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Infrared Photo-Activation Reduces Peptide Folding and Hydrogen Atom Migration Following ETD Tandem Mass Spectrometry**

Ledvina, Aaron R.; McAlister, Graeme C.; Gardener, Myles W.; Smith, Suncerae I.; Madsen, James A.; Schwartz, Jae C.; Stafford, George C.; Syka, John E. P.; Brodbelt, Jennifer S.; Coon, Joshua J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em //2009 Português
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An Initial Characterization of the Serum Phosphoproteome

Zhou, Weidong; Ross, Mark M.; Tessitore, Alessandra; Ornstein, David; VanMeter, Amy; Liotta, Lance A.; Petricoin, Emanuel F.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/2009 Português
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Phosphorylation is a dynamic post-translational protein modification that is the basis of a general mechanism for maintaining and regulating protein structure and function, and of course underpins key cellular processes through signal transduction. In the last several years, many studies of large-scale profiling of phosphoproteins and mapping phosphorylation sites from cultured human cells or tissues by mass spectrometry technique have been published; however, there is little information on general (or global) phosphoproteomic characterization and description of the content of phosphoprotein analytes within the circulation. Circulating phosphoproteins and phosphopeptides could represent important disease biomarkers because of their well-known importance in cellular function, and these analytes frequently are mutated and activated in human diseases such as cancer. Here we report an initial attempt to characterize the phosphoprotein content of serum. To accomplish this, we developed a method in which phosphopeptides are enriched from digested serum proteins and analyzed by LC-MS/MS using LTQ-Orbitrap (CID) and LTQ-ETD mass spectrometers. Using this approach we identified ~100 unique phosphopeptides with stringent filtering criteria and a lower than 1% false discovery rate.

Tailored-waveform Collisional Activation of Peptide Ion Electron Transfer Survivor Ions in Cation Transmission Mode Ion/Ion Reaction Experiments

Han, Hongling; Londry, Frank A.; Erickson, David E.; McLuckey, Scott A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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Broad-band resonance excitation via a tailored waveform in a high pressure collision cell (Q2) on a hybrid quadrupole/time-of-flight (QqTOF) tandem mass spectrometer has been implemented for cation transmission mode electron transfer ion/ion reactions of tryptic polypeptides. The frequency components in the broadband waveform were defined to excite the first generation intact electron transfer products for relatively large tryptic peptides. The optimum amplitude of the arbitrary waveform applied has been determined empirically to be 3.0 Vp-p, which is effective for relatively high mass-to-charge (m/z) ratio precursor ions with little elimination of sequence information for low m/z ions. The application of broadband activation during the transmission mode ion/ion reaction obviates frequency and amplitude tuning normally associated with ion trap collision induced dissociation (CID). This approach has been demonstrated with triply and doubly charged tryptic peptides with and without post-translational modifications. Enhanced structural information was achieved by production of a larger number of informative c- and z-type fragments using the tailored waveform on unmodified and modified (phosphorylated and glycosylated) peptides when the first generation intact electron transfer products fell into the defined frequency range. This approach can be applied to a wide range of tryptic peptide ions...

C-Terminal Phosphorylation of Murine Testis-Specific Histone H1t in Elongating Spermatids

Rose, Kristie L.; Li, Andra; Zalenskaya, Irina; Zhang, Yun; Unni, Emmanuel; Hodgson, Kim C.; Yu, Yaping; Shabanowitz, Jeffrey; Meistrich, Marvin L.; Hunt, Donald F.; Ausió, Juan
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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Previous studies gave differing results as to whether the testis-specific histone H1t was phosphorylated during rodent spermatogenesis. We show here that histones extracted from germ cell populations enriched with spermatids at different stages of development in rat testes reveal an electrophoretic shift in the position of H1t to slower mobilities in elongating spermatids as compared to that from preceding stages. Alkaline phosphatase treatment and radioactive labeling with 32P demonstrated that the electrophoretic shift is due to phosphorylation. Mass spectrometric analysis of histone H1t purified from sexually mature mice and rat testes confirmed the occurrence of singly, doubly, and triply phosphorylated species, with phosphorylation sites predominantly found at the C-terminal end of the molecule. Furthermore, using collision-activated dissociation (CAD) and electron transfer dissociation (ETD), we have been able to identify the major phosphorylation sites. These include a new, previously unidentified putative H1t-specific cdc2 phosphorylation site in linker histones. The presence of phosphorylation at the C-terminal end of H1t and the timing of its appearance suggest that this post-translational modification is involved in the reduction of H1t binding strength to DNA. It is proposed that this could participate in the opening of the chromatin fiber in preparation for histone displacement by transition proteins in the next phase of spermiogenesis.

