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Protein conformations can be probed in top-down HDX MS experiments utilizing electron transfer dissociation of protein ions without hydrogen scrambling

Abzalimov, Rinat R.; Kaplan, Desmond A.; Easterling, Michael L.; Kaltashov, Igor A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Electron transfer dissociation (ETD) is evaluated as a technique to provide local information on higher order structure and dynamics of a whole protein molecule. Isotopic labeling of highly flexible segments of a model 18 kDa protein is carried out in solution under mildly denaturing conditions by means of hydrogen/deuterium exchange (HDX), followed by transfer of intact protein ions to the gas phase by means of electrospray ionization and mass-selection of a precursor ion for subsequent reactions with fluoranthene radical anions. The ETD process gives rise to abundant fragment ions, whose deuterium content can be measured as a function of duration of the HDX reaction in solution. No backbone protection is detected for all protein segments spanning the twenty-five residue long N-terminal part of the protein, which is known to lack structure in solution. At the same time, noticeable protection is evident for segments representing the structured regions of the protein. The results of this work suggest that ETD of intact protein ions is not accompanied by detectable hydrogen scrambling and can be used in tandem with HDX to probe protein conformation in solution.

Application of Electron Transfer Dissociation Mass Spectrometry in Analyses of Non-enzymatically Glycated Peptides

Zhang, Qibin; Frolov, Andrej; Tang, Ning; Hoffmann, Ralf; van de Goor, Tom; Metz, Thomas O.; Smith, Richard D.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em //2007 Português
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Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the context of development of diabetic complications. The fragmentation behavior of glycated peptides produced from reaction of D-glucose with lysine residues was investigated by electron transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. It was found that high abundance ions corresponding to various degrees of neutral water losses, as well as furylium ion production, dominate the CID spectra, and that the sequence informative b and y ions were rarely observed when Amadori-modified peptides were fragmented. Contrary to what was observed under CID conditions, ions corresponding to neutral losses of water or furylium ion production were not observed in the ETD spectra. Instead, abundant and almost complete series of c and z type ions were observed regardless of whether the modification site was located in the middle of the sequence or close to the N-terminus, greatly facilitating the peptide sequencing. This study strongly suggests that ETD is a better technique for proteomics studies of non-enzymatically glycated peptides and proteins.

Correct Identification of Oxidized Histidine Residues Using Electron Transfer Dissociation

Srikanth, Rapole; Wilson, Jonathan; Vachet, Richard W.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/2009 Português
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Oxidative modification to the side chain of histidine can noticeably change the collision-induced dissociation (CID) pathways of peptides containing this oxidized residue. In cases where an oxidized peptide consists of two or more isomers differing only in the site of modification, oxidation to histidine usually causes the other oxidized sites to be mis-assigned in CID spectra. These spectral mis-assignments can sometimes be avoided by using multiple stages of MS/MS (MSn) or via specially-optimized liquid chromatographic separation conditions. In this manuscript, we demonstrate that these mis-assignments can be more readily and easily avoided by using electron-transfer dissociation (ETD) to dissociate the oxidized peptides. Furthermore, we find that the relative insensitivity of ETD to side chain chemistry allows the extent of oxidative modification to be determined readily for peptide isomers having more than one site of oxidation. The current results along with previous studies of oxidized peptides suggest that ETD is probably a better technique than CID for obtaining correct sequence and modification information for oxidized peptides.

Metabolic inhibition increases activity of connexin-32 hemichannels permeable to Ca2+ in transfected HeLa cells

Sánchez, Helmuth A.; Orellana, Juan A.; Verselis, Vytas K.; Sáez, Juan C.
Fonte: American Physiological Society Publicador: American Physiological Society
Tipo: Artigo de Revista Científica
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Numerous cell types express functional connexin (Cx) hemichannels (HCs), and membrane depolarization and/or exposure to a divalent cation-free bathing solution (DCFS) have been shown to promote HC opening. However, little is known about conditions that can promote HC opening in the absence of strong depolarization and when extracellular divalent cation concentrations remain at physiological levels. Here the effects of metabolic inhibition (MI), an in vitro model of ischemia, on the activity of mouse Cx32 HCs were examined. In HeLa cells stably transfected with mouse Cx32 (HeLa-Cx32), MI induced an increase in cellular permeability to ethidium (Etd). The increase in Etd uptake was directly related to an increase in levels of Cx32 HCs present at the cell surface. Moreover, MI increased membrane currents in HeLa-Cx32 cells. Underlying these currents were channels exhibiting a unitary conductance of ∼90 pS, consistent with Cx32 HCs. These currents and Etd uptake were blocked by HC inhibitors. The increase in Cx32 HC activity was preceded by a rapid reduction in mitochondrial membrane potential and a rise in free intracellular Ca2+ concentration ([Ca2+]i). The increase in free [Ca2+]i was prevented by HC blockade or exposure to extracellular DCFS and was virtually absent in parental HeLa cells. Moreover...

