bluuebunny/crossref_metadata_2025_split huggingface dataset:
Database searching using mass spectrometry data
Authors: Yates John R. 
Published: May 2005

AbstractLarge‐scale DNA sequencing is creating a sequence infrastructure of great benefit to protein biochemistry. Concurrent with the application of large‐scale DNA sequencing to whole genome analysis, mass spectrometry has attained the capability to rapidly, and with remarkable sensitivity, determine weights and amino acid sequences of peptides. Computer algorithms have been developed to use the two different types of data generated by mass spectrometers to search sequence databases. When a protein is digested with a site‐specific protease, the molecular weights of the resulting collection of peptides, the mass map or fingerprint, can be determined using mass spectrometry. The molecular weights of the set of peptides derived from the digestion of a protein can then be used to identify the protein. Several different approaches have been developed. Protein identification using peptide mass mapping is an effective technique when studying organisms with completed genomes. A second method is based on the use of data created by tandem mass spectrometers. Tandem mass spectra contain highly specific information in the fragmentation pattern as well as sequence information. This information has been used to search databases of translated protein sequences as well as nucleotide databases such as expressed sequence tag (EST) sequences. The ability to search nucleotide databases is an advantage when analyzing data obtained from organisms whose genomes are not yet completed, but a large amount of expressed gene sequence is available (e.g., human and mouse). Furthermore, a strength of using tandem mass spectra to search databases is the ability to identify proteins present in fairly complex mixtures.
Capillary column chromatography improves sample preparation for mass spectrometric analysis: Complete characterization of human α‐enolase from two‐dimensional gels following in situ proteolytic digestion
Authors: Reid Gavin E. , Rasmussen Richele K. , Dorow Donna S. , Simpson Richard J. 
Published: May 2005

AbstractTwo‐dimensional polyacrylamide gel electrophoresis (2‐DE) in combination with mass spectrometry is an extremely powerful tool for characterizing complex mixtures of proteins. In many cases, the success of this approach relies upon the ability to recover peptides at high concentrations and free of interfering artifacts from in‐gel and/or on‐membrane enzymatic digests. In previous studies, we demonstrated that capillary or microcolumn ( ≤ 350 μm ID) rever‐sed‐phase high performance liquid chromatography (RP‐HPLC) is a powerful microseparation technique for proteins and peptides (Moritz, R. L. and Simpson, R. J., J. Chromatogr. 1992, 599, 119–130). Here we evaluate various capillary column RP‐HPLC/mass spectrometric approaches for identifying and characterizing 2‐DE resolved proteins. For these studies, stable and efficient 0.20 mm and 0.32 mm internal diameter (ID) fused‐silica columns with hydrophilic polyvinylidene difluoride (PVDF) frits were fabricated and slurry packed with 7 μm spherical, 300 Å pore size, C8 bonded phase silica particles. We show that capillary column chromatography is a rapid and efficient desalting/concentrating (ON/OFF) technique for sample cleanup prior to protein identification by peptide‐mass fingerprinting using matrix‐assisted laser desorption ionization (MALDI)‐time‐of‐flight mass spectrometry. While marginally more peptide mass information can be obtained by stepped elution of the peptide mixture with increasing concentrations of organic solvent, best results were obtained by fractionation of the peptide mixture using a linear 60 min gradient. One salient feature of this study was the observation that, in contrast to the stepped elution and gradient approaches, the ionization of peptide T1 (m/z 2402.2 SGETEDTFIADLVV(PeCys)TGQIK) was almost completely suppressed using the ON/OFF approach. Maximal amino acid sequence coverage, a necessary prerequisite for complete characterization of a protein, was accomplished using a capillary column (0.2 mm ID) directly coupled with an electro‐spray ionization (ESI) ion‐trap tandem mass spectrometer. For example, from an in situ tryptic digest of α‐enolase isolated by 2‐DE from the human breast carcinoma cell line MDA‐MB231, 71% of the amino acid sequence was obtained. In addition to identifying two possible N‐terminal acetylated α‐enolase variants, Asn153Asp and Ile152Asp/Asn153Ile, the tandem mass spectrometric data revealed the presence of a number of process‐induced modifications of α‐enolase such as methionine oxidation and cysteine amidoe‐thylationPart of this work was presented at ICES '97, International Council of Electrophoretic Societies Symposium, held March 23–27, 1997 in Seattle, WA, USA.
Application of free‐flow electrophoresis to the purification of trichosanthin from a crude product of acetone fractional precipitation
Authors: Song Jin‐Fang , Liu Tao , Shen Xin , Wu Gao‐de , Xia Qi‐chang 
Published: May 2005

