chromosome.
Polymerase chain reaction‐mediated typing of microorganisms: Tracking dissemination of genes and genomes (minireview)
Authors: van Belkum Alex , Hermans Peter W. M. , Licciardello Lidia , Stefani Stefania , Grubb Warren , van Leeuwen Willem , Goessens Wil H. F.
Published: May 2005

The polymerase chain reaction (PCR) is a powerful molecular biology tool which can be used for the identification of species and strains of diverse microorganisms. By aimed amplification of characteristic genes (i.e., genes encoding ribosomal RNA molecules) and subsequent genetic analysis of amplified fragments, information on microbiological systematics and phylogeny can be obtained in a fast and efficient manner. Similar types of gene identification can be used to verify or detect genes responsible for phenotypic characteristics, whereas modified forms of the PCR enable whole genome searches for genetic polymorphisms among strains of a given species. In medical sciences, both strategies, gene and genome variability analysis by PCR, have an increasing impact on the study of the spread of especially those microbes that are multiply resistant to clinically used antibiotics. In this communication we will exemplify the usefulness of PCR‐mediated typing of microorganisms from a clinical perspective while focusing on gene‐ versus genome‐scanning. Special emphasis will be placed on analysis of the dissemination and characteristics of methicillin‐resistant Staphylococcus aureus (MRSA) strains and bacterial factors providing resistance to penicillin and other β‐lactam antibiotics. Technical limitations and possibilities for improvement will be discussed.
Enantioseparation of anaestethic drugs by capillary zone electrophoresis using cyclodextrin‐containing background electrolytes
Authors: Amini Ahmad , Sörman Ulla Paulsen , Lindgren Björn H. , Westerlund Douglas
Published: May 2005

The enantiomers of five racemic anaestethic drugs were resolved with cyclodextrins using capillary zone electrophoresis. Parameters which affected the chiral resolution, such as type and concentration of cyclodextrin, temperature, and addition of organic modifier were investigated. The results show that the enantiomeric discrimination of the solutes is influenced by the structural shape of the solute molecules, separation temperature, and type of cyclodextrin. It was found that α‐cyclodextrin was the best enantioselector for resolution of prilocaine and ketamine, while the enantiomers of mepivacaine, ropivacaine, and bupivacaine were resolved with β‐cyclodextrin and/or modified β‐cyclodextrins, i.e., methyl‐ and 2‐hydroxypropyl‐β‐cyclodextrin, as chiral selectors. The length of the alkyl chain on the amino group of the drug molecule had a strong effect on the enantioresolution of mepivacaine, ropivacaine, and bupivacaine. Baseline separation of racemic ketamine was achieved with α‐and methyl‐β‐cyclodextrin at 15°C. Addition of 5 M urea to the running buffer containing β‐cyclodextrin at high concentrations resulted in the enantioseparation of prilocaine, mepivacaine, and ketamine. Enantioresolution was improved upon the addition of 10% methanol to the buffer containing urea and β‐cyclodextrin. Generally, the complex formed between the S‐enantiomers and modified β‐cyclodextrins was stronger than the corresponding R‐forms. An exception was prilocaine where the R‐form gave a more stable complex both with α‐ and β‐cyclodextrin.
Two‐dimensional map of basic proteins of Haemophilus influenzae
Authors: Fountoulakis Michael , Takács Béla , Langen Hanno
Published: May 2005

Haemophilus influenzae is a bacterium of medical interest of which the entire genome has been sequenced. The proteome of the microorganism has been analyzed by two‐dimensional gel electrophoresis, during which immobilized pH 3–10 gradient strips were used and approximately 300 proteins were identified. In order to detect additional, basic proteins, we analyzed the soluble protein fraction of H. influenzae and the proteins of fractions collected from affinity chromatography on heparin, by two‐dimensional gel electrophoresis, using for the first‐dimensional separation immobilized pH gradient strips comprising the pH region of 6–11. The protein spots were analyzed by matrix‐assisted laser desorption ionization‐mass spectrometry. One hundred and two proteins were identified, of which 58 were identified for the first time. A large percentage of the basic proteins represent nucleic acid binding and, in particular, ribosomal proteins. The locations of the identified basic proteins of H. influenzae are indicated in a two‐dimensional map.
Two‐dimensional zymography for analysis of proteolytic enzymes in human pure pancreatic juice
Authors: Kaino Seiji , Furui Toshifumi , Hatano Satoko , Kaino Miyuki , Okita Kiwamu , Nakamura Kazuyuki
Published: May 2005

