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External tube drainage or omentoplasty in the management of residual hepatic hydatid cyst cavity: a prospective randomized controlled study

Wani, Ajaz A.; Rashid, Arshad; Laharwal, Asim R.; Kakroo, Showkat M.; Abbas, M.; Chalkoo, Manzoor A.
Fonte: German Medical Science GMS Publishing House Publicador: German Medical Science GMS Publishing House
Tipo: Artigo de Revista Científica
Publicado em 29/07/2013 Português
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Background: Surgical procedures advocated for management of residual hepatic hydatid cyst cavity have been a subject of controversy. The aim of this study was to compare omentoplasty (OP) and external tube drainage (ETD).

Spatially-Directed Protein Identification from Tissue Sections by Top-Down LC-MS/MS with Electron Transfer Dissociation

Schey, Kevin L.; Anderson, David M.; Rose, Kristie L.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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MALDI imaging mass spectrometry (MALDI-IMS) has become a powerful tool for localizing both small molecules and intact proteins in a wide variety of tissue samples in both normal and diseased states. Identification of imaged signals in MALDI-IMS remains a bottleneck in the analysis and limits the interpretation of underlying biology of tissue specimens. In this work, spatially-directed tissue microextraction of intact proteins followed by LC-MS/MS with electron transfer dissociation (ETD) was used to identify proteins from specific locations in three tissue types; ocular lens, brain, and kidney. Detection limits were such that a 1 microliter extraction volume was sufficient to deliver proteins to the LC-MS/MS instrumentation with sufficient sensitivity to detect 50–100 proteins in a single experiment. Additionally, multiple modified proteins were identified; including truncated lens proteins that would be difficult to assign to an imaged mass using a bottom-up approach. Protein separation and identification are expected to improve with advances in intact protein fractionation/chromatography and advances in interpretation algorithms leading to increased depth of proteome coverage from distinct tissue locations.

Discovery and Confirmation of O-GlcNAcylated Proteins in Rat Liver Mitochondria by Combination of Mass Spectrometry and Immunological Methods

Cao, Weiqian; Cao, Jing; Huang, Jiangming; Yao, Jun; Yan, Guoquan; Xu, Haoqi; Yang, Pengyuan
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 02/10/2013 Português
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O-linked β-N-acetylglucosamine (O-GlcNAc) is an important post-translational modification (PTM) consisting of a single N-acetylglucosamine moiety attached via an O-β-glycosidic linkage to serine and threonine residues. Glycosylation with O-GlcNAc occurs on myriad nuclear and cytosolic proteins from almost all functional classes. However, with respect to O-GlcNAcylated proteins special in mitochondria, little attention has been paid. In this study, we combined mass spectrometry and immunological methods to perform global exploration of O-GlcNAcylated proteins specific in mitochondria of rat liver. First, highly purified mitochondrial proteins were obviously shown to be O-GlcNAcylated by immunoblot profiling. Then, β-elimination followed by Michael Addition with Dithiothreitol (BEMAD) treatment and LC-MS/MS were performed to enrich and identify O-GlcNAcylated mitochondrial proteins, resulting in an unambiguous assignment of 14 O-GlcNAcylation sites, mapping to 11 O-GlcNAcylated proteins. Furthermore, the identified O-GlcNAcylated mitochondrial proteins were fully validated by both electron transfer dissociation tandem mass spectrometry (ETD/MS/MS) and western blot. Thus, for the first time, our study definitely not only identified but also validated that some mitochondrial proteins in rat liver are O-GlcNAcylated. Interestingly...

Cation Recombination Energy/Coulomb Repulsion Effects in ETD/ECD as Revealed by Variation of Charge per Residue at Fixed Total Charge

Mentinova, Marija; Crizer, David M.; Baba, Takashi; McGee, William M.; Glish, Gary L.; McLuckey, Scott A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Automated Hydrogen/Deuterium Exchange Electron Transfer Dissociation High Resolution Mass Spectrometry Measured at Single-Amide Resolution

Landgraf, Rachelle R.; Chalmers, Michael J.; Griffin, Patrick R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a well established method for the measurement of solution-phase deuterium incorporation into proteins, which can provide insight into protein conformational mobility. However, most HDX measurements are constrained to regions of the protein where pepsin proteolysis allows detection at peptide resolution. Recently, single-amide resolution deuterium incorporation has been achieved by limiting gas-phase scrambling in the mass spectrometer. This was accomplished by employing a combination of soft ionization and desolvation conditions coupled with the radical-driven fragmentation technique electron transfer dissociation (ETD). Here, a hybrid LTQ-Orbitrap XL is systematically evaluated for its utility in providing single-amide deuterium incorporation for differential HDX analysis of a nuclear receptor upon binding small molecule ligands. We are able to show that instrumental parameters can be optimized to minimize scrambling and can be incorporated into an established and fully automated HDX platform making differential single-amide HDX possible for bottom-up analysis of complex systems. We have applied this system to determine differential single amide resolution HDX data for the peroxizome proliferator activated receptor bound with two ligands of interest.

