bluuebunny/crossref_metadata_2025_split huggingface dataset:
High sensitivity identification of proteins by electrospray ionization tandem mass spectrometry: Initial com‐ parison between an ion trap mass spectrometer and a triple quadrupole mass spectrometer
Authors: Figeys Daniel , Aebersold Ruedi 
Published: May 2005

AbstractRecently, we have shown that a solid‐phase‐microextraction/capillary electrophoresis device coupled to an electrospray ionization triple quadrupole mass spectrometer through a microelectrospray interface represents a powerful analytical system for the rapid, conclusive and sensitive identification of proteins separated by gel electrophoresis. Here we report on the successful coupling of the same device to an electrospray ionization ion trap mass spectrometer and on the comparative evaluation of the performance of the triple quadrupole and ion‐trap‐based systems. In the ion trap mass spectrometer‐based system, using a tryptic digest of a calibrated bovine serum albumin sample, we achieved limits of detection in the single mass spectrometry (MS) and tandem MS mode, respectively, of 400 amol (if 20 μL of solution at a concentration of 20 amol/μL was applied). The system was also successfully used to identify six yeast proteins isolated from a single analytical two‐dimensional polyacrylamide gel. For the detection of unfragmented peptide ions both systems showed comparable sensitivity, whereas the ion‐trap‐based system showed superior performance with fragment ion spectra.
Comparison of in‐gel and on‐membrane digestion methods at low to sub‐pmol level for subsequent peptide and fragment‐ion mass analysis using matrix‐assisted laser‐desorption/ionization mass spectrometry
Authors: Courchesne Paul L. , Luethy Roland , Patterson Scott D. 
Published: May 2005

AbstractThe success of the mass spectrometric‐based approaches for the identification of gel‐separated proteins relies upon recovery of peptides, without high levels of ionization‐suppressing contaminants, in solvents compatible with the mass spectrometer being employed. We sought to determine whether in‐gel or on‐membrane digestion provided a significant advantage when low to sub‐pmol quantities of gel‐separated proteins were analyzed by matrix‐assisted laser‐desorption/ionization mass spectrometry (MALDI‐MS) with respect to the number and size of released peptides. Serial dilutions of five standard proteins of Mr 17 000 to 97 000 (from 16 pmol to 125 fmol) were electrophoresed and subjected to in‐gel digestion (using a microcolumn clean‐up protocol, Courchesne, P.L. and Patterson, S. D., BioTechniques, 1997, in press) or on‐membrane digestion following blotting to the PVDF‐based membranes, Immobilon‐P and Immobilon‐CD. Peptide maps were able to be obtained for all proteins at the detection limit of each method (Immobilon‐P and Immobilon‐CD, 0.5 pmol; and in‐gel, 125 fmol), and searches of Swiss‐Prot or a non‐redundant database (>193 000 entries) successfully identified all of the proteins, except β‐casein. Fragment‐ion spectra using a curved‐filed reflector MALDI‐MS were obtained from more than one peptide per protein at loads down to 250 fmol (except β‐casein). Using the uninterpreted data, a search of the nonredundant database and a six‐way translation of GenBank dbEST (> 2 208 000 entries total) was able to identify myoglobin, carbonic anhydrase II, and phosphorylase b.
Rapid mass spectrometric identification of proteins from two‐dimensional polyacrylamide gels after in gel proteolytic digestion
Authors: Li Guang , Waltham Mark , Anderson N. Leigh , Unsworth Edward , Treston Anthony , Weinstein John N. 
Published: May 2005

AbstractWe report a rapid method for identifying proteins resolved by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) using matrix‐assisted laser desorption ionization‐mass spectrometry (MALDI‐MS). In‐gel digestion was performed in a way such that the volume ratio of trypsin solution to gel plug was quantitatively controlled to promote reproducible digestion and to maximize the digestion yield. To make the digestion samples more compatible with MALDI‐MS, the volatile salt ammonium bicarbonate in the digestion buffer was largely removed prior to peptide extraction. Samples of mixed tryptic peptides from in‐gel digestion were used without purification to obtain molecular weights by MALDI‐MS with α‐cyano, 4‐hydroxy‐cinnamic acid as the matrix. Modifications of MALDI sample loading procedures improved the detetion sensitivity by one half to one order of magnitude. The peptide mass peaks in MALDI‐MS spectra were distinguished from those of impurities by using several types of controls, and masses were corrected by using trypsin autodigestion fragments as internal calibration standards. Two different peptidematching computer programs were used to interrogate sequence databases and identify proteins. Identification was enhanced by generation of orthogonal data sets (by using different proteases) and by including experimental values of isoelectric point (pI) and molecular weight to exclude false entries in the candidate lists. Approximately 1% of the material from a spot was used in each sample loading, and nine protein spots from rat liver 2‐D PAGE gels were identified correctly, as judged by comparison with identification results previously obtained from Edman sequencing. A previously identified low‐abundance spot was not identified by MALDI‐MS, presumably because there was insufficient material in a single gel. The sample handling procedure reported here should permit us to identify many 2‐D PAGE protein spots of medium abundance.
Immobilized pH gradient two‐dimensional gel electrophoresis and mass spectrometric identification of cytokine‐regulated proteins in ME‐180 cervical carcinoma cells
Authors: Matsui Neil M. , Smith Diana M. , Clauser Karl R. , Fichmann Jenny , Andrews Lori E. , Sullivan Christine M. , Burlingame Alma L. , Epstein Lois B. 
Published: May 2005