Compact integration factor methods in high spatial dimensions

Nie, Qing; Wan, Frederic Y.M.; Zhang, Yong-Tao; Liu, Xin-Feng
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em //2008 Português
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The dominant cost for integration factor (IF) or exponential time differencing (ETD) methods is the repeated vector–matrix multiplications involving exponentials of discretization matrices of differential operators. Although the discretization matrices usually are sparse, their exponentials are not, unless the discretization matrices are diagonal. For example, a two-dimensional system of N × N spatial points, the exponential matrix is of a size of N2 × N2 based on direct representations. The vector–matrix multiplication is of O(N4), and the storage of such matrix is usually prohibitive even for a moderate size N. In this paper, we introduce a compact representation of the discretized differential operators for the IF and ETD methods in both two- and three-dimensions. In this approach, the storage and CPU cost are significantly reduced for both IF and ETD methods such that the use of this type of methods becomes possible and attractive for two- or three-dimensional systems. For the case of two-dimensional systems, the required storage and CPU cost are reduced to O(N2) and O(N3), respectively. The improvement on three-dimensional systems is even more significant. We analyze and apply this technique to a class of semi-implicit integration factor method recently developed for stiff reaction–diffusion equations. Direct simulations on test equations along with applications to a morphogen system in two-dimensions and an intra-cellular signaling system in three-dimensions demonstrate an excellent efficiency of the new approach.

High Throughput Characterization of Combinatorial Histone Codes*

Young, Nicolas L.; DiMaggio, Peter A.; Plazas-Mayorca, Mariana D.; Baliban, Richard C.; Floudas, Christodoulos A.; Garcia, Benjamin A.
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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We present a novel method utilizing “saltless” pH gradient weak cation exchange-hydrophilic interaction liquid chromatography directly coupled to electron transfer dissociation (ETD) mass spectrometry for the automated on-line high throughput characterization of hypermodified combinatorial histone codes. This technique, performed on a low resolution mass spectrometer, displays an improvement over existing methods with an ∼100-fold reduction in sample requirements and analysis time. The scheme presented is capable of identifying all of the major combinatorial histone codes present in a sample in a 2-h analysis. The large N-terminal histone peptides are eluted by the pH and organic solvent weak cation exchange-hydrophilic interaction liquid chromatography gradient and directly introduced via nanoelectrospray ionization into a benchtop linear quadrupole ion trap mass spectrometer equipped with ETD. Each polypeptide is sequenced, and the modification sites are identified by ETD fragmentation. The isobaric trimethyl and acetyl modifications are resolved chromatographically and confidently distinguished by the synthesis of mass spectrometric and chromatographic information. We demonstrate the utility of the method by complete characterization of human histone H3.2 and histone H4 from butyrate-treated cells...

Quantitative Mass Spectrometry of Histones H3.2 and H3.3 in Suz12-deficient Mouse Embryonic Stem Cells Reveals Distinct, Dynamic Post-translational Modifications at Lys-27 and Lys-36*

