The Experts below are selected from a list of 55035 Experts worldwide ranked by ideXlab platform
Joshua J Coon - One of the best experts on this subject based on the ideXlab platform.
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mapping physiological adp ribosylation using activated ion electron transfer Dissociation
Cell Reports, 2020Co-Authors: Sara C Buchlarsen, Joshua J Coon, Michael S Westphall, Jean M Lodge, Ivo A Hendriks, Martin Rykaer, Benjamin Furtwangler, Evgenia Shishkova, Michael L NielsenAbstract:Summary ADP-ribosylation (ADPr) is a post-translational modification that plays pivotal roles in a wide range of cellular processes. Mass spectrometry (MS)-based analysis of ADPr under physiological conditions, without relying on genetic or chemical perturbation, has been hindered by technical limitations. Here, we describe the applicability of activated ion electron transfer Dissociation (AI-ETD) for MS-based proteomics analysis of physiological ADPr using our unbiased Af1521 enrichment strategy. To benchmark AI-ETD, we profile 9,000 ADPr peptides mapping to >5,000 unique ADPr sites from a limited number of cells exposed to oxidative stress and identify 120% and 28% more ADPr peptides compared to contemporary strategies using ETD and Electron-Transfer higher-energy collisional Dissociation (EThcD), respectively. Under physiological conditions, AI-ETD identifies 450 ADPr sites on low-abundant proteins, including in vivo cysteine modifications on poly(ADP-ribosyl)polymerase (PARP) 8 and tyrosine modifications on PARP14, hinting at specialist enzymatic functions for these enzymes. Collectively, our data provide insights into the physiological regulation of ADPr.
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Top-Down Characterization of Proteins with Intact Disulfide Bonds Using Activated-Ion Electron Transfer Dissociation
2018Co-Authors: Matthew J. P. Rush, Michael S Westphall, Nicholas M Riley, Joshua J CoonAbstract:Here we report the fragmentation of disulfide linked intact proteins using activated-ion electron transfer Dissociation (AI-ETD) for top-down protein characterization. This fragmentation method is then compared to the alternative methods of beam-type collisional activation (HCD), electron transfer Dissociation (ETD), and electron transfer and higher-energy collision Dissociation (EThcD). We analyzed multiple precursor charge states of the protein standards bovine insulin, α-lactalbumin, lysozyme, β-lactoglobulin, and trypsin inhibitor. In all cases, we found that AI-ETD provides a boost in protein sequence coverage information and the generation of fragment ions from within regions enclosed by disulfide bonds. AI-ETD shows the largest improvement over the other techniques when analyzing highly disulfide linked and low charge density precursor ions. This substantial improvement is attributed to the concurrent irradiation of the gas phase ions while the Electron-Transfer reaction is taking place, mitigating nondissociative electron transfer, helping unfold the gas phase protein during the electron transfer event, and preventing disulfide bond reformation. We also show that AI-ETD is able to yield comparable sequence coverage information when disulfide bonds are left intact relative to proteins that have been reduced and alkylated. This work demonstrates that AI-ETD is an effective fragmentation method for the analysis of proteins with intact disulfide bonds, dramatically enhancing sequence ion generation and total sequence coverage compared to HCD and ETD
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The Value of Activated Ion Electron Transfer Dissociation for High-Throughput Top-Down Characterization of Intact Proteins
2018Co-Authors: Nicholas M. Riley, Michael S Westphall, Jacek W. Sikora, Henrique S. Seckler, Joseph B. Greer, Ryan T. Fellers, Richard D. Leduc, Paul M. Thomas, Neil L. Kelleher, Joshua J CoonAbstract:High-throughput top-down proteomic experiments directly identify proteoforms in complex mixtures, making high quality tandem mass spectra necessary to deeply characterize proteins with many sources of variation. Collision-based Dissociation methods offer expedient data acquisition but often fail to extensively fragment proteoforms for thorough analysis. Electron-driven Dissociation methods are a popular alternative approach, especially for precursor ions with high charge density. Combining infrared photoactivation concurrent with electron transfer Dissociation (ETD) reactions, i.e., activated ion ETD (AI-ETD), can significantly improve ETD characterization of intact proteins, but benefits of AI-ETD have yet to be quantified in high-throughput top-down proteomics. Here, we report the first application of AI-ETD to LC-MS/MS characterization of intact proteins (