Interactions Affected by Arginine Methylation in the Yeast Protein–Protein Interaction Network*

Erce, Melissa A.; Abeygunawardena, Dhanushi; Low, Jason K. K.; Hart-Smith, Gene; Wilkins, Marc R.
Fonte: The American Society for Biochemistry and Molecular Biology Publicador: The American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Protein–protein interactions can be modulated by the methylation of arginine residues. As a means of testing this, we recently described a conditional two-hybrid system, based on the bacterial adenylate cyclase (BACTH) system. Here, we have used this conditional two-hybrid system to explore the effect of arginine methylation in modulating protein–protein interactions in a subset of the Saccharomyces cerevisiae arginine methylproteome network. Interactions between the yeast hub protein Npl3 and yeast proteins Air2, Ded1, Gbp2, Snp1, and Yra1 were first validated in the absence of methylation. The major yeast arginine methyltransferase Hmt1 was subsequently included in the conditional two-hybrid assay, initially to determine the degree of methylation that occurs. Proteins Snp1 and Yra1 were confirmed as Hmt1 substrates, with five and two novel arginine methylation sites mapped by ETD LC-MS/MS on these proteins, respectively. Proteins Ded1 and Gbp2, previously predicted but not confirmed as substrates of Hmt1, were also found to be methylated with five and seven sites mapped respectively. Air2 was found to be a novel substrate of Hmt1 with two sites mapped. Finally, we investigated the interactions of Npl3 with the five interaction partners in the presence of active Hmt1 and in the presence of Hmt1 with a G68R inactivation mutation. We found that the interaction between Npl3 and Air2...

The Histone Code of Toxoplasma gondii Comprises Conserved and Unique Posttranslational Modifications

Nardelli, Sheila C.; Che, Fa-Yun; Silmon de Monerri, Natalie C.; Xiao, Hui; Nieves, Edward; Madrid-Aliste, Carlos; Angel, Sergio O.; Sullivan, William J.; Angeletti, Ruth H.; Kim, Kami; Weiss, Louis M.
Fonte: American Society of Microbiology Publicador: American Society of Microbiology
Tipo: Artigo de Revista Científica
Publicado em 10/12/2013 Português
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Epigenetic gene regulation has emerged as a major mechanism for gene regulation in all eukaryotes. Histones are small, basic proteins that constitute the major protein component of chromatin, and posttranslational modifications (PTM) of histones are essential for epigenetic gene regulation. The different combinations of histone PTM form the histone code for an organism, marking functional units of chromatin that recruit macromolecular complexes that govern chromatin structure and regulate gene expression. To characterize the repertoire of Toxoplasma gondii histone PTM, we enriched histones using standard acid extraction protocols and analyzed them with several complementary middle-down and bottom-up proteomic approaches with the high-resolution Orbitrap mass spectrometer using collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and/or electron transfer dissociation (ETD) fragmentation. We identified 249 peptides with unique combinations of PTM that comprise the T. gondii histone code. T. gondii histones share a high degree of sequence conservation with human histones, and many modifications are conserved between these species. In addition, T. gondii histones have unique modifications not previously identified in other species. Finally...

Performance Comparisons of Nano-LC Systems, Electrospray Sources and LC–MS-MS Platforms

Liu, Qian; Cobb, Jennifer S.; Johnson, Joshua L.; Wang, Qi; Agar, Jeffrey N.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
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Selecting a suitable nano-liquid chromatography system (LC), ionization source and mass spectrometer for LC–tandem mass spectrometry (MS-MS) studies is complicated by numerous competing technologies. This study compares four popular nano-LC systems, four ionization sources and three MS facilities that use completely different LC–MS-MS systems. Statistically significant differences in LC performance were identified with similarly performing Proxeon, Waters and Eksigent nanoLC-Ultra systems [retention time routinely at 0.7–0.9% relative standard deviation (RSD)], and all outperformed the Eksigent nanoLC-2D (RSD ∼2%). In addition, compatibility issues were identified between the Bruker HCT ion trap mass spectrometer and both the Eksigent nanoLC-2D and the Bruker nanoelectrospray source. The electrospray source itself had an unexpected and striking effect on chromatographic reproducibility on the Bruker HCT ion trap. The New Objective nanospray source significantly outperformed the Bruker nanospray source in retention time RSD (1% RSD versus 14% RSD, respectively); and the Bruker nebulized nanospray source outperformed both of these traditional, non-nebulized sources (0.5% RSD in retention time). Finally, to provide useful benchmarks for overall proteomics sensitivity...

