Probing Protein Structure and Folding in the Gas Phase by Electron Capture Dissociation

Schennach, Moritz; Breuker, Kathrin

doi:10.1007/s13361-015-1088-z

Cited by 24 publications

(39 citation statements)

References 83 publications

(125 reference statements)

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“…Comparing the possible salt bridges and ionic hydrogen bonds (Figure 1b ) to the site-specific yields of c (black bars) and z • (open bars) fragments from ECD of KIX (M + 7H) 7+ ions (Figure 1c , 0 eV) reveals that the possible interactions of R7, K8, H11, and H13 (SB: R7/E12, R7/E60, K8/E60, H11/E60, H13/E12, IHB: R7/N63, K8/W10) were, at least in a significant fraction of the ions, not present as this would have prevented separation of fragments from cleavage at sites 7, 8, and 12 [ 30 ]. However, after vibrational activation for unfolding of the KIX (M + 7H) 7+ ions (Figure 1c , 133 eV), the yield of separated c and z • fragments from cleavage at sites 7 and 12 increased substantially, and fragments from sites 10 and 11 appeared, consistent with an increase in the fraction of ions in which the interactions of R7, K8, H11, and H13 were broken.…”

Section: Resultsmentioning

confidence: 99%

“…For example, the transition from one to two charges for c ions (left axis, the corresponding transition for complementary z • ions is from four to five charges on the right axis) is around site 16 for (M + 7H) 7+ ions but around site 13 for (M + 9H) 9+ ions, and that from three to four charges is around site 45 for (M + 8H) 8+ ions but around site 38 for (M + 9H) 9+ ions. It is generally difficult to pinpoint the exact location of all charged sites in protein (M + nH) n+ ions from ECD data [ 30 , 71 ] as capture of an electron neutralizes a positive charge, and because the presence of zwitterionic motifs that comprise both positively and negatively charged sites cannot be excluded. Moreover, even in unfolded structures, protons can be shared between adjacent residues in homodimeric (e.g., K⋅⋅⋅H + ⋅⋅⋅K) or heterodimeric (e.g., K⋅⋅⋅H + ⋅⋅⋅Q) ionic hydrogen bonds [ 51 ].…”

Section: Resultsmentioning

confidence: 99%

“…Native mass spectrometry (MS) has, over the past 25 years, developed from interpreting mass spectra from electrospray ionization (ESI) of different solutions to approaches by which the dissociation of biomolecules such as proteins and nucleic acids and their noncovalent complexes is studied, or their rotationally averaged collision cross section is probed by ion mobility MS [ 1 – 29 ]. Obtaining information of relevance to biological problems by native ESI MS relies, as a matter of course, on the preservation of solution structure after transfer into the gas phase, where it can be probed by a number of techniques, including electron capture dissociation (ECD) [ 30 , 31 ]. However, the use of native ESI “is not without complications” [ 9 ], not least because the stability of a solution structure in the gas phase can presently not be reliably predicted.…”

Section: Introductionmentioning

confidence: 99%

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Unfolding and Folding of the Three-Helix Bundle Protein KIX in the Absence of Solvent

Schennach

Schneeberger

Breuker

2016

J. Am. Soc. Mass Spectrom.

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Electron capture dissociation was used to probe the structure, unfolding, and folding of KIX ions in the gas phase. At energies for vibrational activation that were sufficiently high to cause loss of small molecules such as NH3 and H2O by breaking of covalent bonds in about 5% of the KIX (M + nH)n+ ions with n = 7–9, only partial unfolding was observed, consistent with our previous hypothesis that salt bridges play an important role in stabilizing the native solution fold after transfer into the gas phase. Folding of the partially unfolded ions on a timescale of up to 10 s was observed only for (M + nH)n+ ions with n = 9, but not n = 7 and n = 8, which we attribute to differences in the distribution of charges within the (M + nH)n+ ions.Graphical Abstractᅟ

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Unfolding and Folding of the Three-Helix Bundle Protein KIX in the Absence of Solvent