Proteolytic enzymes in human pure pancreatic juice (PPJ), which was collected by cannulating the main pancreatic duct using endoscopy, were investigated by two‐dimensional zymography (2‐DZ). 2‐DZ was carried out by combining isoelectric focusing (IEF) in the first dimension with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) in the second dimension, using gels containing casein or gelatin as a substrate for the proteolytic enzymes. After electrophoresis, the gels were incubated in Triton X‐100 followed by incubation at 37°C in Tris buffer (pH 8.5) containing CaCl2. By staining the gels with Coomassie Brilliant Blue (CBB R‐250), proteolytic enzymes were detected as clear spots and zones against a blue background. Proteinase inhibitors, such as a cysteine proteinase inhibitor (E‐64), a metalloproteinase inhibitor (EDTA), and a serine proteinase inhibitor (Pefabloc® SC), were added to PPJ in order to determine the types of proteinases. In patients with pancreatic cancer, spots of molecular weight (Mr) 70 000 and isoelectric points (pI) 5.3–5.5 were clearly detected on the gels containing casein and gelatin, while these spots were not detected in the PPJ from healthy subjects. The proteolytic activities of these spots were strongly inhibited by EDTA and Pefabloc SC but not E‐64. These results suggest that the spots of Mr 70 000 and pI 5.3–5.5 in PPJ of pancreatic cancer might be matrix metalloproteinase 2, which is a candidate for tumor‐associated proteinase. 2‐DZ proved to be a tool for analysis of proteolytic enzymes in PPJ and for the clinical diagnosis of pancreatic cancer.
Two‐dimensional polyacrylamide gel electrophoresis map of bull seminal plasma proteins
Authors: Mortarino Michele , Tedeschi Gabriella , Negri Armando , Ceciliani Fabrizio , Gottardi Luciano , Maffeo Giovanni , Ronchi Severino
Published: May 2005

A two‐dimensional polyacrylamide gel electrophoresis map of bull seminal plasma proteins has been established. About 250 spots were detected after silver staining and polypeptides from 24 spots have been N‐terminally sequenced. Major proteins already described in bull seminal plasma, like PDC‐109 and aSFP, have been located on the map; proteins not yet reported in male reproductive tracts have been evidenced; for some polypeptides showing a previously unknown N‐terminal sequence, structural similarities with proteins described in other organisms have been found. A reference map of seminal plasma proteins could be useful in relating protein pattern changes to physiopathological events influencing the reproductive sphere.
Development of a two‐dimensional gel electrophoresis database of human lysosomal proteins
Authors: Chataway Timothy K. , Whittle Alison M. , Lewis Martin D. , Bindloss Colleen A. , Moritz Robert L. , Simpson Richard J. , Hopwood John J. , Meikle Peter J.
Published: May 2005

Two‐dimensional gel electrophoresis databases have been generated for a range of tissue cell and fluid types, from a number of species. A major difficulty in the development of such databases is the large number of proteins present in even a single cell type (> 10 000) and the low levels of many of these proteins. One approach to overcome these difficulties is to fractionate the cell into its organelles and generate individual databases for each subcellular component. This has the added advantage of assigning a cellular location to each protein identified. Here we report the development of a two‐dimensional gel electrophoresis database of lysosomal proteins.
Additional possible tools for identification of proteins on one‐ or two‐dimensional electrophoresis
Authors: Tsugita Akira , Kamo Masaharu , Miyazaki Kenji , Takayama Mitsuo , Kawakami Takao , Shen Ruqun , Nozawa Takehiro
Published: May 2005