Jung, Hye Ryung; Pasini, Diego; Helin, Kristian; Jensen, Ole N.
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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SUZ12 is a core component of the polycomb repressive complex 2 (PRC2) and is required for the differentiation of mouse embryonic stem cells (ESCs). PRC2 is associated with transcriptional repression via methylation of H3 Lys-27. We applied quantitative mass spectrometry to investigate the effects of Suz12 deficiency on H3.2 and H3.3 from mouse ESCs. Using high mass accuracy MS combined with CID or electron transfer dissociation (ETD) tandem mass spectrometry, we identified a total of 81 unique modified peptides from H3.2 and H3.3 and assigned 46 modifications at 22 different positions, including distinct coexisting modifications. In certain cases, high mass accuracy LTQ-Orbitrap MS/MS allowed precise localization of near isobaric coexisting PTMs such as trimethylation and acetylation within individual peptides. ETD MS/MS facilitated sequencing and annotation of phosphorylated histone peptides. The combined use of ETD and CID MS/MS increased the total number of identified modified peptides. Comparative quantitative analysis of histones from wild type and Suz12-deficient ESCs using stable isotope labeling with amino acids in cell culture and LC-MS/MS revealed a dramatic reduction of H3K27me2 and H3K27me3 and an increase of H3K27ac, thereby uncovering an antagonistic methyl/acetyl switch at H3K27. The reduction in H3K27 methylation and increase in H3K27 acetylation was accompanied by H3K36 acetylation and methylation. Estimation of the global isoform percentage of unmodified and modified histone peptides (amino acids 27–40) showed the relative distribution of distinct coexisting histone marks. Our study revealed limitations of antibody-based Western blotting methods for detection of coexisting protein modifications and demonstrated the utility of quantitative tandem mass spectrometry for detailed analysis of the dynamics of coexisting post-translational modifications in proteins.

Charge remote fragmentation in electron capture and electron transfer dissociations

Li, Xiaojuan; Lin, Cheng; Han, Liang; Costello, Catherine E.; O’Connor, Peter B.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Secondary fragmentations of three synthetic peptides (human αA crystallin peptide 1-11, the deamidated form of human βB2 crystallin peptide 4-14, and amyloid β peptide 25-35) were studied in both electron capture dissociation (ECD) and electron transfer dissociation (ETD) mode. In ECD, in addition to c and z• ion formations, charge remote fragmentations (CRF) of z• ions were abundant, resulting in internal fragment formation or partial/entire side chain losses from amino acids, sometimes several residues away from the backbone cleavage site, and to some extent multiple side chain losses. The internal fragments were observed in peptides with basic residues located in the middle of the sequences, which was different from most tryptic peptides with basic residues located at the C-terminus. These secondary cleavages were initiated by hydrogen abstraction at the α-, β-, or γ-position of the amino acid side chain. In comparison, ETD generates fewer CRF fragments than ECD. This secondary cleavage study will facilitate ECD/ETD spectra interpretation, and help de novo sequencing and database searching.

Improving Software Performance for Peptide Electron Transfer Dissociation Data Analysis by Implementation of Charge State- and Sequence-Dependent Scoring*

Baker, Peter R.; Medzihradszky, Katalin F.; Chalkley, Robert J.
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 use of electron transfer dissociation (ETD) fragmentation for analysis of peptides eluting in liquid chromatography tandem mass spectrometry experiments is increasingly common and can allow identification of many peptides and proteins in complex mixtures. Peptide identification is performed through the use of search engines that attempt to match spectra to peptides from proteins in a database. However, software for the analysis of ETD fragmentation data is currently less developed than equivalent algorithms for the analysis of the more ubiquitous collision-induced dissociation fragmentation spectra. In this study, a new scoring system was developed for analysis of peptide ETD fragmentation data that varies the ion type weighting depending on the precursor ion charge state and peptide sequence. This new scoring regime was applied to the analysis of data from previously published results where four search engines (Mascot, Open Mass Spectrometry Search Algorithm (OMSSA), Spectrum Mill, and X!Tandem) were compared (Kandasamy, K., Pandey, A., and Molina, H. (2009) Evaluation of several MS/MS search algorithms for analysis of spectra derived from electron transfer dissociation experiments. Anal. Chem. 81, 7170–7180). Protein Prospector identified 80% more spectra at a 1% false discovery rate than the most successful alternative searching engine in this previous publication. These results suggest that other search engines would benefit from the application of similar rules.

