Dissociation of different conformations of ubiquitin ions

Badman, Ethan R.; Hoaglund-Hyzer, Cherokee S.; Clemmer, David E.

doi:10.1016/s1044-0305(02)00374-4

Cited by 53 publications

(56 citation statements)

References 53 publications

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“…This conformational dependence of ECD spectra is consistent with the strict geometry requirements for excited-state internal conversion before nonergodic dissociation. In contrast, Clemmer and coworkers (48) found that compact and elongated conformers of ubiquitin 8ϩ and 9ϩ ions yield identical collisionally activated dissociation spectra, consistent with lower dissociation energies for noncovalent than for covalent bonds in ergodic dissociation.…”

Section: Ecd Insensitivity To Ion Internal Energymentioning

confidence: 95%

Nonergodic and conformational control of the electron capture dissociation of protein cations

Breuker

Lin

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

162

188

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Electron capture dissociation (ECD) MS is proving to be unusually valuable for ''top down'' protein sequencing and identification͞ localization of posttranslational modifications, because the ECD product ions can represent cleavages between most of a protein's amino acids. As proposed, this unusual reactivity results from immediate local utilization, before randomization, of much of the relatively large (Ϸ6 eV) energy from the electron reaction with the multiply charged protein ion, minimizing the effect of differences in the backbone bond dissociation energies. However, others conclude that e ؊ capture produces a labile free radical species for which backbone cleavage is the lowest energy reaction. Supporting the nonergodic mechanism, ECD of ubiquitin (M ؉ 12H) 12؉ ions also yields thermalized radical (M ؉ 12H) 11؉⅐ ions that instead lose H· when activated. Also, the ECD spectrum of ubiquitin (M ؉ 13H) 13؉ ions is nearly unchanged by heating from 25°C to 125°C, demonstrating that this increase in thermal energy is small compared to the energy driving the reaction. These results support initial capture of the electron in a long-lived high-n Rydberg state, followed by internal conversion to the product valence state at an energy well above the dissociation barriers. The instantaneous conformation of the valence state is critical, with the observed products supporting an ␣-helical structure in which the protonated side chain of each basic residue is intercalated to hydrogen-bond to as many as three amide carbonyls. Activation (e.g., heat, collisions, lowered charge) can disrupt this conformation to allow additional H ؉ -side-chain interactions and provide more complete sequence coverage. E lectron capture dissociation (ECD) mass spectra provide far more extensive characterization of the primary structure of proteins, such as posttranslational modifications, than conventional methods for the dissociation of multiply charged protein cations (1-6). ECD products represent single cleavages between an unusually high proportion of the backbone amino acids (7, 8) (Fig. 1). To explain how hundreds of such reactions are apparently competitive, we have postulated (1-4) that ECD is a nonergodic process, a unimolecular dissociation that occurs before the excitation energy is randomized (9-13). Laser techniques can make such studies in vibrational time periods (11), although energy randomization can require 200 ps (12). Nonergodic dissociation has been observed only for small molecules with a relatively few internal degrees of freedom (9-11); the largest ion found to undergo nonergodic dissociation is the enolic acetone cation, CH 3 OC(OH)ACH 2 ϩ⅐ (12-15). Thus it is not surprising that substantial doubts have been expressed concerning our nonergodicity claim for protein ECD, instead presenting evidence that backbone cleavage of peptide ions from e Ϫ capture is just a free radical reaction of unusually low activation energy (16-21). Here we describe additional experiments that provide evidence for the high energy required and f...

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Section: Ecd Insensitivity To Ion Internal Energymentioning

confidence: 95%

Nonergodic and conformational control of the electron capture dissociation of protein cations

Breuker

Lin

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

162

188

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“…This suggests a complete isomerization and equilibration of the structures prior to dissociation, which is comparable to the conclusions from a previous CID study on mobility-selected ubiquitin conformers. 36 …”

Section: Collisional Activation Of Mobility-selected Ionsmentioning

confidence: 99%

On the Dynamics of Fragment Isomerization in Collision-Induced Dissociation of Peptides

et al. 2008

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The structures of peptide collision-induced dissociation (CID) product ions are investigated using ion mobility/ mass spectrometry techniques combined with theoretical methods. The cross-section results are consistent with a mixture of linear and cyclic structures for both b 4 and a 4 fragment ions. Direct evidence for cyclic structures is essential in rationalizing the appearance of fragments with scrambled (i.e., permutated) primary structures, as the cycle may not open up where it was initially formed. It is demonstrated here that cyclic and linear a 4 structures can interconvert freely as a result of collisional activation, implying that isomerization takes place prior to dissociation.