AbstractThe application of free‐flow electrophoresis (FFE) to the purification of trichosanthin (TCS) from a crude product of acetone fractional precipitation was investigated. An electrophoresis technique, combining field step electrophoresis (FSE) and zone electrophoresis (ZE) to a one‐step procedure, was optimized until a satisfactory purification factor (1.35), high resolution, and purity (>99%) were achieved. Testing several separation buffer systems revealed that a throughput of 14.2 mg/h can be obtained when the very basic TCS (pI 10.1) was dissolved and electrophoresed in a phosphate buffer system of pH 4. The purity of electrophoresed trichosanthin was proved by a variety of analytical methods, such as sodium dodecyl sulfate (SDS)‐gel electrophoresis, capillary isoelectric focusing (CIEF), and sequencing of N‐ and C‐termini. The high purity and large throughput achieved at low cost by using FFE indicates that this method can be employed for TCS purification.
Isolation of peroxisome subpopulations from rat liver by means of immune free‐flow electrophoresis
Authors: Völkl Alfred , Mohr Heribert , Weber Gerhard , Dariush Fahimi H. 
Published: May 2005

AbstractImmune free‐flow electrophoresis (IFFE) has been applied to the separation of peroxisomes (PO). IFFE is a modification of antigen‐specific electrophoretic cell separation (ASECS), and combines the advantages of electrophoretic separation with the high selectivity of an immune reaction. It differs from the latter in the pH of the electrophoresis buffer, which was shifted from the physiological range (ASECS) to the pI of IgG molecules (pH ∼ 8.0), thus further decreasing the mobility produced by the binding of a specific antibody. This enhances the mobility differences between IgG‐coupled particles and those nondecorated, with resultant improved separation. We have now succeeded in isolating different subpopulations of PO by applying IFFE to heavy, light, and post‐mitochondrial fractions separated by differential centrifugation of a rat liver homogenate. The obtained PO subfractions differed in their composition of matrix and membrane proteins, as revealed by immunoblotting. This indicates that they indeed represent distinct subpopulations of rat hepatic PO.
High performance density gradient electrophoresis of subcellular organelles, protein complexes and proteins
Authors: Tulp Abraham , Verwoerd Desirée , Benham Adam , Jalink Kees , Sier Cees , Neefies Jacques 
Published: May 2005

AbstractA density gradient electrophoresis (DGE) apparatus (2.2 Ø, 14 cm) was constructed for the rapid separation of milligram quantities of proteins. By using binary buffers according to Bier (Electrophoresis 1993, 14, 1011–1018) proteins were rate‐zonally separated in less than 60 min. Acidic proteins were separated in a pH 8.6, 56 μS/cm buffer, and basic proteins in a pH 5.4, 76 μS/cm buffer. Thus the A (pI 5.15) and B (pI 5.30) forms of β‐lactoglobulin as well as the sialylated glycoforms of apotransferrin were well separated at pH 8.6. The isoforms of myoglobin (pI 6.9 and 7.35, respectively), RNAse A (pI 9.45) and cytochrome c (pI 10.0) and lysozyme (pI 11) were separated at pH 5.4 within 80 min. On a 7 cm DGE column, subcellular organelles derived from HeLa cells were separated in standard electrophoresis buffer (655 μS/cm) for 90 min at 10 mA. Using a new low conductivity buffer (193 μS/cm) 20 min was sufficient to separate late endosomes, lysosomes, endoplasmic reticulum, early endosomes, plasma membrane, clathrin‐coated pits, proteasomes, and clathrincoated vesicles within a single run directly from a postnuclear supernatant.
Transcriptional regulation of mammalian cytochrome c oxidase genes
Authors: Grossman Lawrence I. , Seelan R. Sathiagana , Jaradat Saied A. 
Published: May 2005

AbstractThe cytochrome c oxidase (COX) holoenzyme is a 13‐subunit complex that carries out the terminal step in the electron transport chain. Three of the subunits, which contain the electron transfer function, are coded by mitochondrial DNA and the other ten subunits by nuclear DNA. Since the holoenzyme contains equivalent amounts of each subunit, we and others have examined transcriptional regulation of COX nuclear subunits to explore whether there is a common basis for co‐regulation. Each gene is seen to have a unique pattern of recognition by regulatory factors; although some factors bind to more than one gene, not all COX genes seem to be regulated by the same set of factors. Current information about the COX promoters that have been examined is summarized, and the relation of promoter regulation to coordinate gene expression is discussed.
Bipolar clamping improves the sensitivity of mutation detection by temperature gradient gel electrophoresis
Authors: Gille Christoph , Gille Andreas , Booms Patrick , Robinson Peter N. , Nürnberg Peter 
Published: May 2005