Data Processing Algorithms for Analysis of High Resolution MSMS Spectra of Peptides with Complex Patterns of Posttranslational Modifications*

Guan, Shenheng; Burlingame, Alma L.
Fonte: The American Society for Biochemistry and Molecular Biology Publicador: The American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
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The emergence of efficient fragmentation methods such as electron capture dissociation (ECD) and electron transfer dissociation (ETD) provides the opportunity for detailed structural characterization of heavily covalently modified large peptides and small proteins such as intact histones. Even with effective gas phase ion isolation so that a single molecular precursor ion is selected, the MSMS spectrum of a heavily modified peptide may reveal the presence of a mixture of peptides with the same amino acid sequence and the same total number of posttranslational modification (PTM) moieties (same PTM composition) but with different PTM configurations or site-specific occupancy isoforms. Currently available data analysis methods depend on a deisotoping procedure, which becomes less effective when spectra (fragmentation patterns) contain many overlapping isotopic distributions. Peptide database search engines can only identify the most abundant PTM configuration (PTM arrangement on different residues) in such mixtures. To identify all the PTM configurations present in these mixtures and to estimate their relative abundances, we extended our fragment assignment by visual assistance program to search for ions representing all possible configurations...

Mapping of Lysine Methylation and Acetylation in Core Histones of Neurospora crassa

Xiong, Lei; Adhvaryu, Keyur K.; Selker, Eric U.; Wang, Yinsheng
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 29/06/2010 Português
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Core histones are susceptible to a variety of post-translational modifications (PTMs), among which methylation and acetylation play critical roles in various chromatin-dependent processes. The nature and biological functions of these PTMs have been extensively studied in plants, animals and yeasts. In contrast, the histone modifications in Neurospora crassa, a convenient model organism for multicellular eukaryotes, remained largely undefined. In the present study, we used several mass spectrometric techniques, coupled with HPLC separation and multiple protease digestion, to identify the methylation and acetylation sites in core histones isolated from Neurospora. Electron transfer dissociation (ETD) was employed to fragment the heavily modified long N-terminal peptides. In addition, accurate mass measurement of fragment ions allowed for unambiguous differentiation of modification by acetylation or tri-methylation. Many modification sites conserved in other organisms were identified in Neurospora. In addition, some unique modification sites in histone H2B, including N-terminal α methylation, methylation at K3 and acetylation at K19, K28 and K29, were observed. Our analysis provides a potentially comprehensive picture of methylation and acetylation of core histones in Neurospora...

A Novel Workflow for Glycopeptide Analysis Using Cellulose-Based Separation Cartridges, TMT-Labeling and LTQ Orbitrap ETD

Viner, R.I.; Snovida, S.; Bodnar, E.; Perreault, H.; Saba, J.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /09/2010 Português
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RP-16

An Ion Trap with Increased Resolution and Scan Speed for Top-Down Proteomics with ETD/PTR

Albers, C.; Brekenfeld, A.; Gebhardt, C.; Hartmer, R.; Fox, J.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /09/2010 Português
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RP-52

Mining the Human Placenta Proteome > 5000 Proteins Deep Using CID/ETD on a Novel Ion Trap

Lemeer, S.; Schneider, A.; Lubeck, M.; Kuester, B.; Jabs, W.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /09/2010 Português
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RP-112

Increased Sequence Coverage in Complex Protein Digests by Consecutive, Targeted LC-MSMS Runs with Both CID and ETD

Schneider, A.; Lubeck, M.; Almeida, R.; Clemens, P.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /09/2010 Português
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RP-110

Complete Protein Characterization using ETD and PTR with the Combination of Top-Down and Bottom-Up Strategies in an Ion Trap

Ingendoh, A.; Hartmer, R.; Brekenfeld, A.; Schneider, A.; Kiehne, A.; Gonzales, B.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /09/2010 Português
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RP-111

The Implementation and Characterization of Electron Transfer Dissociation (ETD) on an Ion Mobility Enabled Q-TOF Mass Spectrometer

Campuzano, I.; Brown, J.; Williams, J.; Langridge, J.; Compson, K.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /09/2010 Português
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RP-51