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negative electron transfer Dissociation sequencing of increasingly sulfated glycosaminoglycan oligosaccharides on an orbitrap mass spectrometer
Journal of the American Society for Mass Spectrometry, 2017Co-Authors: Franklin E Leach, Joshua J Coon, Nicholas M Riley, Michael S Westphall, Jonathan I AmsterAbstract:The structural characterization of sulfated glycosaminoglycan (GAG) carbohydrates remains an important target for analytical chemists attributable to challenges introduced by the natural complexity of these mixtures and the defined need for molecular-level details to elucidate biological structure–function relationships. Tandem mass spectrometry has proven to be the most powerful technique for this purpose. Previously, electron detachment Dissociation (EDD), in comparison to other methods of ion activation, has been shown to provide the largest number of useful cleavages for de novo sequencing of GAG oligosaccharides, but such experiments are restricted to Fourier transform ion cyclotron resonance mass spectrometers (FTICR-MS). Negative electron transfer Dissociation (NETD) provides similar fragmentation results, and can be achieved on any mass spectrometry platform that is designed to accommodate ion–ion reactions. Here, we examine for the first time the effectiveness of NETD-Orbitrap mass spectrometry for the structural analysis of GAG oligosaccharides. Compounds ranging in size from tetrasaccharides to decasaccharides were dissociated by NETD, producing both glycosidic and cross-ring cleavages that enabled the location of sulfate modifications. The highly-sulfated, heparin-like synthetic GAG, ArixtraTM, was also successfully sequenced by NETD. In comparison to other efforts to sequence GAG chains without fully ionized sulfate constituents, the occurrence of sulfate loss peaks is minimized by judicious precursor ion selection. The results compare quite favorably to prior results with electron detachment Dissociation (EDD). Significantly, the duty cycle of the NETD experiment is sufficiently short to make it an effective tool for on-line separations, presenting a straightforward path for selective, high-throughput analysis of GAG mixtures.
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Implementation of Activated Ion Electron Transfer Dissociation on a Quadrupole-Orbitrap-Linear Ion Trap Hybrid Mass Spectrometer
2017Co-Authors: Nicholas M. Riley, Alexander S Hebert, Michael S Westphall, Joshua J CoonAbstract:Using concurrent IR photoactivation during electron transfer Dissociation (ETD) reactions, i.e., activated ion ETD (AI-ETD), significantly increases Dissociation efficiency resulting in improved overall performance. Here we describe the first implementation of AI-ETD on a quadrupole-Orbitrap-quadrupole linear ion trap (QLT) hybrid MS system (Orbitrap Fusion Lumos) and demonstrate the substantial benefits it offers for peptide characterization. First, we show that AI-ETD can be implemented in a straightforward manner by fastening the laser and guiding optics to the instrument chassis itself, making alignment with the trapping volume of the QLT simple and robust. We then characterize the performance of AI-ETD using standard peptides in addition to a complex mixtures of tryptic peptides using LC–MS/MS, showing not only that AI-ETD can nearly double the identifications achieved with ETD alone but also that it outperforms the other available supplemental activation methods (ETcaD and EThcD). Finally, we introduce a new activation scheme called AI-ETD+ that combines AI-ETD in the high pressure cell of the QLT with a short infrared multiphoton Dissociation (IRMPD) activation in the low-pressure cell. This reaction scheme introduces no addition time to the scan duty cycle but generates MS/MS spectra rich in b/y-type and c/z•-type product ions. The extensive generation of fragment ions in AI-ETD+ substantially increases peptide sequence coverage while also improving peptide identifications over all other ETD methods, making it a valuable new tool for hybrid fragmentation approaches
Katalin F Medzihradszky - One of the best experts on this subject based on the ideXlab platform.
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improving software performance for peptide electron transfer Dissociation data analysis by implementation of charge state and sequence dependent scoring
Molecular & Cellular Proteomics, 2010Co-Authors: Peter R Baker, Katalin F Medzihradszky, Robert J ChalkleyAbstract: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.