In Vivo Bioavailability and Therapeutic Assessment of Host-Guest Inclusion Phenomena for the Hydrophobic Molecule Etodolac: Pharmacodynamic and Pharmacokinetic Evaluation

Sinha, Vivek Ranjan; Amita, ; Goel, Honey
Fonte: Österreichische Apotheker-Verlagsgesellschaft Publicador: Österreichische Apotheker-Verlagsgesellschaft
Tipo: Artigo de Revista Científica
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The aim of present investigation was 1) to evaluate the in vivo bioavailability of an Etodolac (ETD)-β-cyclodextrin (β-CD) inclusion complex system prepared by kneading and spray drying techniques in rats, 2) to study the pharmacodynamic parameters in various animal models for analyzing the therapeutic response and, 3) to evaluate the pharmacokinetic profile of the drug administered. Inclusion complexation with β-CD enhanced the solubility of the drug, improved bioavailability and reduced ulcerogenicity of ETD in rats. Pharmacodynamic studies were carried out in normal LACA mice and pharmacokinetic evaluation was done in male Wistar rats. Pharmacokinetic parameters evaluated for the inclusion complexes revealed good correlation. The minimum dose necessary to produce analgesic or anti-arthritic activity was also decreased, indicating that the host-guest strategy that uses β-CD and ETD was very effective and could be successfully employed in the preparation of pharmaceutical formulations of anti-arthritics and analgesics.

Phosphoproteome Analysis Reveals Regulatory Sites in Major Pathways of Cardiac Mitochondria*

Deng, Ning; Zhang, Jun; Zong, Chenggong; Wang, Yueju; Lu, Haojie; Yang, Pengyuan; Wang, Wenhai; Young, Glen W.; Wang, Yibin; Korge, Paavo; Lotz, Christopher; Doran, Philip; Liem, David A.; Apweiler, Rolf; Weiss, James N.; Duan, Huilong; Ping, Peipei
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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Mitochondrial functions are dynamically regulated in the heart. In particular, protein phosphorylation has been shown to be a key mechanism modulating mitochondrial function in diverse cardiovascular phenotypes. However, site-specific phosphorylation information remains scarce for this organ. Accordingly, we performed a comprehensive characterization of murine cardiac mitochondrial phosphoproteome in the context of mitochondrial functional pathways. A platform using the complementary fragmentation technologies of collision-induced dissociation (CID) and electron transfer dissociation (ETD) demonstrated successful identification of a total of 236 phosphorylation sites in the murine heart; 210 of these sites were novel. These 236 sites were mapped to 181 phosphoproteins and 203 phosphopeptides. Among those identified, 45 phosphorylation sites were captured only by CID, whereas 185 phosphorylation sites, including a novel modification on ubiquinol-cytochrome c reductase protein 1 (Ser-212), were identified only by ETD, underscoring the advantage of a combined CID and ETD approach. The biological significance of the cardiac mitochondrial phosphoproteome was evaluated. Our investigations illustrated key regulatory sites in murine cardiac mitochondrial pathways as targets of phosphorylation regulation...

Characterization and Diagnostic Value of Amino Acid Side Chain Neutral Losses Following Electron-Transfer Dissociation

Xia, Qiangwei; Lee, M. Violet; Rose, Christopher M.; Marsh, Alyce J.; Hubler, Shane L.; Wenger, Craig D.; Coon, Joshua J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Using a large set of high mass accuracy and resolution ETD tandem mass spectra, we characterized ETD-induced neutral losses. From these data we deduced the chemical formula for 20 of these losses. Many of them have been previously observed in electron-capture dissociation (ECD) spectra, such as losses of the side chains of arginine, aspartic acid, glutamic acid, glutamine, asparagine, leucine, histidine, and carbamidomethylated cysteine residues. With this information, we examined the diagnostic value of these amino acid-specific losses. Among 1285 peptide–spectrum matches, 92.5% have agreement between neutral loss-derived peptide amino acid composition and the peptide sequences. Moreover, we show that peptides can be uniquely identified by using only the accurate precursor mass and amino acid composition based on neutral losses; the median number of sequence candidates from an accurate mass query is reduced from 21 to 8 by adding side chain loss information. Besides increasing confidence in peptide identification, our findings suggest the potential use of these diagnostic losses in ETD spectra to improve false discovery rate estimation and to enhance the performance of scoring functions in database search algorithms.