Discovery and Characterization of a Photo-Oxidative Histidine-Histidine Cross-Link in IgG1 Antibody Utilizing 18O-Labeling and Mass Spectrometry

Liu, Min; Zhang, Zhongqi; Cheetham, Janet; Ren, Da; Zhou, Zhaohui Sunny
Fonte: American Chemical Society Publicador: American Chemical Society
Tipo: Artigo de Revista Científica
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A novel photo-oxidative cross-linking between two histidines (His-His) has been discovered and characterized in an IgG1 antibody via the workflow of XChem-Finder, 18O labeling and mass spectrometry (Anal. Chem.2013, 85, 5900−590823634697). Its structure was elucidated by peptide mapping with multiple proteases with various specificities (e.g., trypsin, Asp-N, and GluC combined with trypsin or Asp-N) and mass spectrometry with complementary fragmentation modes (e.g., collision-induced dissociation (CID) and electron-transfer dissociation (ETD)). Our data indicated that cross-linking occurred across two identical conserved histidine residues on two separate heavy chains in the hinge region, which is highly flexible and solvent accessible. On the basis of model studies with short peptides, it has been proposed that singlet oxygen reacts with the histidyl imidazole ring to form an endoperoxide and then converted to the 2-oxo-histidine (2-oxo-His) and His+32 intermediates, the latter is subject to a nucleophilic attack by the unmodified histidine; and finally, elimination of a water molecule leads to the final adduct with a net mass increase of 14 Da. Our findings are consistent with this mechanism. Successful discovery of cross-linked His-His again demonstrates the broad applicability and utility of our XChem-Finder approach in the discovery and elucidation of protein cross-linking...

Glycan Side Reaction May Compromise ETD-Based Glycopeptide Identification

Darula, Zsuzsanna; Medzihradszky, Katalin F.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Characterization of Green Fluorescent Proteins by 193 nm Ultraviolet Photodissociation Mass Spectrometry

Cannon, Joe R.; Kluwe, Christien; Ellington, Andrew; Brodbelt, Jennifer S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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We investigate the utility of 193 nm ultraviolet photodissociation (UVPD) in comparison to collision induced dissociation (CID), higher energy CID (HCD), and electron transfer dissociation (ETD) for top down fragmentation of highly homologous green fluorescent proteins (GFP) in the gas phase. Several GFP variants were constructed via mutation of surface residues to charged moieties, demonstrating different isoelectric points and presenting a challenge for identification by mass spectroscopy. Presented is a comparison of fragmentation techniques utilized for top down characterization of four variants with varying levels of surface charge. UVPD consistently resulted in identification of more fragment ions relative to other tandem mass spectrometry (MS/MS) methods, allowing higher confidence identification. In addition to the high number of fragment ions, the sites of fragmentation were more evenly spread throughout the protein backbone, which proved key for localizing the point mutations.

Charge-Site Dependent Dissociation of Hydrogen-Rich Radical Peptide Cations upon Vacuum UV Photoexcitation

Madsen, James A.; Cheng, Ryan R.; Kaoud, Tamer S.; Dalby, Kevin N.; Makarov, Dmitrii E.; Brodbelt, Jennifer S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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193 nm vacuum ultraviolet photodissociation (VUVPD) was used to investigate the fragmentation of hydrogen-rich radical peptide cations generated by electron transfer reactions. VUVPD offers new insight into the factors that drive radical- and photon-directed processes. The location of a basic Arg site influences photon-activated Cα-C(O) bond cleavages of singly charged peptide radical cations, an outcome attributed to the initial conformation of the peptide as supported by molecular dynamics simulated annealing and the population of excited states upon UV excitation. This hybrid ETD/VUVPD method was employed to identify phosphorylation sites of the kinase domain of human TRPM7/ChaK1.