AbstractTemperature gradient gel electrophoresis (TGGE) is a rapid and sensitive screening method for point mutations and other small DNA alterations. Usually a polymerase chain reaction (PCR)‐product of 150 to 500 bp that has been clamped at one end by a psoralen molecule or a “GC‐clamp” is tested for abnormal melting characteristics by electrophoresis in a temperature gradient. Under optimal conditions, a heterozygous mutation within the fragment is detected through the presence of three additional bands in the TGGE gel, the mutant homoduplex and two heteroduplex bands. However, the ideal pattern of four sharp bands is not always found due to inconsistencies in melting behavior along the sequence of the DNA fragment under study. Some of these fragments show fuzzy bands that may impede or even prevent the detection of a mutation. Here, we describe a method to overcome this problem by utilizing one psoralen clamp at each end of the PCR product. Using TGGE assays established for exons 16, 17, and 18 of the NF1 gene and for exon 14 of the FBN1 gene as examples, we show that bipolar clamping may transform blurred bands into sharp ones and may visualize mutations that could not be detected by conventional single‐sided clamping.
Single‐strand conformation polymorphism (SSCP)‐based mutation scanning approaches to fingerprint sequence variation in ribosomal DNA of ascaridoid nematodes
Authors: Zhu Xing Quan , Gasser Robin B. 
Published: May 2005

AbstractIn this study, we assessed single‐strand conformation polymorphism (SSCP)‐based approaches for their capacity to fingerprint sequence variation in ribosomal DNA (rDNA) of ascaridoid nematodes of veterinary and/or human health significance. The second internal transcribed spacer region (ITS‐2) of rDNA was utilised as the target region because it is known to provide species‐specific markers for this group of parasites. ITS‐2 was amplified by PCR from genomic DNA derived from individual parasites and subjected to analysis. Direct SSCP analysis of amplicons from seven taxa (Toxocara vitulorum, Toxocara cati, Toxocara canis, Toxascaris leonina, Baylisascaris procyonis, Ascaris suum and Parascaris equorum) showed that the single‐strand (ss) ITS‐2 patterns produced allowed their unequivocal identification to species. While no variation in SSCP patterns was detected in the ITS‐2 within four species for which multiple samples were available, the method allowed the direct display of four distinct sequence types of ITS‐2 among individual worms of T. cati. Comparison of SSCP/sequencing with the methods of dideoxy fingerprinting (ddF) and restriction endonuclease fingerprinting (REF) revealed that also ddF allowed the definition of the four sequence types, whereas REF displayed three of four. The findings indicate the usefulness of the SSCP‐based approaches for the identification of ascaridoid nematodes to species, the direct display of sequence variation in rDNA and the detection of population variation. The ability to fingerprint microheterogeneity in ITS‐2 rDNA using such approaches also has implications for studying fundamental aspects relating to mutational change in rDNA.
Diploid Musa acuminata genetic diversity assayed with sequence‐tagged microsatellite sites
Authors: Grapin Agnès , Noyer Jean‐Louis , Carreel Françoise , Dambier Dominique , Baurens Franc‐Christophe , Lanaud Claire , Lagoda Pierre J. L. 
Published: May 2005

AbstractThe sequence‐tagged microsatellite site (STMS) discrimination potential was explored using nine microsatellite primer pairs. STMS polymorphism was assayed by nonradioactive urea‐polyacrylamide gel electrophoresis. Genetic relationships were examined among 59 genotypes of wild or cultivated accessions of diploid Musa acuminata. The organization of the subspecies was confirmed and some clone relationships were clarified.
Preconcentration and separation of antisense oligonucleotides by on‐column isotachophoresis and capillary electrophoresis in polymer‐filled capillaries
Authors: Barmé Iris , Bruin Gerard J. M. , Paulus Aran , Ehrat Markus 
Published: May 2005

