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The Utility of ETD Mass Spectrometry in Proteomic Analysis: BIOCHIMICA ET BIOPHYSICA ACTA Proteins and Proteomics Posttranslational Modifications in Proteomics Special Issue

Mikesh, Leann M; Ueberheide, Beatrix; Chi, An; Coon, Joshua J; Syka, John E P; Shabanowitz, Jeffrey; Hunt, Donald F
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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Mass spectrometry has played an integral role in the identification of proteins and their post-translational modifications (PTM). However, analysis of some PTMs, such as phosphorylation, sulfonation, and glycosylation, is difficult with collision-activated dissociation (CAD) since the modification is labile and preferentially lost over peptide backbone fragmentation, resulting in little to no peptide sequence information. The presence of multiple basic residues also makes peptides exceptionally difficult to sequence by conventional CAD mass spectrometry. Here we review the utility of electron transfer dissociation (ETD) mass spectrometry for sequence analysis of post-translationally modified and/or highly basic peptides. Phosphorylated, sulfonated, glycosylated, nitrosylated, disulfide bonded, methylated, acetylated, and highly basic peptides have been analyzed by CAD and ETD mass spectrometry. CAD fragmentation typically produced spectra showing limited peptide backbone fragmentation. However, when these peptides were fragmented using ETD, peptide backbone fragmentation produced a complete or almost complete series of ions and thus extensive peptide sequence information. In addition, labile PTMs remained intact. These examples illustrate the utility of ETD as an advantageous tool in proteomic research by readily identifying peptides resistant to analysis by CAD. A further benefit is the ability to analyze larger...

P38-M Improved Characterization of Glycopeptides Using CID and ETD in a Non-Linear Ion Trap MS

Baessmann, C.; Wuhrer, M.; Lubeck, M.; Hartmer, R.; Brekenfeld, A.; Koeleman, C. A. M.; Deelder, A. M.
Fonte: The Association of Biomolecular Resource Facilities Publicador: The Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /02/2007 Português
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Characterization of glycoproteins by electrospray ionization tandem mass spectrometry of glycosylated peptides using collisionally induced dissociation (CID) results in the preferential fragmentation of the glycosidic bonds, while the peptide bonds are more stable. As low-energy CID of glycopeptides mainly provides information on the glycan moiety, a selective analysis of the peptide chain itself usually requires enzymatic removal of the carbohydrate part. The recently introduced electron transfer dissociation (ETD) option for ion-trap mass spectrometers shows preferred fragmentation of the peptide backbone, leaving posttranslational modifications widely intact. Thus, a combination of ETD and CID is a highly promising tool for the analysis of glycopeptides.

Post-acquisition ETD spectral processing for increased peptide identifications

Good, David M.; Wenger, Craig D.; McAlister, Graeme C.; Coon, Joshua J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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Tandem mass spectra (MS/MS) produced using electron transfer dissociation (ETD) differ from those derived from collision-activated dissociation (CAD) in several important ways. Foremost, the predominant fragment ion series are different: c-and z•-type ions are favored in ETD spectra while b-and y-type ions comprise the bulk of the CAD spectra. Additionally, ETD spectra possess specific neutral losses and charge-reduced precursors . Most database search algorithms were designed to analyze CAD spectra, and have only recently been adapted to accomodate c-and z•-ions; however, inclusion of these additional spectral features can hinder identification, leading to lower confidence scores and decreased sensitivity. Therefore, it is important to pre-process spectral data prior to submission to a database search to remove those features which cause complications. Here, we demonstrate the effect of removing these features on the number of identifications at a 1% false discovery rate (FDR) using the open mass spectrometry search algorithm (OMSSA). When analyzing two biological replicates of a yeast protein extract in three total analyses, the number of identifications with a ~1% FDR increased from ~4611 to ~5931 upon spectral pre-processing – an increase of ~28.6%. We outline the most effective pre-processing methods...

A New Probabilistic Database Search Algorithm for ETD Spectra

Sadygov, Rovshan G.; Good, David M.; Swaney, Danielle L.; Coon, Joshua J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/2009 Português
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Peptide characterization using electron transfer dissociation (ETD) is an important analytical tool for protein identification. The fragmentation observed in ETD spectra is complementary to that seen when using the traditional dissociation method, collision activated dissociation (CAD). Applications of ETD enhance the scope and complexity of the peptides that can be studied by mass spectrometry-based methods. For example, ETD is shown to be particularly useful for the study of post-translationally modified peptides.