Additional, essentially chemical, identification methods of proteins in polyacry‐lamide gel electrophoresis are described. Two cleavages of peptide bonds were used at the C‐side of aspartic acid with a 0.2% pentafluoropropionic acid (PFPA) aqueous vapor at 90° for 4–16 h, and the N‐side of serine/threonine with an S‐ethyl trifluorothioacetate vapor at 50° for 6–24 h. The products were analyzed by mass spectrometry– peptide mass fingerprinting. A new type of C‐terminal sequencing at multisites of protein was introduced. An aqueous vapor of 90% PFPA at 90° for 2–16 h provided cleavages at the C‐side of aspartic acid and the N‐side of serine/threonine and simultaneous successive truncation at the C‐termini of the cleaved fragments. The product resulted in C‐terminal sequences at multisites in proteins by mass spectrometric analysis. The following chemical deblocking methods were used. Anhydrous hydrazine vapor at–5° for 8 h deblocked the N‐formyl group, and the vapor at 20° for 4 h deblocked pyrrolidone carboxylate. N‐acetylserine/threonine was deblocked by aqueous vapor of 75% PFPA at 50° for 1 h, followed by reaction with p‐suifophenylisothiocyanate at pH 6.0. These methods were applied to a variety of protein spots on polyacrylamide gels. A new stepwise C‐terminal sequencing of protein from polyacrylamide gels is also described.
Analyzing glycoproteins separated by two‐dimensional gel electrophoresis
Authors: Packer Nicolle H. , Lawson Margaret A. , Jardine Daniel R. , Sanchez J.‐C. , Gooley Andrew A.
Published: May 2005

Two‐dimensional (2‐D) electrophoresis is the preferred method for separating the glycoforms of proteins. The isoforms usually present as ‘trains’ of spots in the first dimension and may also differ in molecular weight. The primary goal for analyzing the carbohydrate content of glycoprotein spots is to understand the ‘rules’ which govern the migration of glycoproteins in 2‐D electrophoresis. These rules can then be used to produce predictive vectors to interpret changes in glycosylation patterns. Techniques for the analysis of oligosaccharides released from glycoproteins which have been electroblotted to PVDF membrane after one‐dimensional (1‐D) and 2‐D preparative gel electrophoresis are described. The oligosaccharides are removed enzymatically (PNGase F of N‐linked oligosaccharides) or chemically (β‐elimination of O‐linked oligosaccharides) and separated by high performance anion exchange chromatography (HPAEC‐PAD) and identified by electrospray ionization mass spectrometry (ESI‐MS) or analyzed directly by ESI‐MS. After enzymic removal of the N‐linked oligosaccharides the protein spots can be further analyzed by Edman sequence tagging for identification and quantitation of the protein and by acid hydrolysis for monosaccharide analysis of the O‐linked oligosaccharides. These approaches have been proved on 1‐D PAGE electroblotted bovine fetuin and human glycophorin A and then used to analyze two abundant proteins which separate as glycoforms on 2‐D PAGE preparative narrow range (pH 4.5–5.5) blots of human plasma: α2‐HS glycoprotein (human fetuin) and α1‐antitrypsin (α1‐protease inhibitor). It is apparent that both the macroheterogeneity (site occupation) and microheterogeneity (diversity of structures) of the glycosylation contribute to the separation of protein isoforms in 2‐D PAGE.
Identification of phosphorylated proteins from thrombin‐activated human platelets isolated by two‐dimensional gel electrophoresis by electrospray ionization‐tandem mass spectrometry (ESI‐MS/MS) and liquid chromatography‐electrospray ionization‐mass spectrometry (LC‐ESI‐MS)
Authors: Immler Dorian , Gremm Dagmar , Kirsch Dieter , Spengler Bernhard , Presek Peter , Meyer Helmut E.
Published: May 2005