Electron Transfer Dissociation with Supplemental Activation to Differentiate Aspartic and Isoaspartic Residues in Doubly Charged Peptide Cations

Chan, Wai Yi Kelly; Chan, T. W. Dominic; O’Connor, Peter B.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Electron-transfer dissociation (ETD) with supplemental activation of the doubly charged deamidated tryptic digested peptide ions allows differentiation of isoaspartic acid and aspartic acid residues using c + 57 or z• − 57 peaks. The diagnostic peak clearly localizes and characterizes the isoaspartic acid residue. Supplemental activation in ETD of the doubly charged peptide ions involves resonant excitation of the charge reduced precursor radical cations and leads to further dissociation, including extra backbone cleavages and secondary fragmentation. Supplemental activation is essential to obtain a high quality ETD spectrum (especially for doubly charged peptide ions) with sequence information. Unfortunately, the low-resolution of the ion trap mass spectrometer makes detection of the diagnostic peak for the aspartic acid residue difficult due to interference with side-chain loss from arginine and glutamic acid residues.

4-HNE Adduct Stability Characterized by Collision-Induced Dissociation and Electron Transfer Dissociation Mass Spectrometry

Fritz, Kristofer S.; Kellersberger, Katherine A.; Gomez, Jose D.; Petersen, Dennis R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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4-hydroxynonenal (4-HNE) alters numerous proteomic and genomic processes. Understanding chemical mechanisms of 4-HNE interactions with biomolecules and their respective stabilities may lead to new discoveries in biomarkers for numerous diseases of oxidative stress. Collision-induced dissociation (CID) and electron transfer dissociation (ETD) MS/MS were utilized to examine the stability of a 4-HNE-Cys Michael adduct. CID conditions resulted in the neutral loss of 4-HNE, also known as a retro-Michael addition reaction (RMA). Consequently, performing ETD fragmentation on this same adduct did not result in RMA. Interestingly, 4-HNE adduct reduction via sodium borohydride (NaBH4) treatment stabilized against the CID induced RMA. In a direct comparison of three forms of 4-HNE adducts, computational modeling revealed sizeable shifts in the shape and orientation of the lowest unoccupied molecular orbital (LUMO) density around the 4-HNE-Cys moiety. These findings demonstrate that ETD MS/MS analysis can be used to improve the detection of 4-HNE-protein modifications by preventing RMA reactions from occurring.

Accurate Localization and Relative Quantification of Arginine Methylation Using Nanoflow Liquid Chromatography Coupled to Electron Transfer Dissociation and Orbitrap Mass Spectrometry

Wang, Hao; Straubinger, Robert M.; Aletta, John M.; Cao, Jin; Duan, Xiaotao; Yu, Haoying; Qu, Jun
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Protein arginine (Arg) methylation serves an important functional role in eukaryotic cells, and typically occurs in domains consisting of multiple Arg in close proximity. Localization of methylarginine (MA) within Arg-rich domains poses a challenge for mass spectrometry (MS)-based methods; the peptides are highly-charged under electrospray ionization (ESI), which limits the number of sequence-informative products produced by collision induced dissociation (CID), and loss of the labile methylation moieties during CID precludes effective fragmentation of the peptide backbone. Here the fragmentation behavior of Arg-rich peptides was investigated comprehensively using electron transfer dissociation (ETD) and CID for both methylated and unmodified glycine-/Arg-rich peptides (GAR), derived from residues 679-695 of human nucleolin, which contains methylation motifs that are widely-represented in biological systems. ETD produced abundant information for sequencing and MA localization, whereas CID failed to provide credible identification for any available charge state (z=2-4). Nevertheless, CID produced characteristic neutral losses that can be employed to distinguish among different types of MA, as suggested by previous works and confirmed here with product ion scans of high accuracy/resolution by an LTQ/Orbitrap. To analyze MA-peptides in relatively complex mixtures...

A Simple Approach to Assign Disulfide Connectivity Using Extracted Ion Chromatograms of Electron Transfer Dissociation Spectra

Clark, Daniel F; Go, Eden P; Desaire, Heather
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Increasing interest in production of protein-based pharmaceuticals (biotherapeutics) is accompanied by an increased need for verification of protein folding and correct disulfide bonding. Recombinant protein expression may produce aberrant disulfide bonds and could result in safety concerns or decreased efficacy. Thus, the thorough analysis of disulfide bonding is a necessity for protein therapeutics. The use of ETD facilitates this analysis because disulfide bonds are preferentially cleaved when subjected to ETD. Here, we make use of this well-characterized reaction to assign disulfide bonding networks by coupling the use of extracted ion chromatograms (XICs) of cysteine-containing peptides with ETD analysis to produce an efficient assignment approach for disulfide bonding. This method can be used to assign a disulfide pattern in a de novo fashion, to detect disulfide shuffling, and to provide information on heterogeneity, when more than one disulfide bonding pattern is present. The method was applied for assigning the disulfide-bonding network of a recombinant monomer of the HIV envelope protein gp120. It was found that one region of the protein, the V1/V2 loops, had significant heterogeneity in the disulfide bonds.