The Effect of Interfering Ions on Search Algorithm Performance for ETD Data

Good, David M.; Wenger, Craig D.; Coon, Joshua J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/2010 Português
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Collision-activated dissociation (CAD) and electron-transfer dissociation (ETD) each produce spectra containing unique features. Though several database search algorithms (e.g., SEQUEST, Mascot, and OMSSA) have been modified to search ETD data, this consists chiefly of the ability to search for c- and z•-ions; additional ETD-specific features are often unaccounted for, and may hinder identification. Removal of these features via spectral processing increased total search sensitivity by ∼20% for both human and yeast datasets; unique identifications increased by ∼17% for the yeast datasets and ∼16% for the human dataset.

Identification of the Unpaired Cysteine Status and Complete Mapping the 17 Disulfides of Recombinant Tissue Plasminogen Activator Using LC-MS with ETD/CID

Wu, Shiaw Lin; Jiang, Haitao; Hancock, William S.; Karger, Barry L.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/06/2010 Português
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Recombinant tissue plasminogen (rt-PA) with 35 cysteine residues has been completely assigned by mapping the 17 disulfide linkages and the unpaired cysteine. The result is consistent with the prediction from homology except for the unassigned cysteine, which was identified at Cys83. This cysteine was found to be blocked and paired with either a glutathione or cysteine residue in a ~ 60 :40 ratio, respectively. The analysis was conducted using a multi-fragmentation approach consisting of ETD and CID, in combination with a multi-enzyme digestion strategy (Lys-C, trypsin, and Glu-C). The disulfide-linked peptides, even those containing N or O-linked glycosylation, could be assigned since the disulfide bonds were still preferably cleaved over the glycosidic cleavages under ETD fragmentation. The use of a multiple and sequential enzymatic digestion strategy was important in producing fragment sizes suitable for analysis. For the analysis of complex intertwined disulfides, the use of CID MS3 to target partially disulfide dissociated peptides from the ETD fragmentation was necessary for linkage assignment. The ability to identify the exact location and status of the unpaired cysteine (free or blocked with a glutathione or cysteine) could shed light on the activation of rt-PA...

Ultrasensitive Characterization of Site-Specific Glycosylation of Affinity Purified Haptoglobin from Lung Cancer Patient Plasma Using 10 μm i.d. Porous Layer Open Tubular (PLOT) LC-LTQ-CID/ETD-MS

Wang, Dongdong; Hincapie, Marina; Rejtar, Tomas; Karger, Barry L.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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Site-specific analysis of protein glycosylation is important for biochemical and clinical research efforts. Glycopeptide analysis using liquid chromatography - collision induced dissociation/electron transfer dissociation - mass spectrometry (LC-CID/ETD-MS) allows simultaneous characterization of glycan structure and attached peptide site. However, due to the low ionization efficiency of glycopeptides during electrospray ionization (ESI), 200–500 fmol of sample per injection is needed for a single LC-MS run, which makes it challenging for the analysis of limited amounts of glycoprotein purified from biological matrices. To improve the sensitivity of LC-MS analysis for glycopeptides, an ultra-narrow porous layer open tubular (PLOT) LC column (2.5 m × 10 μm i.d.) was coupled to a linear ion trap mass spectrometer (LTQ-CID/ETD-MS) to provide sensitive analysis of N-linked protein glycosylation heterogeneity. The potential of the developed method is demonstrated by the characterization of site-specific glycosylation using haptoglobin (Hpt) as a model protein. To limit the amount of haptoglobin to low pmole amounts of protein, we affinity purified it from 1 μL of pooled lung cancer patients plasma. A total of 26 glycoforms/glycan compositions on three Hpt tryptic glycopeptides were identified and quantified from 10 LC-MS runs with a consumption of 100 fmol Hpt digest (13 ng protein...

Improved identification of O-linked glycopeptides from ETD data with optimized scoring for different charge states and cleavage specificities

Darula, Zsuzsa; Chalkley, Robert J.; Lynn, Aenoch; Baker, Peter R.; Medzihradszky, Katalin F.
Fonte: Springer Vienna Publicador: Springer Vienna
Tipo: Artigo de Revista Científica
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This article describes the effect of re-interrogation of electron-transfer dissociation (ETD) data with newly developed analytical tools. MS/MS-based characterization of O-linked glycopeptides is discussed using data acquired from a complex mixture of O-linked glycopeptides, featuring mucin core 1-type carbohydrates with and without sialic acid, as well as after partial deglycosylation to leave only the core GalNAc units (Darula and Medzihradszky in Mol Cell Proteomics 8:2515, 2009). Information content of collision-induced dissociation spectra generated in collision cell (in QqTOF instruments) and in ion traps is compared. Interpretation of the corresponding ETD data using Protein Prospector is also presented. Search results using scoring based on the frequency of different fragment ions occurring in ETD spectra of tryptic peptides are compared with results obtained after ion weightings were adjusted to accommodate differential ion frequencies in spectra of differing charge states or cleavage specificities. We show that the improved scoring is more than doubled the glycopeptide assignments under very strict acceptance criteria. This study illustrates that “old” proteomic data may yield significant new information when re-interrogated with new...