Two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) is a powerful tool to separate complex protein mixtures including whole cell lysates. In combination with immunoblotting techniques or radioactive labeling techniques it is a fast and convenient way to demonstrate the presence of certain proteins or protein modifications. With the development of extremely sensitive analytical techniques such as matrix‐assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS) or electrospray ionization (ESI)‐MS, it has become possible to use 2‐D gels not only as an analytical but also as a preparative tool. Starting with a number of spots excised from 2‐D gels, a protein can be identified using different strategies involving enzymatic cleavage of the protein in the gel matrix, elution of the resulting peptides and analysis of these peptides by mass spectrometry. The obtained peptide mass fingerprint or fragment ion spectra from peptides can be used to screen protein or nucleic acid databases in order to identify the protein. We have used the techniques described above to identify proteins from human platelets which change their phosphorylation state following activation of platelets by thrombin. Platelets were radioactively labeled with [32P]orthophosphate and stimulated. Several protein spots in the observed range of 10–80 kDa and an isoelectric point of 3–10 showed a significant increase or decrease in phosphorylation. We present the results from the investigation of a spot group representing different isoforms and phosphorylation states of myosin light chain.
An isoelectrically trapped enzyme reactor operating in an electric field
Authors: Righetti Pier Giorgio , Bossi Allessandra
Published: May 2005

Membrane enzyme reactors constitute an attempt at integrating catalytic conversion, product separation and/or concentration and catalyst recovery into a single operation. Whereas conventional membrane reactors confine an enzyme, in a free form, to one side of a membrane by size exclusion, electrostatic repulsion, or physical or chemical immobilization onto an intermediate support (gel, liposome), the membrane reactor here described is shown to operate under an entirely new principle: enzyme confinement into an isoelectric trap located in a multicompartment electrolyzer operating in an electric field. Two isoelectric membranes, having pIvalues encompassing both the enzyme pIand the pH of its optimum of activity, act by continuously titrating the enzyme trapped inside, thus preventing it from escaping the reaction chamber. Charged products generated by the enzyme catalysis are continuously electrophoretically transported away from the reaction chamber and collected into other chambers stacked either towards the cathodic or anodic sides. In a urease reactor, ammonia is continuously harvested towards the cathode, thus allowing >95% substrate consumption with maintenance of enzyme integrity over much longer time periods than in a batch reactor. In a trypsin reactor, casein is digested and biologically active peptides are continuously harvested in a pure form into appropriate isoelectric traps. In a third example, pureD‐phenylglycine is produced from a racemate mixture, via an acylation reaction onto a cosubstrate (the ester methyl‐4‐hydroxyphenyl acetate), brought about by the enzyme penicillin G acylase.
Counterbalancing hydrodynamic sample distortion effects increases resolution of free‐flow zone electrophoresis
Authors: Weber Gerhard , Bauer Johann
Published: May 2005

On fractionation of highly heterogeneous protein mixtures, optimal resolution was achieved by forcing proteins to migrate through a preestablished pH gradient, until they entered a medium with a pH similar but not equal to their pIs. For this purpose, up to seven different media were pumped through the electrophoresis chamber so that they were flowing adjacently to each other, forming a pH gradient declining stepwise from the cathode to the anode. This gradient had a sufficiently strong band‐focusing effect to counterbalance sample distortion effects of the flowing medium as proteins approached their isoelectric medium closer than 0.5 pH units. Continuous free‐flow zone electrophoresis (FFZE) with high throughput capability was applicable if proteins did not precipitate or aggregate in these media. If components of heterogeneous protein mixtures had already started to precipitate or aggregate, in a medium with a pH exceeding their pI by more than 0.5 pH units, the application of interval modus and media forming flat pH gradients appeared advantageous.
Analysis of trypanosomal endocytic organelles using preparative free‐flow electrophoresis
Authors: Grab Dennis J. , Webster Paul , Lonsdale‐Eccles John D.
Published: May 2005

