Detailed Unfolding and Folding of Gaseous Ubiquitin Ions Characterized by Electron Capture Dissociation

Breuker, Kathrin; Oh, Han Bin; Horn, David M.; Cerda, Blas A.; McLafferty, Fred W.

doi:10.1021/ja012267j

Cited by 314 publications

(500 citation statements)

References 45 publications

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“…The loss of SH 2 is somewhat surprising, as the loss of a sulfur would be expected to lead to the separation of the two peptide chains. The observed loss of only SH 2 might be explained by either a rearrangement to form a monosulfide bond between the two peptide chains, or by the existence of noncovalent bonding between the chains that might hold them together°after°disulfide°bond°cleavage° [43].°The°dearth of backbone cleavages and the relatively abundant chain ions observed in the reaction product ions of Figure°1°indicate°that°the°disulfide°bonds°are°cleaved preferentially to the backbone bonds upon electron transfer in this reaction. Previous work with ECD has also shown a preference for cleavage of the disulfide bond° [24].…”

Section: Electron Transfer With Dissociationmentioning

confidence: 96%

“…Two hypotheses can be proposed to account for how electron transfer can occur without leading to discrete fragments. In the first, a covalent bond is broken, but the fragments remain bound via noncovalent°interactions° [43].°In°the°second,°a°bond°may°be significantly°weakened°by°the°electron°transfer° [47],°but the dissociation rate is sufficiently slow relative to cooling rates in the ion trap to allow for at least some of the electron transfer products to be stabilized. The latter interpretation assumes that the covalent bonds in the electron transfer products are sufficiently strong to survive under the normal ion trap storage conditions.…”

Section: Electron Transfer Without Dissociationmentioning

confidence: 99%

“…Upon subsequent activation, they fragment to give primarily loss of SH 2 ] ϩ· ions, with respect to the two hypotheses mentioned above. In the ECD cases for which covalent bond cleavage with stabilization by noncovalent bonds has been°proposed° [43],°the°ions°in°question°are°typically much larger whole protein ions in which there are likely to be a substantial number of noncovalent interactions. The relatively small size of the fragments involved here would give rise to relatively few noncovalent interactions between them.…”

Section: Electron Transfer Without Dissociationmentioning

confidence: 99%

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SO₂^−· electron transfer ion/ion reactions with disulfide linked polypeptide ions

Chrisman

Pitteri

Hogan

et al. 2005

J. Am. Soc. Mass Spectrom.

104

122

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Multiply-charged peptide cations comprised of two polypeptide chains (designated A and B) bound via a disulfide linkage have been reacted with SO 2 Ϫ· in an electrodynamic ion trap mass spectrometer. These reactions proceed through both proton transfer (without dissociation) and electron transfer (with and without dissociation). Electron transfer reactions are shown to give rise to cleavage along the peptide backbone, loss of neutral molecules, and cleavage of the cystine bond. Disulfide bond cleavage is the preferred dissociation channel and both Chain A (or B)OS · and Chain A (or B)OSH fragment ions are observed, similar to those observed with electron capture dissociation (ECD) of disulfide-bound peptides. Electron transfer without dissociation produces [M ϩ 2H] ϩ· ions, which appear to be less kinetically stable than the proton transfer [M ϩ H] ϩ product. When subjected to collision-induced dissociation (CID), the [M ϩ 2H] ϩ· ions fragment to give products that were also observed as dissociation products during the electron transfer reaction. However, not all dissociation channels noted in the electron transfer reaction were observed in the CID of the [M ϩ 2H] ϩ· ions. The charge state of the peptide has a significant effect on both the extent of electron transfer dissociation observed and the variety of dissociation products, with higher charge states giving more of each. ( T andem mass spectrometry currently plays a major role in the identification and characterization of proteins [1][2][3]. This application is enabled by the ability to form gaseous ions from peptides and proteins, typically via electrospray ionization [4,5] or matrix-assisted laser desorption ionization [6,7], the ability to form fragments from peptides and proteins of interest that reveal primary structure information, and the ability to measure and detect the fragments. Amino acid sequence information is usually sought for the identification of a protein. However, the identities and locations of post-translational modifications arising from, for example, phosphorylation, glycosylation, and disulfide bonding are also of interest for the complete characterization of the protein of interest. Unimolecular dissociation is the predominant chemical means for deriving primary structure information from a peptide or protein in the gas phase. The extent to which structural information can be derived from a posttranslationally modified peptide or protein depends upon many factors including, for example, charge state and nature of the ion (e.g., protonated versus metal cationized), nature of the modification, and ion activation conditions. The nature of the modification itself can play a major role in directing dissociation chemistry. For this reason, it is of interest to explore various means for both forming and activating post-translationally modified ions. In this study, we focus on the behavior of polypeptide chains bound by a cystine bridge. The formation of such bridges takes place as a protein folds into its native conformation, and the bri...