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“…Previous attempts at analysis of protein mixtures by CID after IM separation suffered because protein ions in high charge states were not resolved in the mobility separation [62]. Thus, mobility labeling for proteins is not as useful as for peptides.…”

Section: And [M ϫ Cf 3 ]mentioning

confidence: 99%

An ion trap-ion mobility-time of flight mass spectrometer with three ion sources for ion/ion reactions

Zhao

Soyk

Schieffer

et al. 2009

J. Am. Soc. Mass Spectrom.

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This instrument combines the capabilities of ion/ion reactions with ion mobility (IM) and time-of-flight (TOF) measurements for conformation studies and top-down analysis of large biomolecules. Ubiquitin ions from either of two electrospray ionization (ESI) sources are stored in a three dimensional (3D) ion trap (IT) and reacted with negative ions from atmospheric sampling glow discharge ionization (ASGDI). The proton transfer reaction products are then separated by IM and analyzed via a TOF mass analyzer. In this way, ubiquitin ϩ7 ions are converted to lower charge states down to ϩ1; the ions in lower charge states tend to be in compact conformations with cross sections down to ϳ880 Å 2 . The duration and magnitude of the ion ejection pulse on the IT exit and the entrance voltage on the IM drift tube can affect the measured distribution of conformers for ubiquitin ϩ7 and ϩ6. Alternatively, protein ions are fragmented by collision-induced dissociation (CID) in the IT, followed by ion/ion reactions to reduce the charge states of the CID product ions, thus simplifying assignment of charge states and fragments using the mobility-resolved tandem mass spectrum. Instrument characteristics and the use of a new ion trap controller and software modifications to control the entire instrument are described. Ion mobility (IM) [4,5] has become a very useful technique for analysis of biological ions in the gas phase [6]. IM provides information about ion size and structure [7], as it rapidly separates ions based on collision cross-section, rather than just m/z ratio. The use of IM to disperse a mixture of ions in time before analysis via a time-of-flight (TOF) MS, i.e., nested drift (flight) time measurements, is an important recent advance. These experiments were pioneered by Clemmer and coworkers in the mid-1990s [8], and have now been used by several other groups [9 -13]. The recent Synapt ESI-IMS-MS by Waters Corp. provides a commercially available instrument for gas-phase ion conformation studies by IM using a novel traveling wave approach [9].In a series of instrumental designs, the Clemmer group has made various modifications to the initial ESI-IM-TOF, including the insertion of a collision cell between the IM drift tube and the TOF for mobility labeling experiments [14,15], and the addition of an IT before the mobility drift tube to improve the duty cycle from the continuous ESI source [16,17]. One publication demonstrated MS/MS capabilities with an ion trap before IM-TOF [18], but the entire instrument was not under computer control. Therefore, only relatively simple experiments were possible.IM has been used to analyze the products of ionmolecule reactions [19], including proton transfer [20,21], H/D exchange [22], and solvation [23][24][25][26]. In these studies, the desired reactions take place either in the atmospheric pressure ion source interface region or in the drift tube itself. Thus, only short reaction times and certain reagent ions can be used. In addition, performing reactions in the IM cell can make spe...

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Dissociation of different conformations of ubiquitin ions

Cited by 53 publications

References 53 publications

Nonergodic and conformational control of the electron capture dissociation of protein cations

Nonergodic and conformational control of the electron capture dissociation of protein cations

On the Dynamics of Fragment Isomerization in Collision-Induced Dissociation of Peptides

An ion trap-ion mobility-time of flight mass spectrometer with three ion sources for ion/ion reactions

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