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Helen J Cooper - One of the best experts on this subject based on the ideXlab platform.

Roman A. Zubarev - One of the best experts on this subject based on the ideXlab platform.

  • Radical a-Ions in Electron Capture Dissociation: On the Origin of Species
    Journal of the American Society for Mass Spectrometry, 2012
    Co-Authors: Roman A. Zubarev, David M. Good, Mikhail M. Savitski
    Abstract:

    Radical a* ions appear in electron capture Dissociation mass spectra sporadically, but sometimes with high intensity. Mechanistically, radical a ions are hypothesized to arise due to thermodynamically disadvantaged charge solvation on the backbone nitrogen (instead of carbonyl), which upon neutralization produces a hypervalent group instantly fragmenting into a radical b* and conventional y' ion. The former species is unstable and, after releasing a CO molecule, decays to an a* ion. Here we validate this scenario by direct observation of the complementarity of a* and y' ions by interrogation of an ECD MS/MS database of >10,000 doubly and >5,000 triply charged tryptic peptides. Intriguingly, the most abundant a*/y' pairs are found to come from the cleavage of the same backbone link as the most abundant c' and z* complementary ions. This result gives strong support to the “local” N-Cα bond cleavage mechanism, in which the Dissociation occurs at the site of charge solvation. However, a second strong peak is observed in the c'/z* fragment distribution four residues away from the a*/y' cleavage, which supports the indirect N-Cα bond cleavage mechanism. The size distribution of a ions from doubly (but not triply!) charged precursors shows deficit of a3 ions, and possibly a6 ions.

  • Effects of peptide backbone amide-to-ester bond substitution on the cleavage frequency in electron capture Dissociation and collision-activated Dissociation.
    Journal of the American Society for Mass Spectrometry, 2011
    Co-Authors: Frank Kjeldsen, Roman A. Zubarev
    Abstract:

    Probing the mechanism of electron capture Dissociation on variously modified model peptide polycations has resulted in discovering many ways to prevent or reduce \( {\text{N}} - {{\text{C}}_α } \) bond fragmentation. Here we report on a rare finding of how to increase the backbone bond Dissociation rate. In a number of model peptides, amide-to-ester backbone bond substitution increased the frequency of \( {\text{O}} - {{\text{C}}_α } \) bond cleavage (an analogue of \( {\text{N}} - {{\text{C}}_α } \) bonds in normal peptides) by several times, at the expense of reduced frequency of cleavages of the neighboring \( {\text{N}} - {{\text{C}}_α } \) bonds. In contrast, the ester linkage was only marginally broken in collisional Dissociation. These results further highlight the complementarity of the reaction mechanisms in electron capture Dissociation (ECD) and collision-activated Dissociation (CAD). It is proposed that the effects of amide-to-ester bond substitution on fragmentation are mainly due to the differences in product ion stability (ECD, CAD) as well as proton affinity (CAD). This proposal is substantiated by calculations using density functional theory. The implications of these results in relation to the current understanding of the mechanisms of electron capture Dissociation and electron transfer Dissociation are discussed.

  • effects of peptide backbone amide to ester bond substitution on the cleavage frequency in electron capture Dissociation and collision activated Dissociation
    Journal of the American Society for Mass Spectrometry, 2011
    Co-Authors: Frank Kjeldsen, Roman A. Zubarev
    Abstract:

    Probing the mechanism of electron capture Dissociation on variously modified model peptide polycations has resulted in discovering many ways to prevent or reduce \( {\text{N}} - {{\text{C}}_α } \) bond fragmentation. Here we report on a rare finding of how to increase the backbone bond Dissociation rate. In a number of model peptides, amide-to-ester backbone bond substitution increased the frequency of \( {\text{O}} - {{\text{C}}_α } \) bond cleavage (an analogue of \( {\text{N}} - {{\text{C}}_α } \) bonds in normal peptides) by several times, at the expense of reduced frequency of cleavages of the neighboring \( {\text{N}} - {{\text{C}}_α } \) bonds. In contrast, the ester linkage was only marginally broken in collisional Dissociation. These results further highlight the complementarity of the reaction mechanisms in electron capture Dissociation (ECD) and collision-activated Dissociation (CAD). It is proposed that the effects of amide-to-ester bond substitution on fragmentation are mainly due to the differences in product ion stability (ECD, CAD) as well as proton affinity (CAD). This proposal is substantiated by calculations using density functional theory. The implications of these results in relation to the current understanding of the mechanisms of electron capture Dissociation and electron transfer Dissociation are discussed.