In this paper we demonstrate the power of preparative free‐flow electrophoresis (FFE) for the study of endocytosis by African trypanosomes. Endocytosis of extracellular macromolecules by these parasites occurs through a specialized region of the parasite called the flagella pocket. The uptake of fluid phase markers such as horseradish peroxidase (HRP) into the various compartments of the endocytic pathway of bloodstream forms of Trypanosoma brucei brucei was manipulated by regulating the external environment (e.g., by altering the temperature of incubation). The various subcellular compartments were then separated by free‐flow electrophoresis (FFE) or isopycnic density gradient centrifugation and analyzed for marker uptake. At low temperatures, HRP was found predominantly in the flagellar pocket. Increasing the temperature resulted in a time‐dependent uptake of HRP into more positively charged endosomal fractions. However, little HRP activity was detected in lysosomal compartments, suggesting that either HRP had not yet entered the lysosome or was degraded immediately upon entry. Through the use of FFE we were able to identify and analyze compartments of the endosomal pathway that were not possible to identify by density gradient centrifugation alone. Although the differences in FFE separation of the endocytic compartments as seen in HRP uptake were striking, the minor changes seen within the lysosomal system were more subtle, as depicted in the protease profiles. In conlusion, we show that preparative FFE is a powerful technique for the analysis and separation of flagellar pocket‐derived membranes from other endosomal and lysosomal compartments of African trypanosomes.
Characterization of a subpopulation of mouse red blood cells as preferential target for malarial invasion
Authors: Sabolovic Domagoj , Sestier Claude
Published: May 2005

Cell electrophoresis was used to study a distinct subpopulation of murine red blood cells (RBC). These RBC are normally found in the spleen and bone marrow. They appear in the peripheral blood when mice are mildly bled or sucked by mosquitos. These cells have been characterized as having larger size, light density, lower electrophoretic mobility, and being more resistant to lysis with unsaturated fatty acids and in glycerol‐containing medium than mature erythrocytes. All their features suggest that their differentiation status represents an intermediate stage between reticulocytes and adult RBC. In vitro Plasmodium invasion tests showed their increased sensitivity to invasion by the parasites. The extent of their spreading in the blood was found to be strain‐dependent, being more pronounced in C57B1/6 mice, highly susceptible to developing cerebral malaria after infection with Plasmodium berghei, as compared to Balb/c, a nonsusceptible strain of mice.
Capillary electrophoretic analysis of ginseng polypeptide
Authors: Kajiwara Hideyuki , Hemmings Andrew M.
Published: May 2005

A ginseng polypeptide (GPP) found in ginseng roots and its modified peptides were tested by capillary zone electrophoresis (CZE) under acidic as well as basic conditions. Modified peptides were synthesized for three purposes: (i) to analyze their functions in the first three acidic amino acid residues, (ii) to analyze their functions in three sequenced glycines, and (iii) to analyze the length of side chains in acidic amino acids. The roles of glycines, acidic amino acids and amino acid side chains in the binding of Mg2+ were studied at pH values less than 7.0. The migration times of GPP varied with the pH of various electrophoresis buffers, and electrophoresis patterns were significantly changed between pH 7.0–7.5. Based on the electrophoretic analysis, it was concluded that the binding mechanisms for Mg2+ or conformations of GPP changed between low pH and high pH.
Immunoblot analysis of components of barley recognized by IgE antibodies in sera from pig farm workers
Authors: Chiung Yin‐Mei , Shen Horng‐Der , Huang Ju‐Wei
Published: May 2005

Components of the crude extract of barley, recognized by IgE antibodies in sera from 132 pig farm‐workers in southern Taiwan, were investigated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and immunoblotting. Among the sera tested, 43 (32.6%) showed IgE reactivity to 15 components of barley with molecular mass ranging from 148000 to 14000 daltons (Da). Heterogeneity in the IgE‐binding patterns of the samples tested was observed. However, the major antigen appears to be the component with about 37 kDa, in 32 (74.4%) of the 43 immunoblot‐positive serum samples. Furthermore, components with about 55 and 67 kDa which reacted with 26 (60.5%) and 18 (41.9%) of the positive samples, respectively. These might also be considered as important allergens of the flour made from entire seeds of barley. The results suggest that barley may be considered to play a role in the IgE‐mediated occupational respiratory disorders and the 37 kDa component has also been shown to be the major allergen.
Two‐dimensional gel electrophoresis for proteome projects: The effects of protein hydrophobicity and copy number
Authors: Wilkins Marc R. , Gasteiger Elisabeth , Sanchez Jean‐Charles , Bairoch Amos , Hochstrasser Denis F.
Published: May 2005