AbstractTwo‐dimensional (2‐D) polyacrylamide gel electrophoresis combined with mass spectrometry is a powerful combination of technologies that allows high resolution separation of proteins and their rapid identification. Immobilized pH gradient (IPG) first‐dimensional gels have several advantages over carrier ampholyte isoelectric focusing, including a high degree of reproducibility, good protein spot resolution, and a selection of pH range. Here we demonstrate the utility and efficacy of combining IPG 2‐D gel electrophoresis with mass spectrometry to identify interferon‐γ‐ (IFN) and tumor necrosis factor (TNF)‐regulated proteins in ME‐180 cervical carcinoma cells. Three cytokine‐regulated proteins have been identified, using imidazole‐zinc‐stained preparative IPG 2‐D gels and in‐gel tryptic digestion followed by matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometry for determination of peptide masses and sequences: (1) triosephosphate isomerase, a glycolytic pathway enzyme, (2) proteasome subunit C3, which is important in protein degradation, and (3) Ran, a GTP‐binding protein important in cell cycle regulation, protein import into the nucleus, and RNA export from the nucleus.
The Dictyostelium discoideum proteome – the SWISS‐2DPAGE database of the multicellular aggregate (slug)
Authors: Yan Jun X. , Tonella Luisa , Sanchez Jean‐Charles , Wilkins Marc R. , Packer Nicolle H. , Gooley Andrew A. , Hochstrasser Denis F. , Williams Keith L. 
Published: May 2005

AbstractThe cellular slime mold Dictyostelium discoideum is a eukaryotic microorganism which has developmental life stages attractive to the cell and molecular biologist. By displaying the two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) protein map of different developmental stages, the key molecules can be identified and characterised, allowing a detailed understanding of the D. discoideum proteome. Here we describe the preparation of reference gel of the D. discoideum multicellular aggregate, the slug. Proteins were separated by 2‐D PAGE with immobilised pH gradients (pH 3.5–10) in the first dimension and sodium dodecyl sulfate (SDS)‐PAGE in the second dimension. Micropreparative gels were electroblotted onto polyvinylidene difluoride (PVDF) membranes and 150 spots were visualised by amido black staining. Protein spots were excised and 31 were putatively identified by matching their amino acid composition, estimated isoelectric point (pI) and molecular weight (Mr) against the SWISS‐PROT database with the ExPASy AAcompID tool (http://expasy.hcuge.ch/ch2d/aacompi.html). A total of 25 proteins were identified by matching against database entries for D. discoideum, and another six by crossspecies matching against database entries for Saccharomyces cerevisiae proteins. This map will be available in the SWISS‐2DPAGE database.
Large‐scale protein modelling and integration with the SWISS‐PROT and SWISS‐2DPAGE databases: The example of Escherichia coli
Authors: Peitsch Manuel C. , Wilkins Marc R. , Tonella Luisa , Sanchez Jean‐Charles , Appel Ron D. , Hochstrasser Denis F. 
Published: May 2005

AbstractKnowledge‐based molecular modelling of proteins has proven useful in many instances, including the rational design of mutagenesis experiments, but it has generally been limited by the availability of expensive computer hardware and software. To overcome these limitations, we developed the SWISS‐MODEL sever for automated knowledge‐based protein modelling. The SWISS‐MODEL server uses the Brookhaven Protein Data Bank as a source of structural information and automatically generates protein models for sequences which share significant similarities with at least one protein of known three‐dimensional structure. We have now used the software framework of the server to generate large collections of protein models, and established the SWISS‐MODEL Repository, a new database for automatically generated and theoretical protein models. This repository is directly integrated with the SWISS‐PROT and SWISS‐2DPAGE databases through the ExPASy World Wide Web server (URL is http://expasy.hcuge.ch). Here we present an illustration of this process by an application to the Escherichia coli sequences.
Characterization of target antigens from anti‐neutrophil cytoplasmic antibodies in autoimmune hepatitis type‐I
Authors: Kellner Roland , Orth Thomas , Mayet Werner‐Johannes 
Published: May 2005