  • Side-chain losses in electron capture Dissociation to improve peptide identification.
    Analytical chemistry, 2007
    Co-Authors: Mikhail M. Savitski, Michael L. Nielsen, Roman A. Zubarev
    Abstract:

    Analysis of a database of some 20 000 conventional Electron-Capture Dissociation (ECD) mass spectra of doubly charged ions belonging to tryptic peptides revealed widespread appearance of w ions and related u ions that are due to partial side chain losses from radical z• ions. Half of all z• ions that begin with Leu or Ile produce w ions in conventional one-scan ECD mass spectra, which differentiates these isomeric residues with >97% reliability. Other residues exhibiting equally frequent side chain losses are Gln, Glu, Asp, and Met (cysteine was not included in this work). Unexpectedly, Asp lost not a radical group like other amino acids but a molecule CO2, thus giving rise to a radical w• ion with the possibility of a radical cascade. Losses from amino acids as distant as seven residues away from the cleavage site were detected. The mechanism of such losses seems to be related to radical migration from the original site at the αCn atom in a zn• ion to other αC and βC atoms. The side chain losses confirm ...

  • characterization of an n acylated glucagon like peptide 1 derivative by electron capture Dissociation
    Journal of the American Society for Mass Spectrometry, 2005
    Co-Authors: Kim F Haselmann, Per F Nielsen, Roman A. Zubarev
    Abstract:

    An N-acylated glucagon-like peptide 1 derivative was characterized by Fourier transform ion cyclotron resonance mass spectrometry. Both electron capture Dissociation (ECD) and sustained off-resonance irradiation collisionally activated Dissociation (SORI-CAD) were employed. While ECD revealed full sequence coverage, site of modification, branching point, structure of the palmitoylated modification, SORI-CAD produced less complete and more ambiguous information attributable to facile losses of the fatty acid group from both parent and fragments. Thus, ECD showed a superior characterization performance over SORI-CAD in analysis of N-acylated polypeptides.

Per Håkansson - One of the best experts on this subject based on the ideXlab platform.

Yury O. Tsybin - One of the best experts on this subject based on the ideXlab platform.

  • Charge Location Directs Electron Capture Dissociation of Peptide Dications
    Journal of the American Society for Mass Spectrometry, 2006
    Co-Authors: Yury O. Tsybin, Kim F Haselmann, Mark R Emmett, Christopher L. Hendrickson, Alan G. Marshall
    Abstract:

    The effect of peptide dication charge location on electron capture Dissociation (ECD) fragmentation pattern is investigated. ECD fragmentation patterns are compared for peptides with amide and free acid C-terminal groups. ECD of free acid compared with C-terminally amidated peptides with basic residues near the N-terminus demonstrates increased formation of a-type ions. Similarly, ECD of free acid compared with C-terminally amidated peptides with basic residues near the C-terminus exhibits increased formation of y-type ions. Alteration of the peptide sequence to inhibit the formation of charged side chains (i.e., amino acid substitution and acetylation) provides further evidence for charge location effect on ECD. We propose that formation of zwitterionic peptide structures increases the likelihood of amide nitrogen protonation (versus basic side chains), which is responsible for the increase in a- and y-type ion formation.

  • Impact of ion magnetron motion on electron capture Dissociation Fourier transform ion cyclotron resonance mass spectrometry
    International Journal of Mass Spectrometry, 2006
    Co-Authors: Yury O. Tsybin, Christopher L. Hendrickson, Steven C. Beu, Alan G. Marshall
    Abstract:

    Abstract Electron capture Dissociation (ECD) efficiency in a 9.4 T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer varies periodically with the time interval between ion and electron injection. The observed modulation frequency correlates to within 1% with ion magnetron frequency, most probably due to misalignment between the ion beam and the electron beam. The optimum ECD conditions are obtained by correctly phasing electron injection with the ion magnetron motion. Displacement of the trapped ion cloud by variation of the ICR trap radial electric field decreases ECD efficiency modulation amplitude. Experiments directly suggest that only ions interacting with electrons at the moment of electron injection participate in ECD reactions.