Fast and Highly Accurate QTOF ETD MS/MS for Top-Down Sequencing

Lubeck, M.; Stoermer, C.; Hartmer, R.; Fox, J.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /09/2010 Português
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RP-92

Analysis of Isoaspartic Acid by Selective Proteolysis with Asp-N and Electron Transfer Dissociation Mass Spectrometry

Ni, Wenqin; Dai, Shujia; Karger, Barry L.; Zhou, Zhaohui Sunny
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/09/2010 Português
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A ubiquitous yet underappreciated protein post-translational modification, isoaspartic acid (isoAsp, isoD or β-Asp), generated via the deamidation of asparagine or isomerization of aspartic acid in proteins, plays a diverse and crucial role in ageing, as well as autoimmune, cancer, neurodegeneration and other diseases. In addition, formation of isoAsp is a major concern in protein pharmaceuticals, as it may lead to aggregation or activity loss. The scope and significance of isoAsp have, up to now, not been fully explored, as an unbiased screening of isoAsp at low abundance remains challenging. This difficulty is due to the subtle difference in the physicochemical properties between isoAsp and Asp, e.g., identical mass. In contrast, endoprotease Asp-N (EC 3.4.24.33) selectively cleaves aspartyl peptides but not the isoaspartyl counterparts. As a consequence, isoaspartyl peptides can be differentiated from those containing Asp and also enriched by Asp-N digestion. Subsequently, the existence and site of isoaspartate can be confirmed by electron transfer dissociation (ETD) mass spectrometry. As little as 0.5 % of isoAsp was detected in synthetic beta amyloid and cytochrome c peptides, even though both were initially assumed to be free of isoAsp. Taken together...

Better score function for peptide identification with ETD MS/MS spectra

Liu, Xiaowen; Shan, Baozhen; Xin, Lei; Ma, Bin
Fonte: BioMed Central Publicador: BioMed Central
Tipo: Artigo de Revista Científica
Publicado em 18/01/2010 Português
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FGF-1 induces ATP release from spinal astrocytes in culture and opens pannexin and connexin hemichannels

Garré, Juan M.; Retamal, Mauricio A.; Cassina, Patricia; Barbeito, Luis; Bukauskas, Feliksas F.; Sáez, Juan C.; Bennett, Michael V. L.; Abudara, Verónica
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
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Spinal astrocytes are coupled by connexin (Cx) gap junctions and express pannexin 1 (Px1) and purinergic receptors. Fibroblast growth factor 1 (FGF-1), which is released in spinal cord injury, activated spinal astrocytes in culture, induced secretion of ATP, and permeabilized them to relatively large fluorescent tracers [ethidium (Etd) and lucifer yellow (LY)] through “hemichannels” (HCs). HCs can be formed by connexins or pannexins; they can open to extracellular space or can form gap junction (GJ) channels, one HC from each cell. (Pannexins may not form gap junctions in mammalian tissues, but they do in invertebrates). HC types were differentiated pharmacologically and by Px1 knockdown with siRNA and by use of astrocytes from Cx43 knockout mice. Permeabilization was reduced by apyrase (APY), an ATPase, and by P2X7 receptor antagonists, implicating secretion of ATP and autocrine and/or paracrine action. Increased permeability of cells exposed to FGF-1 or ATP for 2 h was mediated largely by Px1 HCs activated by P2X7 receptors. After a 7-h treatment, the permeability was mediated by both Cx43 and Px1 HCs. FGF-1 also caused reduction in gap junctional communication. Botulinum neurotoxin A, a blocker of vesicular release, reduced permeabilization when given 30 min before FGF-1 application...

Histone Variants and Their Post-Translational Modifications in Primary Human Fat Cells

Jufvas, Åsa; Strålfors, Peter; Vener, Alexander V.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 07/01/2011 Português
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Epigenetic changes related to human disease cannot be fully addressed by studies of cells from cultures or from other mammals. We isolated human fat cells from subcutaneous abdominal fat tissue of female subjects and extracted histones from either purified nuclei or intact cells. Direct acid extraction of whole adipocytes was more efficient, yielding about 100 µg of protein with histone content of 60% –70% from 10 mL of fat cells. Differential proteolysis of the protein extracts by trypsin or ArgC-protease followed by nanoLC/MS/MS with alternating CID/ETD peptide sequencing identified 19 histone variants. Four variants were found at the protein level for the first time; particularly HIST2H4B was identified besides the only H4 isoform earlier known to be expressed in humans. Three of the found H2A potentially organize small nucleosomes in transcriptionally active chromatin, while two H2AFY variants inactivate X chromosome in female cells. HIST1H2BA and three of the identified H1 variants had earlier been described only as oocyte or testis specific histones. H2AFX and H2AFY revealed differential and variable N-terminal processing. Out of 78 histone modifications by acetylation/trimethylation, methylation, dimethylation, phosphorylation and ubiquitination...