Chemical derivatization of peptide carboxyl groups for highly efficient electron transfer dissociation

Frey, Brian L.; Ladror, Daniel T.; Sondalle, Samuel B.; Krusemark, Casey J.; Jue, April L.; Coon, Joshua J.; Smith, Lloyd M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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The carboxyl groups of tryptic peptides were derivatized with a tertiary or quaternary amine labeling reagent to generate more highly charged peptide ions that fragment efficiently by electron transfer dissociation (ETD). All peptide carboxyl groups—aspartic and glutamic acid side-chains as well as C-termini—were derivatized with an average reaction efficiency of 99%. This nearly complete labeling avoids making complex peptide mixtures even more complex due to partially-labeled products, and it allows the use of static modifications during database searching. Alkyl tertiary amines were found to be the optimal labeling reagent among the four types tested. Charge states are substantially higher for derivatized peptides: a modified tryptic digest of bovine serum albumin (BSA) generates ∼90% of its precursor ions with z > 2, compared to less than 40% for the unmodified sample. The increased charge density of modified peptide ions yields highly efficient ETD fragmentation, leading to many additional peptide identifications and higher sequence coverage (e.g. 70% for modified versus only 43% for unmodified BSA). The utility of this labeling strategy was demonstrated on a tryptic digest of ribosomal proteins isolated from yeast cells. Peptide derivatization of this sample produced an increase in the number of identified proteins...

Confident and sensitive phosphoproteomics using combinations of collision induced dissociation and electron transfer dissociation☆

Collins, Mark O.; Wright, James C.; Jones, Matthew; Rayner, Julian C.; Choudhary, Jyoti S.
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
Publicado em 30/05/2014 Português
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•We report and benchmark a data analysis pipeline for phosphoproteomic data analysis.•Combined use of Mascot Percolator and turbo-SLoMo to compare fragmentation methods•CID and ETD fragmentation for phosphorylation site identification•Demonstrate the utility of data-dependent neutral loss triggered ETD fragmentation•High confidence of phosphoproteomic analysis using ETD/CID spectral pairs

Effector-triggered defence against apoplastic fungal pathogens

Stotz, Henrik U.; Mitrousia, Georgia K.; de Wit, Pierre J.G.M.; Fitt, Bruce D.L.
Fonte: Elsevier Science, Ltd Publicador: Elsevier Science, Ltd
Tipo: Artigo de Revista Científica
Publicado em /08/2014 Português
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•ETD is triggered by RLPs that engage the receptor-like kinase SOBIR1.•ETD triggers cell wall-related defence responses.•ETD does not eliminate apoplastic pathogens.

Rapid sensitive analysis of cysteine rich peptide venom components

Ueberheide, Beatrix M.; Fenyö, David; Alewood, Paul F.; Chait, Brian T.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
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Disulfide-rich peptide venoms from animals such as snakes, spiders, scorpions, and certain marine snails represent one of nature's great diversity libraries of bioactive molecules. The various species of marine cone shells have alone been estimated to produce >50,000 distinct peptide venoms. These peptides have stimulated considerable interest because of their ability to potently alter the function of specific ion channels. To date, only a small fraction of this immense resource has been characterized because of the difficulty in elucidating their primary structures, which range in size between 10 and 80 aa, include up to 5 disulfide bonds, and can contain extensive posttranslational modifications. The extraordinary complexity of crude venoms and the lack of DNA databases for many of the organisms of interest present major analytical challenges. Here, we describe a strategy that uses mass spectrometry for the elucidation of the mature peptide toxin components of crude venom samples. Key to this strategy is our use of electron transfer dissociation (ETD), a mass spectrometric fragmentation technique that can produce sequence information across the entire peptide backbone. However, because ETD only yields comprehensive sequence coverage when the charge state of the precursor peptide ion is sufficiently high and the m/z ratio is low...