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Section: Electron Transfer With Dissociationmentioning

confidence: 96%

Section: Electron Transfer Without Dissociationmentioning

confidence: 99%

Section: Electron Transfer Without Dissociationmentioning

confidence: 99%

See 1 more Smart Citation

SO₂^−· electron transfer ion/ion reactions with disulfide linked polypeptide ions

Chrisman

Pitteri

Hogan

et al. 2005

J. Am. Soc. Mass Spectrom.

104

122

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“…Variations in ECD behavior between proteins have been attributed to conformational differences. 11,38 It has been shown that gas-phase structures of a single protein differ between specific charge states and even differ for the same charge state. 39 Increasing Coulomb repulsion through an increase of the number Figure 5.…”

Section: Scheme 1 Reaction Pathways Of Gp31 21+ Upon Activation Usinmentioning

confidence: 99%

Electron Capture Dissociation as Structural Probe for Noncovalent Gas-Phase Protein Assemblies

et al. 2006

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Electron capture dissociation (ECD) of proteins in Fourier transform ion cyclotron resonance mass spectrometry usually leads to charge reduction and backbone-bond cleavage, thereby mostly retaining labile, intramolecular noncovalent interactions. In this report, we evaluate ECD of the 84-kDa noncovalent heptameric gp31 complex and compare this with sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD) of the same protein. Unexpectedly, the 21+ charge state of the gp31 oligomer exhibits a main ECD pathway resulting in a hexamer and monomer, disrupting labile, intermolecular noncovalent bonds and leaving the backbone intact. Unexpectedly, the charge separation over the two products is highly proportional to molecular weight. This indicates that a major charge redistribution over the subunits of the complex does not take place during ECD, in contrast to the behavior observed when using SORI-CAD. We speculate that the ejected monomer retains more of its original structure in ECD, when compared to SORI-CAD. ECD of lower charge states of gp31 does not lead to dissociation of noncovalent bonds. We hypothesize that the initial gas-phase structure of the 21+ charge state is significantly different from the lower charge states. These structural differences result in the different reaction pathways when using ECD.Since the late 1990s, electron capture dissociation 1 (ECD) has been available as a tool for structural analysis in Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). To date, ECD is routinely used for top-down sequencing and identification of the posttranslational modifications, such as sulfide bridges, phosphorylation, and methylation. [2][3][4][5][6][7] During an ECD experiment, low-energy electrons are injected into the ICR cell and captured by multiply protonated ions, resulting in the release of approximately 2-6 eV of recombination energy into the ion. 1,8 Typically, the capture of one or more electrons leads to the rapid dissociation of covalent backbone bonds, resulting in the formation of c and z‚ fragment ions. 9 The ECD process has so far been studied using peptides and proteins with a molecular mass up to 50 kDa 5,10 but not yet for protein complexes or proteins with a higher molecular mass. There is still an active debate about the exact mechanisms of ECD. 8,[11][12][13][14][15][16][17] The early research demonstrated that ECD leads to rapid dissociation of the backbone close to the site of electron capture, not necessarily fragmenting the weakest bonds in the molecule. In line with this observation, ECD has been found to often preserve labile noncovalent bonds, 10 for instance in cytochrome c 18 and vancomycin complexed with diacetyl-L-Lys-D-Ala- This makes ECD a potentially useful technique for examining interaction sites in larger, noncovalent protein complexes.In this report, we use ECD to study the interaction sites in the large gp31 heptameric protein complex from bacteriophage T4. The protein complex has a molecular mass of 84 kDa and co...