Top-Down and Bottom-Up Identification of Proteins by Liquid Extraction Surface Analysis Mass Spectrometry of Healthy and Diseased Human Liver Tissue

Sarsby, Joscelyn; Martin, Nicholas J.; Lalor, Patricia F.; Bunch, Josephine; Cooper, Helen J.
Fonte: Springer US Publicador: Springer US
Tipo: Artigo de Revista Científica
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Liquid extraction surface analysis mass spectrometry (LESA MS) has the potential to become a useful tool in the spatially-resolved profiling of proteins in substrates. Here, the approach has been applied to the analysis of thin tissue sections from human liver. The aim was to determine whether LESA MS was a suitable approach for the detection of protein biomarkers of nonalcoholic liver disease (nonalcoholic steatohepatitis, NASH), with a view to the eventual development of LESA MS for imaging NASH pathology. Two approaches were considered. In the first, endogenous proteins were extracted from liver tissue sections by LESA, subjected to automated trypsin digestion, and the resulting peptide mixture was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) (bottom-up approach). In the second (top-down approach), endogenous proteins were extracted by LESA, and analyzed intact. Selected protein ions were subjected to collision-induced dissociation (CID) and/or electron transfer dissociation (ETD) mass spectrometry. The bottom-up approach resulted in the identification of over 500 proteins; however identification of key protein biomarkers, liver fatty acid binding protein (FABP1), and its variant (Thr→Ala, position 94)...

Formation and reshuffling of disulfide bonds in bovine serum albumin demonstrated using tandem mass spectrometry with collision-induced and electron-transfer dissociation

Rombouts, Ine; Lagrain, Bert; Scherf, Katharina A.; Koehler, Peter; Delcour, Jan A.
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
Publicado em 20/07/2015 Português
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Thermolysin hydrolyzates of freshly isolated, extensively stored (6 years, 6 °C, dry) and heated (60 min, 90 °C, in excess water) bovine serum albumin (BSA) samples were analyzed with liquid chromatography (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using alternating electron-transfer dissociation (ETD) and collision-induced dissociation (CID). The positions of disulfide bonds and free thiol groups in the different samples were compared to those deduced from the crystal structure of native BSA. Results revealed non-enzymatic posttranslational modifications of cysteine during isolation, extensive dry storage, and heating. Heat-induced extractability loss of BSA was linked to the impact of protein unfolding on the involvement of specific cysteine residues in intermolecular and intramolecular thiol-disulfide interchange and thiol oxidation reactions. The here developed approach holds promise for exploring disulfide bond formation and reshuffling in various proteins under conditions relevant for chemical, biochemical, pharmaceutical and food processing.

Characterization of protein N-glycosylation by tandem mass spectrometry using complementary fragmentation techniques

Ford, Kristina L.; Zeng, Wei; Heazlewood, Joshua L.; Bacic, Antony
Fonte: Frontiers Media S.A. Publicador: Frontiers Media S.A.
Tipo: Artigo de Revista Científica
Publicado em 28/08/2015 Português
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The analysis of post-translational modifications (PTMs) by proteomics is regarded as a technically challenging undertaking. While in recent years approaches to examine and quantify protein phosphorylation have greatly improved, the analysis of many protein modifications, such as glycosylation, are still regarded as problematic. Limitations in the standard proteomics workflow, such as use of suboptimal peptide fragmentation methods, can significantly prevent the identification of glycopeptides. The current generation of tandem mass spectrometers has made available a variety of fragmentation options, many of which are becoming standard features on these instruments. We have used three common fragmentation techniques, namely CID, HCD, and ETD, to analyze a glycopeptide and highlight how an integrated fragmentation approach can be used to identify the modified residue and characterize the N-glycan on a peptide.

Autoria na Web 2.0 no contexto da educação e a ética dos hackers.

SOUZA, M. I. F.; SILVA, L. O.; ARAÚJO, I. C.
Fonte: ETD: Educação Temática Digital, Campinas, v. 12, p. 154-173, mar. 2011. Publicador: ETD: Educação Temática Digital, Campinas, v. 12, p. 154-173, mar. 2011.
Tipo: Artigo em periódico indexado (ALICE)
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RESUMO - Este artigo discute a importância do exercício da autoria em ambiente virtual da Web 2.0, considerando que se trata de fundamento essencial à aprendizagem, no professor e no aluno. Autoria na Web 2.0 é apresentada como estratégia pedagógica para ambientes de aprendizagem virtuais, que se utilizam principalmente de ferramentas como blog, wiki e redes sociais. Embora não sejam determinantes, essas tecnologias digitais são condicionantes para que a aprendizagem e a autoria ocorram. Autoria na Web 2.0, além do estabelecimento de novos aparatos tecnológicos, requer novos modos de produção, nos quais prevaleça a postura ética dos hackers, favorecendo a participação, a colaboração, a liberdade e o compartilhamento.; 2011; Número especial.