Top-Down Protein Characterization Facilitated by Ion/Ion Reactions on a Quadrupole/Time-of-Flight Platform

Huang, Teng-yi; McLuckey, Scott A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/2010 Português
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In comparison to bottom-up proteomics approaches, whereby peptides derived from proteolytic digestion are analyzed, top-down approaches, involving direct analysis of intact proteins, provides higher specificity for protein identification and are better-suited for the characterization of sequence variants. However, top-down protein characterization usually requires more sophisticated instrumentation and methodologies to deal with the more complex tandem mass spectra derived from dissociation of high mass multiply charged intact proteins. Gas-phase ion/ion reactions are universally applicable and have proved to be useful in mixture analysis and top-down biomolecule characterization. The coupling of the ion/ion proton transfer reaction (PTR) in the context of tandem mass spectrometry has been demonstrated to expand informing power in top-down protein characterization, particularly with platforms that employ electrodynamic ion trap and time-of-flight mass analysis. In addition, probing protein primary structure using ion/ion electron transfer dissociation (ETD) usually provides extensive structurally informative fragmentation and also allows for the localization of labile post-translational modifications. Here, the performance of the widely used quadrupole/time-of-flight platform...

Dissociation of Disulfide-Intact Somatostatin Ions: The Roles of Ion Type and Dissociation Method

Mentinova, Marija; Han, Hongling; McLuckey, Scott A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/2009 Português
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The dissociation chemistry of somatostatin-14 was examined using various tandem mass spectrometry techniques including low energy beam type and ion trap CID of protonated and deprotonated forms of the peptide, CID of peptide-gold complexes, and ETD of cations. Most of the sequence of somatostatin-14 is present within a loop defined by the disulfide linkage between Cys-3 and Cys-14. The generation of readily interpretable sequence-related ions from within the loop requires the cleavage of at least one of the bonds of the disulfide linkage and the cleavage of one polypeptide backbone bond. CID of the protonated forms of somatostatin did not appear to give rise to an appreciable degree of dissociation of the disulfide linkage. Sequential fragmentation via multiple alternative pathways tended to generate very complex spectra. CID of the anions proceeded through CH2-S cleavages extensively but relatively few structurally diagnostic ions were generated. The incorporation of Au(I) into the molecule via ion/ion reactions followed by CID gave rise to many structurally relevant dissociation products, particularly for the [M+Au+H]2+ species. The products were generated by a combination of S-S bond cleavage and amide bond cleavage. Electron transfer dissociation of the [M+3H]3+ ion generated rich sequence information...

A Novel Proteomics Approach to Identify SUMOylated Proteins and Their Modification Sites in Human Cells*

Galisson, Frederic; Mahrouche, Louiza; Courcelles, Mathieu; Bonneil, Eric; Meloche, Sylvain; Chelbi-Alix, Mounira K.; Thibault, Pierre
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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The small ubiquitin-related modifier (SUMO) is a small group of proteins that are reversibly attached to protein substrates to modify their functions. The large scale identification of protein SUMOylation and their modification sites in mammalian cells represents a significant challenge because of the relatively small number of in vivo substrates and the dynamic nature of this modification. We report here a novel proteomics approach to selectively enrich and identify SUMO conjugates from human cells. We stably expressed different SUMO paralogs in HEK293 cells, each containing a His6 tag and a strategically located tryptic cleavage site at the C terminus to facilitate the recovery and identification of SUMOylated peptides by affinity enrichment and mass spectrometry. Tryptic peptides with short SUMO remnants offer significant advantages in large scale SUMOylome experiments including the generation of paralog-specific fragment ions following CID and ETD activation, and the identification of modified peptides using conventional database search engines such as Mascot. We identified 205 unique protein substrates together with 17 precise SUMOylation sites present in 12 SUMO protein conjugates including three new sites (Lys-380, Lys-400, and Lys-497) on the protein promyelocytic leukemia. Label-free quantitative proteomics analyses on purified nuclear extracts from untreated and arsenic trioxide-treated cells revealed that all identified SUMOylated sites of promyelocytic leukemia were differentially SUMOylated upon stimulation.