PeaksDB: New Software for Substantially Improved Peptide Identification from Orbitrap ETD Mass Spectrometry

Zhang, J.; Xin, L.; Shan, B.; Chen, W.; Ma, B.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /10/2011 Português
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Objective: To substantially improve the peptide identification sensitivity and accuracy from the Orbitrap ETD data with computational methods. Method: The algorithm takes full advantage of the characteristics of the Orbitrap ETD data, including: (1) high mass resolution of the precursor ions, and (2) the distributions of different fragment ion types in the MS/MS scans. For the first characteristic, a pre-search step is conducted to determine the precursor mass error distribution. This does not only make the precursor mass more accurate by a software recalibration, but also allows the use of the mass error as an important feature in the peptide-spectrum matching score function. For the second characteristic, the frequencies of different fragment ion types at different precursor charge states are statistically learned, and used in the score calculation. Moreover, the precursor-related ions in the MS/MS spectra are removed. Additionally, the score function makes use of the similarity between a database peptide and the de novo sequencing result. Result: PeaksDB was compared against three other search engines: MSGF-DB, Mascot, and ZCore. The same shuffled decoy database was appended to the target database and searched together to estimate the false discovery rate (FDR) of each individual engine. The same search parameters were used for all engines except that MSGFDB does not support variable PTMs. If no variable PTM is allowed...

Improving CID, HCD, and ETD FT MS/MS degradome-peptidome identifications using high accuracy mass information

Shen, Yufeng; Tolić, Nikola; Purvine, Samuel O.; Smith, Richard D.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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MS dissociation methods, including CID, HCD, and ETD, can each contribute distinct peptidome identifications using conventional peptide identification methods (Shen et al. J. Proteome Res. 2011), but such samples still pose significant informatics challenges. In this work, we explored utilization of high accuracy fragment ion mass measurements, in this case provided by FT MS/MS, to improve peptidome peptide dataset size and consistency relative to conventional descriptive and probabilistic scoring methods. For example, we identified 20–40% more peptides than SEQUEST, Mascot, and MS-GF scoring methods using high accuracy fragment ion information and the same FDR (e.g., <10 mass errors) from CID, HCD, and ETD spectra. Identified species covered >90% of the collective identifications obtained using various conventional peptide identification methods, which resolves the issue of different data analysis methods generating different peptide datasets. Choice of peptide dissociation and high-precision measurement-based identification methods presently available for degradomic-peptidomic analyses needs to be based on the coverage and confidence (or specificity) afforded by the method, as well as practical issues (e.g., throughput). By using accurate fragment information...

Complete Mapping of a Cystine Knot and Nested Disulfides of Recombinant Human Arylsulfatase A by Multi-Enzyme Digestion and LC-MS Analysis Using CID and ETD

Ni, Wenqin; Lin, Melanie; Salinas, Paul; Savickas, Philip; Karger, Barry L.; Wu, Shiaw-Lin
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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Cystine knots or nested disulfides are structurally difficult to characterize, despite current technological advances in peptide mapping with high-resolution liquid chromatography coupled with mass spectrometry (LC-MS). In the case of recombinant human arylsulfatase A (rhASA), there is one cystine knot at the C-terminal, a pair of nested disulfides at the middle, and two out of three unpaired cysteines in the N-terminal region. The statuses of these cysteines are critical structure attributes for rhASA function and stability that requires precise examination. We used a unique approach to determine the status and linkage of each cysteine in rhASA, which was comprised of multi-enzyme digestion strategies (from Lys-C, trypsin, Asp-N, pepsin, and PNGase F) and multi-fragmentation methods in mass spectrometry using electron transfer dissociation (ETD), collision induced dissociation (CID), and CID with MS3 (after ETD). In addition to generating desired lengths of enzymatic peptides for effective fragmentation, the digestion pH was optimized to minimize the disulfide scrambling. The disulfide linkages, including the cystine knot and a pair of nested cysteines, unpaired cysteines, and the posttranslational modification of a cysteine to formylglycine...