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“…18 The second employed an on-axis hollow electron beam for high-rate ECD, while the IR laser beam was kept on-axis. 19 The application area of high-rate ECD/FTICR-MS is growing rapidly, currently covering the de novo sequencing and sequence verification of peptides and proteins, 24 characterization of post-translational modifications in peptides and proteins, 24,31 revealing the secondary and tertiary structures of proteins, 32 studies of protein folding/unfolding dynamics, 33,34 and analysis of complex biological mixtures by on-line and off-line combination of ECD and LC(CE)/FTICR-MS. 27 -29,35 The present paper presents an overview of selected applications of high-rate ECD/FTICR-MS in peptide and protein characterization, preceded by a short description of the basic features of ECD and current experimental configurations.…”

Section: Introductionmentioning

confidence: 99%

Peptide and protein characterization by high‐rate electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry

et al. 2004

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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 transform ion cyclotron resonance mass spectrometry (FTICR-MS). The limitations of the first ECD implementations on commercial instruments were eliminated by the employment of low-energy electron-injection systems based on indirectly heated dispenser cathodes. In particular, the ECD rate and reliability were greatly increased, enabling the combination of ECD/FTICR-MS with on-line liquid separation techniques. Further technique development allowed the combination of two rapid fragmentation techniques, high-rate ECD and infrared multiphoton dissociation (IRMPD), in a single experimental configuration. Simultaneous and consecutive irradiations of trapped ions with electrons and photons extended the possibilities for ion activation/dissociation and led to improved peptide and protein characterization. The application of high-rate ECD/FTICR-MS has demonstrated its power and unique capabilities in top-down sequencing of peptides and proteins, including characterization of post-translational modifications, improved sequencing of peptides with multiple disulfide bridges and secondary fragmentation (w-ion formation). Analysis of peptide mixtures has been accomplished using high-rate ECD in bottom-up mass spectrometry based on mixture separation by liquid chromatography and capillary electrophoresis. This paper summarizes the current impact of high-rate ECD/FTICR-MS for top-down and bottom-up mass spectrometry of peptides and proteins.

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Detailed Unfolding and Folding of Gaseous Ubiquitin Ions Characterized by Electron Capture Dissociation

Cited by 314 publications

References 45 publications

SO₂^−· electron transfer ion/ion reactions with disulfide linked polypeptide ions

SO₂^−· electron transfer ion/ion reactions with disulfide linked polypeptide ions

Electron Capture Dissociation as Structural Probe for Noncovalent Gas-Phase Protein Assemblies

Peptide and protein characterization by high‐rate electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry

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Detailed Unfolding and Folding of Gaseous Ubiquitin Ions Characterized by Electron Capture Dissociation

Cited by 314 publications

References 45 publications

SO2−· electron transfer ion/ion reactions with disulfide linked polypeptide ions

SO2−· electron transfer ion/ion reactions with disulfide linked polypeptide ions

Electron Capture Dissociation as Structural Probe for Noncovalent Gas-Phase Protein Assemblies

Peptide and protein characterization by high‐rate electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry

Contact Info

Product

Resources

About

SO₂^−· electron transfer ion/ion reactions with disulfide linked polypeptide ions

SO₂^−· electron transfer ion/ion reactions with disulfide linked polypeptide ions