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affinity enrichment and characterization of mucin core 1 type glycopeptides from bovine serum
Molecular & Cellular Proteomics, 2009Co-Authors: Zsuzsanna Darula, Katalin F MedzihradszkyAbstract:The lack of consensus sequence, common core structure, and universal endoglycosidase for the release of O-linked oligosaccharides makes O-glycosylation more difficult to tackle than N-glycosylation. Structural elucidation by mass spectrometry is usually inconclusive as the CID spectra of most glycopeptides are dominated by carbohydrate-related fragments, preventing peptide identification. In addition, O-linked structures also undergo a gas-phase rearrangement reaction, which eliminates the sugar without leaving a telltale sign at its former attachment site. In the present study we report the enrichment and mass spectrometric analysis of proteins from bovine serum bearing Gal1–3GalNAc (mucin core-1 type) structures and the analysis of O-linked glycopeptides utilizing electron transfer Dissociation and high resolution, high mass accuracy precursor ion measurements. Electron transfer Dissociation (ETD) analysis of intact glycopeptides provided sufficient information for the identification of several glycosylation sites. However, glycopeptides frequently feature precursor ions of low charge density (m/z > 850) that will not undergo efficient ETD fragmentation. Exoglycosidase digestion was utilized to reduce the mass of the molecules while retaining their charge. ETD analysis of species modified by a single GalNAc at each site was significantly more successful in the characterization of multiply modified molecules. We report the unambiguous identification of 21 novel glycosylation sites. We also detail the limitations of the enrichment method as well as the ETD analysis. Molecular & Cellular Proteomics 8:2515–2526, 2009.
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affinity enrichment and characterization of mucin core 1 type glycopeptides from bovine serum
Molecular & Cellular Proteomics, 2009Co-Authors: Zsuzsanna Darula, Katalin F MedzihradszkyAbstract:The lack of consensus sequence, common core structure, and universal endoglycosidase for the release of O-linked oligosaccharides makes O-glycosylation more difficult to tackle than N-glycosylation. Structural elucidation by mass spectrometry is usually inconclusive as the CID spectra of most glycopeptides are dominated by carbohydrate-related fragments, preventing peptide identification. In addition, O-linked structures also undergo a gas-phase rearrangement reaction, which eliminates the sugar without leaving a telltale sign at its former attachment site. In the present study we report the enrichment and mass spectrometric analysis of proteins from bovine serum bearing Gal1–3GalNAc (mucin core-1 type) structures and the analysis of O-linked glycopeptides utilizing electron transfer Dissociation and high resolution, high mass accuracy precursor ion measurements. Electron transfer Dissociation (ETD) analysis of intact glycopeptides provided sufficient information for the identification of several glycosylation sites. However, glycopeptides frequently feature precursor ions of low charge density (m/z > 850) that will not undergo efficient ETD fragmentation. Exoglycosidase digestion was utilized to reduce the mass of the molecules while retaining their charge. ETD analysis of species modified by a single GalNAc at each site was significantly more successful in the characterization of multiply modified molecules. We report the unambiguous identification of 21 novel glycosylation sites. We also detail the limitations of the enrichment method as well as the ETD analysis. Molecular & Cellular Proteomics 8:2515–2526, 2009.
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affinity enrichment and characterization of mucin core 1 type glycopeptides from bovine serum
Molecular & Cellular Proteomics, 2009Co-Authors: Zsuzsanna Darula, Katalin F MedzihradszkyAbstract:The lack of consensus sequence, common core structure, and universal endoglycosidase for the release of O-linked oligosaccharides makes O-glycosylation more difficult to tackle than N-glycosylation. Structural elucidation by mass spectrometry is usually inconclusive as the CID spectra of most glycopeptides are dominated by carbohydrate-related fragments, preventing peptide identification. In addition, O-linked structures also undergo a gas-phase rearrangement reaction, which eliminates the sugar without leaving a telltale sign at its former attachment site. In the present study we report the enrichment and mass spectrometric analysis of proteins from bovine serum bearing Galβ1–3GalNAcα (mucin core-1 type) structures and the analysis of O-linked glycopeptides utilizing electron transfer Dissociation and high resolution, high mass accuracy precursor ion measurements. Electron transfer Dissociation (ETD) analysis of intact glycopeptides provided sufficient information for the identification of several glycosylation sites. However, glycopeptides frequently feature precursor ions of low charge density (m/z > ∼850) that will not undergo efficient ETD fragmentation. Exoglycosidase digestion was utilized to reduce the mass of the molecules while retaining their charge. ETD analysis of species modified by a single GalNAc at each site was significantly more successful in the characterization of multiply modified molecules. We report the unambiguous identification of 21 novel glycosylation sites. We also detail the limitations of the enrichment method as well as the ETD analysis.