GlycoPep Detector: A tool for assigning mass spectrometry data of N-linked glycopeptides based on their ETD spectra

Zhu, Zhikai; Hua, David; Clark, Daniel F.; Go, Eden P.; Desaire, Heather
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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Electron transfer dissociation (ETD) is commonly used in fragmenting N-linked glycopeptides in their mass spectral analyses to complement collision induced dissociation (CID) experiments. The glycan remains intact through ETD, while the peptide backbone is cleaved, providing the sequence of amino acids for a glycopeptide. Nonetheless, data analysis is a major bottleneck to high throughput glycopeptide identification based on ETD data, due to the complexity and diversity of ETD mass spectra compared to CID counterparts. GlycoPep Detector (GPD) is a web-based tool to address this challenge. It filters out noise peaks that interfere with glycopeptide sequencing, correlates input glycopeptide compositions with the ETD spectra, and assigns a score for each candidate. By considering multiple ion series (c-, z- and y-ions) and scoring them separately, the software gives more weighting to the ion series that matches peaks of high intensity in the spectra. This feature enables the correct glycopeptide to receive a high score while keeping scores of incorrect compositions low. GPD has been utilized to interpret data collected on six model glycoproteins (RNase B, avidin, fetuin, asialofetuin, transferrin and AGP) as well as a clade C HIV envelope glycoprotein...

Sulfonium Ion Derivatization, Isobaric Stable Isotope Labeling and Data Dependent CID- and ETD-MS/MS for Enhanced Phosphopeptide Quantitation, Identification and Phosphorylation Site Characterization

Lu, Yali; Zhou, Xiao; Stemmer, Paul M.; Reid, Gavin E.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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An amine specific peptide derivatization strategy involving the use of novel isobaric stable isotope encoded ‘fixed charge’ sulfonium ion reagents, coupled with an analysis strategy employing capillary HPLC, ESI-MS, and automated data dependent ion trap CID-MS/MS, -MS3, and/or ETD-MS/MS, has been developed for the improved quantitative analysis of protein phosphorylation, and for identification and characterization of their site(s) of modification. Derivatization of 50 synthetic phosphopeptides with S,S′-dimethylthiobutanoylhydroxysuccinimide ester iodide (DMBNHS), followed by analysis using capillary HPLC-ESI-MS, yielded an average 2.5-fold increase in ionization efficiencies and a significant increase in the presence and/or abundance of higher charge state precursor ions compared to the non-derivatized phosphopeptides. Notably, 44% of the phosphopeptides (22 of 50) in their underivatized states yielded precursor ions whose maximum charge states corresponded to +2, while only 8% (4 of 50) remained at this maximum charge state following DMBNHS derivatization. Quantitative analysis was achieved by measuring the abundances of the diagnostic product ions corresponding to the neutral losses of ‘light’ (S(CH3)2) and ‘heavy’ (S(CD3)2) dimethylsulfide exclusively formed upon CID-MS/MS of isobaric stable isotope labeled forms of the DMBNHS derivatized phosphopeptides. Under these conditions...

E-THESIS SUPPORT FOR GRADUATE STUDENTS: How prepared are libraries to support students in compiling, formatting and submitting their e-theses?

Kalb, Sam
Fonte: Quens University Publicador: Quens University
Tipo: Conferência ou Objeto de Conferência
Português
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This presentation, presented at the Ontario Library Association, Superconference, Feb. 1-4, 2012, looks at ETD templates, learning resources, and other support services that the library can offer to help students deal with the challenges of ETD preparation and submission.

Sequencing-Grade De novo Analysis of MS/MS Triplets (CID/HCD/ETD) From Overlapping Peptides

Guthals, Adrian; Clauser, Karl R.; Frank, Ari M.; Bandeira, Nuno
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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Full-length de novo sequencing of unknown proteins remains a challenging open problem. Traditional methods that sequence spectra individually are limited by short peptide length, incomplete peptide fragmentation, and ambiguous de novo interpretations. We address these issues by determining consensus sequences for assembled tandem mass (MS/MS) spectra from overlapping peptides (e.g., by using multiple enzymatic digests). We have combined electron-transfer dissociation (ETD) with collision-induced dissociation (CID) and higher-energy collision-induced dissociation (HCD) fragmentation methods to boost interpretation of long, highly charged peptides and take advantage of corroborating b/y/c/z ions in CID/HCD/ETD. Using these strategies, we show that triplet CID/HCD/ETD MS/MS spectra from overlapping peptides yield de novo sequences of average length 70 AA and as long as 200 AA at up to 99% sequencing accuracy.