AbstractSmall, single‐stranded, chemically modified oligonucleotides, complementary to a specific gene section, commonly referred to as antisense compounds, are being investigated as potential therapeutic drugs. A number of modified oligonucleotides, in particular phosphorothioates, are in clinical development. Shorter fragments are found as metabolic products. Isotachophoresis (ITP) allows the introduction of large, diluted sample plugs into the separation capillary. In this work, ITP and capillary electrophoresis (CE) in polymer solutions were successfully coupled in a single capillary in a commercial instrument to increase sensitivity with UV detection and to shorten the time for sample pretreatment. It was shown that ITP‐CE can be used as a preconcentration and clean‐up method for phosphodiester‐ and phosphorothioate‐containing samples. Up to 3 μL sample could be injected into the capillary without significantly disturbing the separation performance. ITP‐CE of phosphodiesters directly out of salt‐ and protein‐containing samples could be demonstrated. For phosphorothioates in serum samples an additional sample clean‐up was necessary, due to oligonucleotide‐protein binding. An optimized replaceable polymer solution was developed to increase the separation performance for heterogeneous phosphorothioates. A dextran‐based sieving medium showed a good separation performance in ITP‐CE of phosphorothioates. A concentration detection limit of 8.10−9 mol/L for the 20‐mer phosphorothioate ISIS5132, isolated from rat serum, was found.
Identifying human platelet glycoproteins IIb and IIIa by capillary electrophoresis
Authors: Vecchione Gennaro , Margaglione Maurizio , Grandone Elvira , Colaizzo Donatella , Cappucci Giuseppe , Giuliani Nicola , d'Addedda Marina , D'Andrea Giovanna , Nobile Michele , Amoriello Aldo , Minno Giovanni Di 
Published: May 2005

AbstractGlanzmann thrombasthenia (GT) is an inherited hemorrhagic defect due to a failure of the platelet membrane glycoprotein (GP) IIb–IIIa complex. Capillary electrophoresis (CE) analysis of solubilized platelet membranes from normal individuals showed the presence of two peaks with a migration time of 27 and 29 min, respectively. An excellent run‐to‐run and day‐to‐day reproducibility of the technique (< 1% variation of the retention time) was documented. Using an automated Ferguson method, the apparent molecular masses were 100.0 kDa and 138.5 kDa, respectively. Immunoprecipitation with monoclonal antibodies anti‐GP IIIa (B59.2.1) and anti‐IIb (61.9.1.3) showed the two peaks as IIIa and IIb, respectively. Electropherograms of a GT young man showed the lack of both peaks. Less than 50% of each peak was present in his parents. Polyacrylamide gel electrophoresis (PAGE), immunoblotting, and flow cytometry analyses showed that GP IIb and IIIa were undetectable in the platelet membranes from the propositus, half of the normal amount being present in both parents. These findings indicate CE to be a rapid, sensitive and reliable tool to investigate patients with abnormalities of the GP IIb–IIIa complex.
Capillary electrophoresis for simultaneous quantification of human proinsulin, insulin and intermediate forms
Authors: Arcelloni Cinzia , Falqui Luca , Martinenghi Sabina , Pontiroli Antonio E. , Paroni Rita 
Published: May 2005

AbstractCapillary electrophoresis (CE) for the simultaneous and precise quantification of human insulin (hI), proinsulin (hPI) and intermediate forms (des 31, 32; split 65–66 and des 64, 65), released in culture media by engineered cells, is described. Analytical conditions for standard proteins were optimized using a bare silica capillary (20 cm × 50 μm internal diameter). Proteins were monitored at 200 nm and separated at constant voltage. Culture supernatants (12–24 mL) were purified on Sep‐Pak Vac C18 cartridges, recovered in 1 mL of acetonitrile:trifluoracetic acid mixture (60:40, v:v), concentrated, ultrafiltered and injected into CE. Protein recovery was 85 ± 14% (n = 5, mean ± standard deviation) with a sensitivity limit of 0.5 nmol/L in the culture media, corresponding to 2 fmol injected in 22 nL. Using the CE method, it was possible to detect and quantify, with precision and accuracy, the release of hPI, hI and intermediate forms directly in the cell culture media, and to compare the proteic pattern released from engineered cells transduced with different hPI gene constructs.
Migration of single‐stranded DNA in polyacrylamide gels during electrophoresis
Authors: Pluen Alain , Tinland Bernard , Sturm Jean , Weill Gilbert 
Published: May 2005