AbstractThe occurrence of anti‐neutrophil cytoplasmic antibodies (ANCA) has been described in sera of patients with autoimmune hepatitis (AIH). The significance of this finding remains uncertain and the nature of the target antigen(s) has not yet been defined. We studied 32 sera from patients with AIH type‐I and prepared extracts of human neutrophils to identify the target antigen(s). A 43 kDa dominant immunoreactive protein was found and identified as the cytoskeletal component actin. Initial studies to define the antigenic determinants identified three different actin domains.
Computer‐supported analysis (MegaBlot) of allopurinol‐induced changes in the autoantibody repertoires of rats suffering from experimental lens‐induced uveitis
Authors: Grus Franz‐H. , Augustin Albert J. , Zimmermann Christoph W. 
Published: May 2005

AbstractUsing a new quantitative immunoblot technique (MegaBlot and artificial neural network) sera of rats with lens‐induced uveitis (LIU) were tested against Western blots (WB) of sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS‐PAGE) separations of protein fractions from rat lenses. The blots were scanned using digital image analysis and evaluated by multivariate analysis of discriminance and an artificial neural network. Five groups of LIU rats (male Wistar rats, n = 28) were investigated: no treatment, single doses of methylprednisolone (MPR; 7.5 mg/kg body weight, intravenous), allopurinol (AL; 50 mg/kg body weight, intravenous), a combination of both drugs (AL and MPR), and repeated application of AL (ALFR; 50 mg/kg body weight intravenous every 2 weeks during the immunization period and a daily dose of approximately 25 mg/kg body weight, orally). ALFR, MPR, and MPRAL show statistically significant immunological effects in LIU treatment Classification of WBs using both discriminance analysis or neural network resulted in 100% correct assignment; and 82% (discriminance analysis) and 86% (neural network) of WBs were correctly assigned using only unknown blots for classification (not studied before). The MegaBlot technique is able to detect therapeutically induced changes in autoantibody repertoires allowing therapeutic control and a prognostic view of treatment.
Characterization of Chlamydia trachomatis L2‐induced tyrosine‐phosphorylated HeLa cell proteins by two‐dimensional gel electrophoresis
Authors: Birkelund Svend , Bini Luca , Pallini Vitaliano , Sanchez‐Campillo Maria , Liberatori Sabrina , Clausen Johannes D. , Østergaard Søren , Holm Arne , Christiansen Gunna 
Published: May 2005

AbstractChlamydia trachomatis is an obligate intracellular bacteria, inducing its own uptake in nonprofessional phagocytes either by phagocytosis or pinocytosis. We have previously shown that C. trachomatis L2 induces tyrosine phosphorylation of eukaryotic proteins upon their entry by phagocytosis. In this paper we characterize the tyrosine‐phosphorylated proteins by two‐dimensional gel electrophoresis. In immunoblotting with anti‐phosphotyrosine antibodies of C. trachomatis L2‐infected HeLa cells, but not with uninfected cells, two rows of spots were observed with a molecular mass of 69 and 71 kDa and pI from 5.0 to 5.2. In addition, a single spot of 100 kDa and pI 6.2 was observed.
A two‐dimensional electrophoresis database of human breast epithelial cell proteins
Authors: Giometti Carol S. , Williams Katherine , Tollaksen Sandra L. 
Published: May 2005