Two‐dimensional (2‐D) gel electrophoresis is often used in proteome projects to provide a global view of the proteins expressed in any cell or tissue type. Here we have investigated the effects of protein hydrophobicity and cellular protein copy number on a protein's presence or absence on a two‐dimensional gel. The average hydropathy values of all known proteins from Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae were calculated, thus defining the range of protein hydrophobicity and hydrophilicity in these organisms. The average hydropathy values were then calculated for a total of 427 proteins from these species, which had been identified elsewhere on 2‐D gels. Strikingly, it was seen that no highly hydrophobic proteins, as defined by average hydrophobicity values, have been found to date on 2‐D gel separations of whole cell lysates. A clear hydrophobicity cutoff point was seen, above which current 2‐D electrophoresis methods appear not to be useful for protein separation. The effect of cellular protein copy number on a protein's presence on a 2‐D gel was investigated by means of a graphical model. This model showed how variations in protein loading and copy number per cell interact to determine the quantity of a protein that will be present on a 2‐D gel. Considering the current maximum in 2‐D gel loading capacity, it was found that 2‐D probably can not visualize or produce analytical quantities of proteins present at less than 1000 copies per cell. We conclude that further developments of 2‐D electrophoresis techniques are desirable to enable the visualization and analysis of all proteins expressed by a cell or tissue.
Effects of carrier and hapten array on the production of anti‐hapten antibodies analyzed by two‐dimensional affinity electrophoresis
Authors: Wang Pei , Nakamura Kazuyuki
Published: May 2005

Effects of a carrier, bovine serum albumin (BSA) and a hapten array, fluorescein isothiocyanate‐conjugated dextran (FITC‐DEX), on the production of anti‐FITC antibodies in BALB/c mice were analyzed by two‐dimensional affinity electrophoresis (2‐D AEP). The mice were immunized with FITC‐BSA, followed by immunizations with FITC‐BSA, saline, BSA, human serum albumin (HSA), fluorescein (Flu), and FITC‐DEX. The heterogeneity and quantity of anti‐FITC antibodies were increased markedly during the second and third immunization with FITC‐BSA. However, the production of anti‐FITC antibodies with low affinity to FITC was suppressed by the second and third immunization with the carrier protein of BSA. The suppression of anti‐FITC antibodies by immunization with BSA may be due to the major histocompatibility complex (MHC)‐mediated competition through antigen processing and the clonal expansion of BSA‐specific B cells because similar suppression was induced by the immunization with HSA. The hapten array of FITC‐DEX generally suppressed the production of anti‐FITC antibodies; however, immunization with free hapten of Flu did not affect the production significantly. This may indicate that cross‐linking of anti‐FITC antibodies on the surface of specific B cells via the binding of hapten array is required for the suppression.
Steady‐state concentration distribution of ampholytes in isoelectric focusing in a linear immobilized pH gradient
Authors: Stoyanov Alexander V. , Righetti Pier Giorgio
Published: May 2005

The equation of balance between electrophoretic and diffusional mass flows in the steady state of isoelectric focusing is analyzed. To create the pH gradient, a model system composed of only one Immobiline is used. The solution is found for the case of a small sample concentration, but without assumptions about linear “focusing force” and constant (linear) conductivity profiles. The effect of sample concentration on the final concentration profile is also evaluated. At high sample concentrations, it is demonstrated that the steady‐state distribution is essentially non‐Gaussian and results in a considerably lower concentration maximum as compared with low sample levels.
On the concept of “normalized buffering power/conductivity ratio” of isoelectric buffers for capillary zone electrophoresis
Authors: Stoyanov Alexander V. , Righetti Pier Giorgio
Published: May 2005

The introduction of a new physico‐chemical parameter is proposed: the “normalized buffering power/conductivity ratio”. It expresses the ratio of buffering power to conductivity (Rβ/λ), normalized by the electrolyte concentration, and gives an opportunity to calculate the properties of buffering ions typically used in capillary electrophoretic separations of biopolymers. This procedure of normalization is possible due to the fact that, in the concentration range practivally used, the relationship of Rβ/λ on concentration is close to linear.
Identification of maize lines via capillary electrophoresis of zeins in isoelectric, acidic buffers
Authors: Righetti Pier Giorgio , Olivieri Erna , Viotti Angelo
Published: May 2005