AbstractBy using fluorescence recovery after photobleaching (FRAP) and electric birefringence, the migration of single‐stranded DNA in polyacrylamide gels and orientation as a response to an electric pulse were investigated. Electrophoretic mobility is in good agreement with the model of biased reptation including fluctuations. The determination of the electrophoretic mobility in solution, μo, allows an estimation of the gel pore diameter seen by the molecule. As previously observed for double‐stranded DNA, the electric birefringence results from two processes: the alignment of the molecule along the electric field and the elongation of the primitive path in the gel, for long single‐stranded DNA (> 2000 bases). The combination of results obtained with the two techniques allows us to propose experimental conditions to improve the separation of single‐stranded DNA with pulsed field techniques.
A nonradioactive method for sizing most D11S533 alleles discretely
Authors: Gaudin Daniela , Schmidtke Jörg 
Published: May 2005

AbstractA method is described which allows accurate nonradioactive sizing of most D11S533 alleles, which are characterized by a highly variable number of 6 bp units. In a small population sample of German origin the heterozygosity of the locus was estimated to be 0.933.
Discontinuities of pH at zone boundaries in isotachophoretic systems with poorly buffering leading electrolytes
Authors: Reijenga Jetse , Kašička Václav 
Published: May 2005

AbstractDiscontinuities of pH at zone boundaries of strong ion sample zones in isotachophoretic systems with poorly buffering leading electrolytes have been discovered by dynamic computer simulations of isotachophoretic separations with uncommon electrolyte systems. When using a salt solution containing a strong cation (anion) as leading ion and a weak anion (cation) as counterion for the isotachophoretic separation of strong cations (anions), severe pH discontinuities are present at zone boundaries of these cations (anions). The magnitude of these pH discontinuities has been investigated as a function of several parameters, namely counter ion/leading ion concentration ratio of the leading electrolyte, pK values, and mobilities of sample ions and counterion.
The band areas of proteins determined by fluorescent scanning in the commercial automated gel electrophoresis apparatus
Authors: Zakharov Sergey F. , Kwok Steven H. , Sokoloff Helen , Chang Huan‐Tsung , Radko Sergey P. , Chrambach Andreas 
Published: May 2005

AbstractAn automated gel electrophoresis apparatus, recently available commercially, allows one to follow the band during electrophoresis in real time, and lends itself therefore to an evaluation of bandwidth as a function of migration time (the dispersion coefficient), resolution and band shape. These determinations assume the constancy of band area with migration time and at various gel concentrations. The purpose of the present study was to verify these assumptions. Representative proteins and sodium dodecyl sulfate (SDS)‐proteins, either natively fluorescent or fluorescein carboxylate labeled, were found to exhibit band areas which approach constancy as a function of migration time in both agarose and polyacrylamide gel electrophoresis, provided that (i) the protein concentration under the band was low enough to obviate self‐quenching of fluorescence; (ii) the separation of the protein of interest from contaminants had progressed sufficiently during the time at which band areas were measured; (iii) the baseline under the peak was sufficiently well defined. However, band areas decrease with increasing gel concentration. Protein peaks exhibited leading and trailing tails. The ratio of the combined tail area to total area appeared to be near‐constant at varying migration times. However, that ratio increases with increasing gel concentration. The tail area does not appear to be an artifact of fluorometric detection since it is reproduced upon fluorimetric analysis of the protein eluted from gel slices after electrophoresis. However, it may be due to photochemical destruction under the conditions of repetitive fluorometric peak detection.
Immunoblotting of multiple antigenic peptides
Authors: Kurien Biji T. , Jackson Kenneth , Scofield R. Hal 
Published: May 2005

AbstractMultiple antigenic peptides (MAPs) can be efficiently separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to a nitrocellulose membrane for subsequent use in immunoblot (IgG and IgM). MAPs can be stained by Coomassie and silver on SDS‐PAGE as well as by Fast Green on an immunoblot. Affinity immunoblotting for analysis of antibody clonotype distribution has also been carried out using MAPs. High performance liquid chromatography purification of the MAPs is mainly responsible for their migration as sharp bands in SDS‐PAGE and immunoblotting.
Sensitivity models in simulation of capillary zone electrophoresis: The effect of buffer power and initial buffer concentration
Authors: Blanco Stéphane , Clifton Michael J. , Joly Jean‐Louis 
Published: May 2005

AbstractSeparation of proteins by capillary zone electrophoresis in an amphoteric medium is investigated by numerical simulation. We explain how sensitivity models can be used to gain further information about this process. The sensitivity models that are derived are used to study the sensitivity of the separation to the specific buffer capacity of the amphoteric molecule that serves as a buffer. The effect of changing the overall concentration of this ampholyte is also examined and it is shown that a change in its concentration is equivalent to a change in its buffer capacity.