Barry L Karger - One of the best experts on this subject based on the ideXlab platform.
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Analysis of Isoaspartic Acid by Selective Proteolysis with Asp-N and Electron Transfer Dissociation Mass Spectrometry
2015Co-Authors: Shujia Dai, Barry L Karger, Zhaohui Sunny ZhouAbstract: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, our approach should expedite the unbiased discovery of isoAsp
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ultrasensitive characterization of site specific glycosylation of affinity purified haptoglobin from lung cancer patient plasma using 10 μm i d porous layer open tubular liquid chromatography linear ion trap collision induced Dissociation electron transfer Dissociation mass spectrometry
Analytical Chemistry, 2011Co-Authors: Dongdong Wang, Marina Hincapie, Tomas Rejtar, Barry L KargerAbstract: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 the glycan structure and attached peptide site. However, due to the low ionization efficiency of glycopeptides during electrospray ionization, 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 matrixes. To improve the sensitivity of LC−MS analysis for glycopeptides, an ultranarrow porous layer open tubular (PLOT) LC column (2.5 m × 10 μm i.d.) was coupled to a linear ion trap (LTQ) collision-induced Dissociation/electron transfer Dissociation mass spectrometer to provide sensitive analysis of N-linked protein glycosylation heterogeneity. The potential of the developed method is demonstrated by ...
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identification of the unpaired cysteine status and complete mapping of the 17 disulfides of recombinant tissue plasminogen activator using lc ms with electron transfer Dissociation collision induced Dissociation
Analytical Chemistry, 2010Co-Authors: Shiaw Lin Wu, William S Hancock, Haitao Jiang, Barry L KargerAbstract: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 an ∼60:40 ratio, respectively. The analysis was conducted using a multifragmentation approach consisting of electron transfer Dissociation (ETD) and collision induced Dissociation (CID), in combination with a multienzyme 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 comple...
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identification of the unpaired cysteine status and complete mapping of the 17 disulfides of recombinant tissue plasminogen activator using lc ms with electron transfer Dissociation collision induced Dissociation
Analytical Chemistry, 2010Co-Authors: Haitao Jiang, William S Hancock, Barry L KargerAbstract: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 an approximately 60:40 ratio, respectively. The analysis was conducted using a multifragmentation approach consisting of electron transfer Dissociation (ETD) and collision induced Dissociation (CID), in combination with a multienzyme 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-MS(3) 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, upon stimulation by either oxidative or ischemic stress.
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mass spectrometric determination of disulfide linkages in recombinant therapeutic proteins using online lc ms with electron transfer Dissociation
Analytical Chemistry, 2009Co-Authors: Haitao Jiang, Shujia Dai, William S Hancock, Barry L KargerAbstract:In the biotechnology industry, the generation of incorrectly folded recombinant proteins, either from an E.coli expression system or from an overexpressed CHO cell line (disulfide scrambling), is often a great concern as such incorrectly folded forms may not be completely removed in the final product. Thus, significant efforts have been devoted to map disulfide bonds to ensure drug quality. Similar to ECD, disulfide bond cleavages are preferred over peptide backbone fragmentation in ETD. Thus, an online LC−MS strategy combining collision-induced Dissociation (CID-MS2), Electron-Transfer Dissociation (ETD-MS2), and CID of an isolated product ion derived from ETD (MS3) has been used to characterize disulfide-linked peptides. Disulfide-linked peptide ions were identified by CID and ETD fragmentation, and the disulfide-dissociated (or partially dissociated) peptide ions were characterized in the subsequent MS3 step. The online LC−MS approach is successfully demonstrated in the characterization of disulfide li...