Modified capillary zone electrophoresis of zeins in strongly acidic, isoelectric buffers is reported for screening of maize (Zea mays L.) lines. The optimized background electrolyte contained 40 mM aspartic acid (pH = pI = 2.77), 8 M urea and 0.5% short‐chain hydroxyethylcellulose (Mn 27000 Da) (apparent pH in 8 M urea: 3.9). Due to the low conductivity of such a buffer (0.7 mmhos), separations can be carried out at 800 V/cm, even in relatively large bore capillaries (50 μ ID), ensuring good sensitivity. The zein patterns thus obtained are species‐specific and allow easy identification of all maize lines tested. No adsorption of proteins to the silica wall is observed and high reproducibility in peak areas and transit times is obtained. The relatively slow migration time (ca. 30 min), even under such a high voltage gradient, is attributed to the paucity of basic amino acid residues typical of zeins, leading to an unfavorable charge/ mass ratio due to the modest net positive charge at the operative pH.
In vitro model system for the identification and characterization of proteins involved in inflammatory processes
Authors: Dax Claudia I. , Lottspeich Friedrich , Müllner Stefan
Published: May 2005

An in vitro model featuring important inflammatory cellular states was established, based on the murine monocyte/macrophage cell line RAW 264.7. Macrophages are key players in chronic inflammation, and major parts of the biochemical reactions taking place in vivo, e.g., the production of proinflammatory cytokines, can be triggered in vitro by stimulation of the cells with bacterial lipopolysaccharide (LPS). A mastergel, representing a synthetic image of the expressed basic set of cellular proteins, was designed by a computer‐assisted overlay of a statistically significant number of two‐dimensional electrophoresis (2‐DE) gels of unstimulated RAW 264.7 cells. This image served as a reference for qualitative and quantitative changes in the protein pattern induced by stimulation of the macrophages with LPS. The optimal conditions for LPS stimulation were evaluated by monitoring the expression and secretion of the proinflammatory cytokine tumor necrosis factor‐α (TNF‐α). The comparison of the mastergel with the 2‐DE gels of LPS‐stimulated cells revealed several changes in the protein pattern. In order to prove the relevance of the presented model system, we focused on two low molecular weight proteins, which showed significant changes in the apparent concentration in a 2‐DE pattern. These proteins were further characterized by microsequencing of internal peptides. A comparison of the obtained sequences with protein databases identified them as cofilin and keratinocyte lipid‐binding protein.
Glycobiology and proteomics: Is mass spectrometry the holy grail?
Authors: Packer Nicolle H. , Harrison Mathew J.
Published: May 2005

One characteristic of glycoproteins is that they are separated by two‐dimensional electrophoresis (2‐D PAGE) into typical ‘trains’ of protein spots which separate on the basis of different isoelectric point (pI) and/or molecular mass. The pattern of these trains often varies in development and disease. While the isoforms differ both in the number of sites of glycosylation and the types of carbohydrate attached to the protein, classical methods of glycan analysis are insensitive at the levels typically separated by 2‐D PAGE. Developments in mass spectrometry technologies have enabled the characterization of most of the oligosaccharide attributes to be determined on picomole amounts of protein. These techniques are beginning to allow the glycoform heterogeneity on 2‐D separated glycoproteins to be analyzed.
New zwitterionic detergents improve the analysis of membrane proteins by two‐dimensional electrophoresis
Authors: Chevallet Mireille , Santoni Véronique , Poinas Alexandra , Rouquié David , Fuchs Alexandra , Kieffer Sylvie , Kieffer Sylvie , Lunardi Joël , Garin Jérome , Rabilloud Thierry
Published: May 2005