AbstractAs sequencing of the human genome progresses, attention is turning to when and where specific genes are being expressed and how that expression is regulated. The human breast, with the highly specific, but transient, function of milk production (lactation), exemplifies human gene regulation. The molecular mechanisms for the dramatic structural and functional changes involved in shifting from lactation‐capable to lactation‐incapable tissue are poorly understood, as are the mechanisms that result in deviation from normal breast cell growth into different types of breast neoplasms. We are using quantitative two‐dimensional electrophoresis (2‐DE) to determine which proteins are present in different types of human breast cells (milk‐producing and ‐nonproducing, estrogen‐receptor‐positive and ‐negative, normal and malignant) and which proteins change in abundance in response to stimuli that trigger cell differentiation, growth, or death. A composite map of proteins found in human breast cells is being generated and used as an index of human genes that are differentially expressed, both qualitatively and quantitatively. Proteins found in 15 different types of human breast cells, two from healthy tissue (from milk and reduction mammoplasty tissue) and 13 from tumor tissue, are now included in the composite map. Copies of the human breast epithelial cell protein map are available on the World Wide Web (URL: www.anl.gov/CMB/PMG/projects/index_hbreast.html) with links to quantitative data and identifications for proteins found to be differentially expressed in these epithelial cells. Links to the Swiss‐Prot and enzyme metabolic pathway databases are also provided. The World Wide Web presentation is designed to allow public access to the available 2‐DE data together with logical connections to databases providing genome‐related information.
Renal cell carcinoma and normal kidney protein expression
Authors: Sarto Cecilia , Marocchi Alessandro , Sanchez Jean‐Charles , Giannone Daniela , Frutiger Séverine , Golaz Olivier , Wilkins Marc R. , Doro Giancarlo , Cappellano Francesco , Hughes Graham , Hochstrasser Denis F. , Mocarelli Paolo 
Published: May 2005

AbstractRenal cell carcinoma (RCC), a human kidney cancer from the proximal tubular epithelium, accounts for about 3% of adult malignancies. Molecular and cytogenetic analysis have highlighted deletions, translocations, or loss of heterozygosity in the 3p21–p26, a putative RCC locus, as well as in 6q, 8p, 9pq, and 14pq. Studies on phenotypic expression of human kidney tissue and on post‐translational modifications in RCC have not yet provided a marker for early renal cell carcinoma diagnosis. Current dignostic methods do not help to detect the tumor before advanced stages. We therefore used two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) to study normal and tumor kidney tissues in ten patients suffering from RCC. A human kidney protein map in the SWISS‐2DPAGE database accessible through the ExPASy WWW Molecular Biology Server was established. Of 2789 separated polypeptides, 43 were identified by gel comparison, amino acid analysis, N‐terminal sequencing, and/or immunodetection. The comparison between normal and tumor kidney tissues showed four polypeptides to be absent in RCC. One of them was identified as ubiquinol cytochrome c reductase (UQCR), whose locus has elsewhere been tentatively assigned to chromosome 19p12 or chromosome 22. A second polypeptide was identified as mitochondrial NADH‐ubiquinone oxido reductase complex I whose locus is located on chromosome 18p 11.2 and chromosome 19q 13.3. These result suggest that the lack of UQCR and of mitochondrial NADH‐ubiquinone oxidoreductase complex I expression in RCC may be caused by unknown deletions, or by changes in gene transcription or translation. It might indicate that mitochondrial disfunction plays a major role in RCC genesis or evolution.
A two‐dimensional gel database of human colon carcinoma proteins
Authors: Ji Hong , Reid Gavin E. , Moritz Robert L. , Eddes James S. , Burgess Antony W. , Simpson Richard J. 
Published: May 2005

AbstractThe master two‐dimensional gel database of human colon carcinoma cells currently lists cellular proteins from normal crypts and the colorectal cancer cell lines LIM 1863, LIM 1215 and LIM 1899 (Ward et al., Electrophoresis 1990, 11, 883–891; Ji et al., Electrophoresis 1994, 15, 391–405). Updated two‐dimensional electrophoretic (2‐DE) maps of cellular proteins from LIM 1215 cells, acquired under both nonreducing and reducing conditions, are presented. Fifteen cellular proteins are identified in the reducing 2‐DE gel map, and seven in the nonreducing gel map, along with a tabular listing of their Mr/pI loci and mode of identification. We also include our mass spectrometric based procedures for identifying 2‐DE resolved proteins. This procedure relies on a combination of capillary column (0.10–0.32 mm internal diameter) reversed‐phase HPLC peptide mapping of in‐gel digested proteins, peptide mass fingerprinting, sequence analysis by either collision‐induced dissociation or post‐source‐decay fragmentation, and protein identification using available database search algorithms. These data, and descriptions of the micro‐techniques employed in this laboratory for identifying 2‐DE resolved proteins can be accessed via the internet URL: http://www.ludwig.edu.au.
Electrophoretic analysis of the “cross‐class” interaction between novel inhibitory serpin, squamous cell carcinoma antigen‐1 and cysteine proteinases
Authors: Nawata Shugo , Nakamura Kazuyuki , Tanaka Tatehiko , Numa Fumitaka , Suminami Yoshinori , Tsunaga Nagato , Kakegawa Hisao , Katunuma Nobuhiko , Kato Hiroshi 
Published: May 2005