Jonathan I Amster - One of the best experts on this subject based on the ideXlab platform.
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online capillary zone electrophoresis negative electron transfer Dissociation tandem mass spectrometry of glycosaminoglycan mixtures
International Journal of Mass Spectrometry, 2019Co-Authors: Morgan Stickney, Franklin E Leach, Robert J Linhardt, Patience Sanderson, Fuming Zhang, Jonathan I AmsterAbstract:Abstract Glycosaminoglycans (GAGs) are important biological molecules that are highly anionic and occur in nature as complex mixtures. A platform that combines capillary zone electrophoresis (CZE) separations with mass spectrometry (MS) and gas-phase sequencing by using negative electron transfer Dissociation (NETD) is shown to be efficacious for the structural analysis of GAG mixtures. CZE is a separation method well suited to the highly negatively charged nature of GAGs. NETD is an electron-based ion activation method that enables the generation of informative fragments with retention of the labile sulfate half-ester modification that determine specific GAG function. Here we combine for the first time NETD and CZE for assigning the structures of GAG oligomers present in mixtures. The speed of ion activation by NETD is found to couple well with the narrow peaks resulting from CZE migration. The platform was optimized with mixtures of GAG tetrasaccharide standards. The potential of the platform is demonstrated by the analysis of enoxaparin, a complex mixture of low molecular weight heparins, which was separated by CZE within 30 min and characterized by NETD MS/MS in one online experiment. 37 unique molecular compositions have been identified in enoxaparin using CZE-MS and 9 structures have been assigned with CZE-NETD-MS/MS.
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negative electron transfer Dissociation sequencing of increasingly sulfated glycosaminoglycan oligosaccharides on an orbitrap mass spectrometer
Journal of the American Society for Mass Spectrometry, 2017Co-Authors: Franklin E Leach, Joshua J Coon, Nicholas M Riley, Michael S Westphall, Jonathan I AmsterAbstract:The structural characterization of sulfated glycosaminoglycan (GAG) carbohydrates remains an important target for analytical chemists attributable to challenges introduced by the natural complexity of these mixtures and the defined need for molecular-level details to elucidate biological structure–function relationships. Tandem mass spectrometry has proven to be the most powerful technique for this purpose. Previously, electron detachment Dissociation (EDD), in comparison to other methods of ion activation, has been shown to provide the largest number of useful cleavages for de novo sequencing of GAG oligosaccharides, but such experiments are restricted to Fourier transform ion cyclotron resonance mass spectrometers (FTICR-MS). Negative electron transfer Dissociation (NETD) provides similar fragmentation results, and can be achieved on any mass spectrometry platform that is designed to accommodate ion–ion reactions. Here, we examine for the first time the effectiveness of NETD-Orbitrap mass spectrometry for the structural analysis of GAG oligosaccharides. Compounds ranging in size from tetrasaccharides to decasaccharides were dissociated by NETD, producing both glycosidic and cross-ring cleavages that enabled the location of sulfate modifications. The highly-sulfated, heparin-like synthetic GAG, ArixtraTM, was also successfully sequenced by NETD. In comparison to other efforts to sequence GAG chains without fully ionized sulfate constituents, the occurrence of sulfate loss peaks is minimized by judicious precursor ion selection. The results compare quite favorably to prior results with electron detachment Dissociation (EDD). Significantly, the duty cycle of the NETD experiment is sufficiently short to make it an effective tool for on-line separations, presenting a straightforward path for selective, high-throughput analysis of GAG mixtures.