Severe quantitative loss of protein is often observed in high‐resolution two‐dimensional electrophoresis of membrane proteins, while the resolution is usually not affected. To improve the solubility of proteins in this technique, we tested denaturing cocktails containing various detergents and chaotropes. Best results were obtained with a denaturing solution containing urea, thiourea, and zwitterionic detergents, synthesized for this purpose. Among the dozen detergents synthesized and tested, amidosulfobetaines with an alkyl tail containing 14–16 carbons proved most efficient, solubilizing previously undetected membrane proteins.
Suppression effects in enzymatic peptide ladder sequencing using ultraviolet ‐ matrix assisted laser desorption/ionization ‐ mass spectrometry
Authors: Kratzer Robert , Eckerskorn Christoph , Karas Michael , Lottspeich Friedrich
Published: May 2005

The techniques of enzymatic and chemical peptide ladder sequencing, coupled with ultraviolet ‐ matrix assisted laser desorption/ionization ‐ mass spectrometry (UV‐MALDI‐MS) have been improving continuously in the last five years and have now become important tools for primary structure identification. In this work, signal suppression effects, appearing in UV‐MALDI‐MS (excitation 337 nm) of ladder peptides, were investigated using the 17‐amino acid peptide dynorphin A. We show, with examples of simple “two‐peptide” systems and more complex “multi‐peptide” systems, that suppression effects do in fact exist. The magnitude of the observed suppression is strongly dependent upon both the nature and the amount of the suppressing peptide. Suppression behavior of individual ladder peptides was investigated on equimolar mixtures of ten ladder peptides. Significant signal suppression was recorded for all ladder peptides, with some of them being approximately 170 times lower in signal intensity than the pure, i.e., unsuppressed peptide at the same concentration. For the investigated system — dynorphin A, 4‐hydroxy‐α‐cyanocinnamic acid (4‐HCCA) matrix, UV excitation — a correlation between the extent of suppression and an intractable combination of peptide hydrophobicity and the presence of several basic amino acids can be seen.
Towards an automated approach for protein identification in proteome projects
Authors: Traini Mathew , Gooley Andrew A. , Ou Keli , Wilkins Marc R. , Tonella Luisa , Sanchez Jean‐Charles , Hochstrasser Denis F. , Williams Keith L.
Published: May 2005

The development of automated, high throughput technologies for the rapid identification of proteins is essential for large‐scale proteome projects. While a degree of automation already exists in some stages of the protein identification process, such as automated acquisition of matrix assisted laser desorption ionisation — time of flight (MALDI‐TOF) mass spectra, efficient interfaces between different stages are still lacking. We report the development of a highly automated, integrated system for large scale identification of proteins separated by two‐dimensional gel electrophoresis (2‐DE), based on peptide mass fingerprinting. A prototype robotic system was used to image and excise 288 protein spots from an amido black stained polyvinylidene difluoride (PVDF) blot. Protein samples were enzymatically digested with a commercial automated liquid handling system. MALDI‐TOF mass spectrometry was used to acquire mass spectra automatically, and the data analysed with novel automated peptide mass fingerprinting database interrogation software. Using this highly automated system, we were able to identify 95 proteins on the basis of peptide mass fingerprinting, isoelectric point and molecular weight, in a period of less than ten working days. Advantages, problems, and future developments in robotic excision systems, liquid handling, and automated database interrogation software are discussed.
Strategies towards a better understanding of antibiotic action: Folate pathway inhibition in Haemophilus influenzae as an example
Authors: Evers Stefan , Padova Karin Di , Meyer Michelle , Fountoulakis Michael , Keck Wolfgang , Gray Christopher P.
Published: May 2005

Two‐dimensional electrophoresis was applied to the global analysis of the cellular response of Haemophilus influenzae to sulfamethoxazole and trimethoprim, both inhibitors of tetrahydrofolate synthesis. Deregulation of the synthesis rate of 118 proteins, involved in different metabolic pathways, was observed. The regulation of the genes involved in the metabolism of the amino acids methionine, threonine, serine, glycine, and aspartate was investigated in detail by analysis of protein synthesis and Northern hybridization. The results suggested that the synthesis of methionine biosynthetic enzymes in H. influenzae is regulated in a similar fashion as in Escherichia coli. A good correlation between the results obtained by Northern hybridization and quantification of protein synthesis was observed. In contrast to trimethoprim, sulfamethoxazole triggered the increased synthesis of the heat shock proteins DnaK, GroEL, and GroES.