Moving Electronic Theses from ETD-db to EPrints : The Best of Both Worlds: A Project Briefing Presented at the CNI Spring 2010 Membership Meeting, Baltimore, MD, April 12, 2010

Coles, Betsy; Johnson, Katherine
Fonte: California Institute of Technology Publicador: California Institute of Technology
Tipo: Report or Paper; NonPeerReviewed Formato: application/vnd.ms-powerpoint; application/pdf
Publicado em 12/04/2010 Português
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Caltech Library Services' first digital archive came online in April 2001 using EPrints software from the University of Southampton. We began collecting electronic theses early as well: voluntary deposit of Ph.D. theses began in 2001, and became mandatory in July 2002. The thesis collection was hosted on the ETD-db software platform developed at Virginia Tech. By 2008 it became clear that we needed to consolidate our repository platforms. We decided to move our electronic theses to EPrints Version 3, the platform in use for our institutional repository. We did not, however, want to lose the many unique features of the Virginia Tech ETD-db software, such as thesis-specific workflow, the ability for staff to communicate with thesis authors via email from within the ETD-db interface, and fine-grained, thesis-specific access controls. We also wanted to add new features that were not available in either platform, such as tracking the progress of a thesis through the complex local approval and release process, and the ability to store related documents, such as signed thesis forms and permissions letters, with the thesis but in a "dark" area of the record visible only to repository administrators. This briefing explains what was involved in the transition from ETD-db to EPrints for our thesis collection and how the Caltech Library took advantage of the flexibility of the EPrints platform to meet our requirements. It also suggests ways that other institutions may be able to adopt and build on what we've done...

ETD Outperforms CID and HCD in the Analysis of the Ubiquitylated Proteome

Porras-Yakushi, Tanya R.; Sweredoski, Michael J.; Hess, Sonja
Fonte: Springer Publicador: Springer
Tipo: Article; PeerReviewed Formato: application/pdf; application/vnd.ms-excel; application/vnd.ms-excel; application/pdf
Publicado em /09/2015 Português
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Comprehensive analysis of the ubiquitylome is a prerequisite to fully understand the regulatory role of ubiquitylation. However, the impact of key mass spectrometry parameters on ubiquitylome analyses has not been fully explored. In this study, we show that using electron transfer dissociation (ETD) fragmentation, either exclusively or as part of a decision tree method, leads to ca. 2-fold increase in ubiquitylation site identifications in K-ε-GG peptide-enriched samples over traditional collisional-induced dissociation (CID) or higher-energy collision dissociation (HCD) methods. Precursor ions were predominantly observed as 3+ charged species or higher and in a mass range 300–1200 m/z. N-ethylmaleimide was used as an alkylating agent to reduce false positive identifications resulting from overalkylation with halo-acetamides. These results demonstrate that the application of ETD fragmentation, in addition to narrowing the mass range and using N-ethylmaleimide yields more high-confidence ubiquitylation site identification than conventional CID and HCD analysis.

ETD Vol. 15, n. 2 (2013)

Costa Junior, Maurício Pereira da
Fonte: Pesquisa Brasileira em Ciência da Informação e Biblioteconomia Publicador: Pesquisa Brasileira em Ciência da Informação e Biblioteconomia
Tipo: info:eu-repo/semantics/article; info:eu-repo/semantics/publishedVersion; Artigo Avaliado pelos Pares
Publicado em 28/06/2014 Português
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ETD Vol. 15, n. 2 (2013) Link de acesso ao número http://www.fae.unicamp.br/revista/index.php/etd/issue/view/272/showToc

ETD Vol. 15, n. 1 (2013)

Costa Junior, Maurício Pereira da
Fonte: Pesquisa Brasileira em Ciência da Informação e Biblioteconomia Publicador: Pesquisa Brasileira em Ciência da Informação e Biblioteconomia
Tipo: info:eu-repo/semantics/article; info:eu-repo/semantics/publishedVersion; Artigo Avaliado pelos Pares
Publicado em 29/03/2014 Português
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ETD Vol. 15, n. 1 (2013) Link de acesso ao número http://www.fae.unicamp.br/revista/index.php/etd/issue/view/271/showToc

ETD Vol. 15, n. 3 (2013)

Costa Junior, Maurício Pereira da
Fonte: Pesquisa Brasileira em Ciência da Informação e Biblioteconomia Publicador: Pesquisa Brasileira em Ciência da Informação e Biblioteconomia
Tipo: info:eu-repo/semantics/article; info:eu-repo/semantics/publishedVersion; Artigo Avaliado pelos Pares
Publicado em 28/06/2014 Português
Relevância na Pesquisa
27.000703%
ETD Vol. 15, n. 3 (2013) Link de acesso ao número http://www.fae.unicamp.br/revista/index.php/etd/issue/view/273/showToc