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negative electron transfer Dissociation of glycosaminoglycans
Analytical Chemistry, 2010Co-Authors: Jeremy J Wolff, Michael L Easterling, Desmond Allen Kaplan, Franklin E Leach, Tatiana N Laremore, Robert J Linhardt, Jonathan I AmsterAbstract:Structural characterization of glycosaminoglycans (GAGs) has been a challenge in the field of mass spectrometry, and the application of electron detachment Dissociation (EDD) Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) has shown great promise to GAG oligosaccharide characterization in a single tandem mass spectrometry experiment. In this work, we apply the technique of negative electron transfer Dissociation (NETD) to GAGs on a commercial ion trap mass spectrometer. NETD of GAGs, using fluoranthene or xenon as the reagent gas, produces fragmentation very similar to previously observed EDD fragmentation. Using fluoranthene or xenon, both glycosidic and cross-ring cleavages are observed, as well as even- and odd-electron products. The loss of SO(3) can be minimized and an increase in cross-ring cleavages is observed if a negatively charged carboxylate is present during NETD, which can be controlled by the charge state or the addition of sodium. NETD effectively dissociates GAGs up to eight saccharides in length, but the low resolution of the ion trap makes assigning product ions difficult. Similar to EDD, NETD is also able to distinguish the epimers iduronic acid from glucuronic acid in heparan sulfate tetrasaccharides and suggests that a radical intermediate plays an important role in distinguishing these epimers. These results demonstrate that NETD is effective at characterizing GAG oligosaccharides in a single tandem mass spectrometry experiment on a widely available mass spectrometry platform.
Cheng Lin - One of the best experts on this subject based on the ideXlab platform.
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characterization and quantification of highly sulfated glycosaminoglycan isomers by gated trapped ion mobility spectrometry negative electron transfer Dissociation ms ms
Analytical Chemistry, 2019Co-Authors: Juan Wei, Catherine E Costello, Yang Tang, Mark E Ridgeway, Melvin A Park, Joseph Zaia, Cheng LinAbstract:Glycosaminoglycans (GAGs) play vital roles in many biological processes and are naturally present as complex mixtures of polysaccharides with tremendous structural heterogeneity, including many structural isomers. Mass spectrometric analysis of GAG isomers, in particular highly sulfated heparin (Hep) and heparan sulfate (HS), is challenging because of their structural similarity and facile sulfo losses during analysis. Herein, we show that highly sulfated Hep/HS isomers may be resolved by gated-trapped ion mobility spectrometry (gated-TIMS) with negligible sulfo losses. Subsequent negative electron transfer Dissociation (NETD) tandem mass spectrometry (MS/MS) analysis of TIMS-separated Hep/HS isomers generated extensive glycosidic and cross-ring fragments for confident isomer differentiation and structure elucidation. The high mobility resolution and preservation of labile sulfo modifications afforded by gated-TIMS MS analysis also allowed relative quantification of highly sulfated heparin isomers. These ...
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Characterization and Quantification of Highly Sulfated Glycosaminoglycan Isomers by Gated-Trapped Ion Mobility Spectrometry Negative Electron Transfer Dissociation MS/MS
2019Co-Authors: Juan Wei, Catherine E Costello, Yang Tang, Mark E Ridgeway, Melvin A Park, Joseph Zaia, Cheng LinAbstract:Glycosaminoglycans (GAGs) play vital roles in many biological processes and are naturally present as complex mixtures of polysaccharides with tremendous structural heterogeneity, including many structural isomers. Mass spectrometric analysis of GAG isomers, in particular highly sulfated heparin (Hep) and heparan sulfate (HS), is challenging because of their structural similarity and facile sulfo losses during analysis. Herein, we show that highly sulfated Hep/HS isomers may be resolved by gated-trapped ion mobility spectrometry (gated-TIMS) with negligible sulfo losses. Subsequent negative electron transfer Dissociation (NETD) tandem mass spectrometry (MS/MS) analysis of TIMS-separated Hep/HS isomers generated extensive glycosidic and cross-ring fragments for confident isomer differentiation and structure elucidation. The high mobility resolution and preservation of labile sulfo modifications afforded by gated-TIMS MS analysis also allowed relative quantification of highly sulfated heparin isomers. These results show that the gated-TIMS-NETD MS/MS approach is useful for both qualitative and quantitative analysis of highly sulfated Hep/HS compounds in a manner not possible with other techniques
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charge remote fragmentation in electron capture and electron transfer Dissociation
Journal of the American Society for Mass Spectrometry, 2010Co-Authors: Cheng Lin, Liang Han, Catherine E Costello, Peter B OconnorAbstract: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.