Autoria na Web 2.0 no contexto da educação e a ética dos hackers.

SOUZA, M. I. F.; AMARAL, S. F. do; SILVA, L. C.; ARAÚJO, I. C.
Fonte: In: AMARAL, S. F. do; PRETTO, N. de L. (Org). Ética, Hacker e a Educação. 2. ed. Campinas: Faculdade de Educação/UNICAMP, 2012. Publicador: In: AMARAL, S. F. do; PRETTO, N. de L. (Org). Ética, Hacker e a Educação. 2. ed. Campinas: Faculdade de Educação/UNICAMP, 2012.
Tipo: Capítulo em livro técnico-científico (ALICE) Formato: p. 48-61.
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Este artigo discute a importância do exercício da autoria em ambiente virtual da Web 2.0, considerando que se trata de fundamento essencial à aprendizagem, no professor e no aluno. Autoria na Web 2.0 é apresentada como estratégia pedagógica para ambientes de aprendizagem virtuais, que se utilizam principalmente de ferramentas como blog, wiki e redes sociais. Embora não sejam determinantes essas, tecnologias digitais são condicionantes para que a aprendizagem e a autoria ocorram. Autoria na Web 2.0, além do estabelecimento de novos aparatos tecnológicos, requer novos modos de produção, nos quais prevaleça a postura ética dos hackers, favorecendo a participação, a colaboração, a liberdade e o compartilhamento.; 2012; Artigo publicado na revista ETD - Educação Temática Digital, Campinas, SP, v. 12, n. esp., p. 154-173, mar. 2011.

IR@UF :: Theses and Dissertations

Taylor, Laurie N.
Fonte: George A. Smathers Libraries, University of Florida; George A. Smathers Libraries, University of Florida ( Gainesville, FL ) Publicador: George A. Smathers Libraries, University of Florida; George A. Smathers Libraries, University of Florida ( Gainesville, FL )
Tipo: mixed material Formato: Documentation
Publicado em //2013 Português
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Texas Digital Library Application Profile for ETDs

Surratt, Brian E.; Little, Alisha; Mitchell, Anne M.; Thomale, Jason; Flannery, Melinda Reagor
Fonte: Universidade Rice Publicador: Universidade Rice
Tipo: Artigo de Revista Científica
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Application profile for ETDs.; This MODS application profile for electronic theses and dissertations (ETDs) describes the best practices for descriptive metadata for member of the Texas Digital Library (TDL). This document defines the mandatory minimum elements for ETDs. Besides these elements, other valid MODS elements may be included in ETD records. Other optional subelements and attributes are described throughout the document.

Caracterización de la modificación por O-GlcNAc de la proteína de la cápside del potyvirus Plum pox virus y su relevancia par la infección viral

Pérez Martínez, José de Jesús
Fonte: Universidade Autônoma de Madrid Publicador: Universidade Autônoma de Madrid
Tipo: Tese de Doutorado
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Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 27-01-2014; The addition of residues of N-acetylglucosamine through O linkages (O-GlcNAc) to target proteins is a post-translational modification widely distributed in eukaryotic cells. A large number of O-GlcNAc modification sites have been mapped in mammalian proteins and the functions of these proteins have been thoroughly studied. However in plants, targets of OGlcNAcylation are largely uncharacterized. Arabidopsis thaliana has two O-GlcNAc transferases (OGTs), Spindly (SPY) and Secret Agent (SEC), and previous results of the laboratory where this PhD thesis was carried out demonstrated that the coat protein (CP) of the potyvirus Plum pox virus (PPV) is O-GlcNAcylated and phosphorylated. In this work, we identified that SEC, and not SPY, is the OGT responsible of the PPV CP O-GlcNAcylation. In null sec- mutants of A. thaliana and in Nicotiana benthamiana transgenic plants in which the SEC-like genes where silenced by RNAi, O-GlcNAcylation of PPV CP was abolished or drastically disturbed, respectively. Virus accumulation was reduced at early times of infection in these plants compared to wild type plants. Site-directed mutagenesis and mass spectrometry analysis demonstrated that threonines 19...