  • Peptide and protein characterization by high-rate electron capture Dissociation fourier transform ion cyclotron resonance mass spectrometry
    Journal of mass spectrometry : JMS, 2004
    Co-Authors: Yury O. Tsybin, Goekhan Baykut, Matthias Witt, Margareta Ramström, Per Håkansson
    Abstract:

    The analytical utility of the electron capture Dissociation (ECD) technique, developed by McLafferty and co-workers, has substantially improved peptide and protein characterization using Fourier tr ...

  • Electron capture Dissociation Fourier transform ion cyclotron resonance mass spectrometry in the electron energy range 0-50 eV.
    Rapid communications in mass spectrometry : RCM, 2004
    Co-Authors: Yury O. Tsybin, Goekhan Baykut, Matthias Witt, Per Håkansson
    Abstract:

    Electron capture Dissociation (ECD) of polypeptide cations was obtained with pencil and hollow electron beams for both sidekick and gas-assisted dynamic ion trapping (GADT) using Fourier transform ...

  • High Rate Electron Capture Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
    2004
    Co-Authors: Yury O. Tsybin
    Abstract:

    Advances in science and technology during the past decade have greatly enhanced the level of the structural investigation of macromolecules – peptides and proteins. Biological mass spectrometry has become one of the most precise and sensitive techniques in peptide and protein analysis. However, increasing demands of biotechnological applications require further progress to be made.In the present thesis the development and improvement of peptide and protein characterization methods and techniques based on ion-electron and ion-photon reactions in electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry are described. The focus is on the development of the electron capture Dissociation method, recently discovered by the group of professor McLafferty, into a high rate, efficient tandem mass spectrometrical technique.The rate and reliability of the electron capture Dissociation technique were greatly increased by implementation of low-energy pencil electron beam injection systems based on indirectly heated dispenser cathodes. Further implementation of a hollow electron beam injection system combined, in a single experimental configuration, two rapid fragmentation techniques, high rate electron capture Dissociation and infrared multiphoton Dissociation. Simultaneous and consecutive irradiations of trapped ions with electrons and photons extended the possibilities for ion activation/Dissociation reaction schemes and lead to improved peptide and protein characterization. Using these improvements, high rate electron capture Dissociation was employed in time-limited experiments, such as liquid chromatography–tandem mass spectrometry and capillary electrophoresis-tandem mass spectrometry.The analytical applications of the developed techniques have been demonstrated in top-down sequencing of peptides and proteins up to 29 kDa, improved sequencing of peptides with multiple disulfide bridges and secondary fragmentation (w-ion formation), as well as extended characterization of peptide mixtures separated by liquid chromatography and capillary electrophoresis. For instance, the Dissociation of peptides resulting from enzymatic digestion of proteins provided complementary structural information on peptides and proteins, as well as their post-translational modifications.

Takashi Baba - One of the best experts on this subject based on the ideXlab platform.

  • Electron Capture Dissociation in a Branched Radio-Frequency Ion Trap
    Analytical chemistry, 2014
    Co-Authors: Takashi Baba, J. Larry Campbell, J. C. Yves Le Blanc, James W. Hager, Bruce A. Thomson
    Abstract:

    We have developed a high-throughput electron capture Dissociation (ECD) device coupled to a quadrupole time-of-flight mass spectrometer using novel branched radio frequency ion trap architecture. With this device, a low-energy electron beam can be injected orthogonally into the analytical ion beam with independent control of both the ion and electron beams. While ions and electrons can interact in a “flow-through” mode, we observed a large enhancement in ECD efficiency by introducing a short ion trapping period at the region of ion and electron beam intersection. This simultaneous trapping mode still provides up to five ECD spectra per second while operating in an information-dependent acquisition workflow. Coupled to liquid chromatography (LC), this LC-ECD workflow provides good sequence coverage for both trypsin and Lys C digests of bovine serum albumin, providing ECD spectra for doubly charged precursor ions with very good efficiency.