AbstractWe investigated the “cross‐class” interaction between cysteine proteinases and a novel inhibitory serpin, recombinant squamous cell carcinoma (rSCC) antigen‐1, which inhibits a serine proteinase, chymotrypsin. rSCC antigen‐1 inhibited the cysteine proteinases, papain, papaya proteinase IV and cathepsin L. Interestingly, although rSCC antigen‐1 formed sodium dodecyl sulfate (SDS)‐ and heat‐stable complexes with chymotrypsin, rSCC antigen‐1 gave the 40 kDa fragment and small molecular mass peptide by incubation with papain without forming an SDS‐ and heat‐stable complex. The cleavage was observed between the Gly353‐Ser354 bond, indicating that rSCC antigen‐1 interacts with cysteine proteinases not at the predicted reactive site P1‐P1′ portion (Ser354‐Ser355), but at the Gly353‐Ser354 of the P2‐P1 portion. These findings promote understanding of the “suicide inhibition” mechanism of SCC antigen‐1 against cysteine proteinases.
Separation of enantiomers of drugs by capillary electrophoresis. Part 4: Hydroxyprpyl‐gammacyclodextrin as chiral solvating agent
Authors: Koppenhoefer Bernhard , Epperlein Ulrich , Xiaofeng Zhu , Bingcheng Lin 
Published: May 2005

AbstractIn an extended chiral drug screening program, enantioseparation of 86 racemic drugs was tested with hydroxypropyl‐γ‐cyclodextrin as chiral solvating agent (CSA). A total of 30 drugs out of 86 could be resolved in this straightforward approach. The number of experiments performed under identical conditions allows a statistical treatment of the data. The enantioseparation of the analytes is correlated with their interaction strength with the CSA. Hence, the concentration of the CSA is a crucial parameter for optimization of the enantioseparation, as shown by a subset of 23 examples.
Chiral interactions in capillary zone electrophoresis: Computer simulation and comparison with experiment
Authors: Ingelse Benno A. , Sarmini Karim , Reijenga Jetse C. , Kenndler Ernst , Everaerts Frans M. 
Published: May 2005

AbstractChiral interaction in capillary electrophoresis can be modeled using pK values, mobilities of analytes, and their formation constants with the chiral selector. An existing steady‐state simulation program for CE (HPCESIM) was recently extended with a chiral submenu involving the chiral parameters listed above. These were experimentally determined in both our laboratories for mandelic acid and terbutaline using hydroxypropylated β‐cyclodextrin as chiral selector. A comparison was made between both sets of parameters and between experimental electropherograms and those obtained from simulation. Error analysis of the results indicate the sensitivity of the obtained results.
Determination of the enantiomeric purity of N‐propionyl‐6,7‐dimethoxy‐2‐aminotetralin by cyclodextrin‐modified micellar electrokinetic chromatography
Authors: Castelnovo Pierfrancesco , Albanesi Carlo 
Published: May 2005

AbstractN‐Propionyl‐6,7‐dimethoxy‐2‐aminotetralin (Z12231A), a useful intermediate in the synthesis of dopaminergic agonists, was resolved into its enantiomers by high‐performance capillary electrophoresis. Cyclodextrin‐modified micellar electrokinetic chromatography was employed with a combination of chiral selectors both in the aqueous phase (a hydroxyalkyl‐β‐cyclodextrin) and in the micellar phase (sodium taurodeoxycholate). The absolute amount of the two chiral selectors as well as their ratio were found to be the most critical parameters in order to optimize separation. A resolution factor >3.5 was obtained, allowing a high amount of solute to be loaded in order to improve the detection limit. The reproducibility of the assay was also evaluated.
Enantiomer separation of denopamine by capillary electrophoresis with charged and uncharged cyclodextrins
Authors: Ishibuchi Kiyotaka , Izumoto Shin‐ichi , Nishi Hiroyuki , Sato Tadashi 
Published: May 2005

AbstractDirect separation of enantiomers of denopamine was investigated by capillary electrophoresis employing charged and uncharged cyclodextrin (CD) derivatives. Uncharged β‐type CDs, having hydrophobic groups, were essential for the enantioseparation of denopamine; of these, especially dimethyl‐β‐CD was effective. Among charged CDs, γ‐type as well as β‐type CDs were found effective for the enantioseparation of denopamine. Reversal of migration order of R‐form (active) and S‐form enantiomers was investigated by using two types of coated capillaries: (i) an amine capillary with an inner wall coated with dimethylamino groups, and (ii) a polyacrylamide‐coated capillary. Manipulation of migration order could be easily performed by selecting suitable capillaries, buffer pH, and CDs.