  • Elucidating the sequence of intact bioactive peptides by using electron capture Dissociation and hot electron capture Dissociation in a linear radio‐frequency quadrupole ion trap
    Rapid communications in mass spectrometry : RCM, 2013
    Co-Authors: Hiroyuki Satake, Naomi Manri, Akihito Kaneko, Atsumu Hirabayashi, Hideki Hasegawa, Yuichiro Hashimoto, Takashi Baba, Takeshi Sakamoto, Katsuyoshi Masuda
    Abstract:

    RATIONALE Electron capture Dissociation (ECD) is useful tool for sequencing of peptides and proteins with post-translational modifications. To increase the sequence coverage for peptides and proteins, it is important to develop ECD device with high fragmentation efficiency. METHODS Sequence analysis of intact undigested bioactive peptides (3000–5000 Da) was performed by use of electron capture Dissociation (rf-ECD) and collision-induced Dissociation (CID) in a linear radio-frequency quadrupole ion trap that was coupled to a time-of-flight mass spectrometer. We applied rf-ECD, hot rf-ECD (rf-ECD with high electron energy), and CID for intact bioactive peptide ions of various charge states and evaluated the sequence coverage of their fragment spectra. RESULTS Hot rf-ECD produced a higher number of c- and z-type fragment ions of modified peptide ions as electron energy increased in lower charged peptide ions, and sequence coverage greater than 80% was obtained compared with the CID case (40–80%). CONCLUSIONS The result indicates that intact bioactive modified peptides (Ghrelin, ANP) were correctly identified by use of hot rf-ECD. Copyright © 2013 John Wiley & Sons, Ltd.

  • simultaneous collision induced Dissociation of the charge reduced parent ion during electron capture Dissociation
    Analytical Chemistry, 2009
    Co-Authors: Jared M Bushey, Takashi Baba, Gary L Glish
    Abstract:

    A method of performing collision induced Dissociation (CID) on the charge-reduced parent ion as it is formed during electron capture Dissociation (ECD), called ECD+CID, is described. In ECD+CID, the charge-reduced parent ion is selectively activated using resonant excitation and collisions with the helium bath gas inside a linear quadrupole ion trap ECD device (ECDLIT). It has been observed that ECD+CID can improve the sequence coverage for β-endorphin over performing ECD alone (i.e., from 72 to 97%). Perhaps just as important, ECD+CID can be used to reduce the extent of multiple electron capture events observed when performing ECD in the ECDLIT. Consequently, the abundance of mass-to-charge ratios corresponding to ECD product ions that contain neutralized protons is decreased, simplifying the interpretation of the product ion spectrum.

  • Simultaneous collision induced Dissociation of the charge reduced parent ion during electron capture Dissociation.
    Analytical chemistry, 2009
    Co-Authors: Jared M Bushey, Takashi Baba, Gary L Glish
    Abstract:

    A method of performing collision induced Dissociation (CID) on the charge-reduced parent ion as it is formed during electron capture Dissociation (ECD), called ECD+CID, is described. In ECD+CID, the charge-reduced parent ion is selectively activated using resonant excitation and collisions with the helium bath gas inside a linear quadrupole ion trap ECD device (ECDLIT). It has been observed that ECD+CID can improve the sequence coverage for β-endorphin over performing ECD alone (i.e., from 72 to 97%). Perhaps just as important, ECD+CID can be used to reduce the extent of multiple electron capture events observed when performing ECD in the ECDLIT. Consequently, the abundance of mass-to-charge ratios corresponding to ECD product ions that contain neutralized protons is decreased, simplifying the interpretation of the product ion spectrum.

  • Fast Multiple Electron Capture Dissociation in a Linear Radio Frequency Quadrupole Ion Trap
    Analytical chemistry, 2007
    Co-Authors: Hiroyuki Satake, Atsumu Hirabayashi, Hideki Hasegawa, Yuichiro Hashimoto, Takashi Baba, Katsuyoshi Masuda
    Abstract:

    We developed a fast electron capture Dissociation (ECD) device using a linear radio frequency-quadrupole (RFQ) ion trap. The device dissociated peptides and proteins using a focused electron beam with an intensity of 0.5 μA and a diameter of 1 mm. The electron capture rate was 13%/ms for doubly charged peptides, and the total amount of ECD products was identical to the theoretical limit, i.e., 50% of incident precursor ions were observed as maximum ECD products by electron irradiation of 7 ms in a pulse counting detection scheme. Coupling this ECD device to a time-of-flight mass spectrometer, we applied multiple ECD. Protonated ubiquitin precursor ions with a charge state of 10 were repeatedly cleaved by ECD, i.e., charge-reduced species and their highly charged fragments were cleaved again and again, creating lower charged products, leaving only singly to triply charged states among the final products. Meanwhile with the amount of electron irradiated, lower charged products increased. Applying an electro...