Schennach

Schneeberger

Breuker

2016

J. Am. Soc. Mass Spectrom.

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“…To the best of our knowledge, no reports of NECD appeared in the literature for the following decade, and it was only very recently that Kelleher and coworkers extended this approach to the 490 kDa ferritin 24mer . In the past decade, continued experimental and computational work by Breuker and McLafferty has provided a more detailed understanding of the evolution of the structure of small proteins in the gas phase, on a timescale that ranges from picoseconds to minutes . Their model, which represents a stepwise structural evolution, is summarized in Figure .…”

Section: Exd For Interrogation Of Protein Higher Order Structurementioning

confidence: 99%

“…223 In the past decade, continued experimental and computational work by Breuker and McLafferty has provided a more detailed understanding of the evolution of the structure of small proteins in the gas phase, on a timescale that ranges from picoseconds to minutes. 211,[224][225][226][227][228][229][230][231][232] Their model, which represents a stepwise structural evolution, is summarized in Figure 6. It should be noted that both the gas-phase stability of the native structure, as well as the timescale of structural rearrangements, depends on a number of factors.…”

Section: Supplemental Activation Facilitates Cleavage Of the C(α)-c(βmentioning

confidence: 99%

Radical solutions: Principles and application of electron‐based dissociation in mass spectrometry‐based analysis of protein structure

Lermyte

Valkenborg

Loo

et al. 2018

Mass Spectrometry Reviews

130

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In recent years, electron capture (ECD) and electron transfer dissociation (ETD) have emerged as two of the most useful methods in mass spectrometry-based protein analysis, evidenced by a considerable and growing body of literature. In large part, the interest in these methods is due to their ability to induce backbone fragmentation with very little disruption of noncovalent interactions which allows inference of information regarding higher order structure from the observed fragmentation behavior. Here, we review the evolution of electron-based dissociation methods, and pay particular attention to their application in "native" mass spectrometry, their mechanism, determinants of fragmentation behavior, and recent developments in available instrumentation. Although we focus on the two most widely used methods-ECD and ETD-we also discuss the use of other ion/electron, ion/ion, and ion/neutral fragmentation methods, useful for interrogation of a range of classes of biomolecules in positive- and negative-ion mode, and speculate about how this exciting field might evolve in the coming years.

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Highly Charged Protein Ions: The Strongest Organic Acids to Date

et al. 2017

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The basicity of highly protonated cytochrome c (cyt c) and myoglobin (myo) ions were investigated using tandem mass spectrometry,i on-molecule reactions (IMRs), and theoretical calculations as af unction of charge state. Surprisingly,h ighly charged protein ions (HCPI) can readily protonate non-polar molecules and inert gases,i ncluding Ar, O 2 ,a nd N 2 in thermal IMRs.T he most HCPIs that can be observed are over 130 kJ mol À1 less basic than the least basic neutral organic molecules known( tetrafluoromethane and methane). Based on theoretical calculations,itispredicted that protonated cyt ca nd myo ions should spontaneously lose ap roton to vacuum for charge states in which every third residue is protonated. In this study,H CPIs are formed where every fourth residue on average is protonated. These results indicate that protein ions in higher charge states can be formed using al ow-pressure ion source to reduce proton-transfer reactions between protein ions and gases from the atmosphere.Electrospray ionization (ESI) is renowned for its ability to form intact, gaseous,m ultiply charged protein ions for rapid and sensitive detection by mass spectrometry. [1] However,the mechanism by which protein ions are formed in ESI is controversial and continues to be actively debated. Thet wo primary competing models to explain ion formation are known as the charge residue model (CRM) [2] and the ion evaporation model (IEM). [3] In both models,a sn eutral molecules evaporate from ac harged droplet, the electric field at the surface of the droplet increases,w hich initiates fission. [4] Such droplet fission events result in the emission of afine stream of smaller droplets that remove less than 1% of the mass but more than 30 %o ft he charge of the precursor droplet. [4,5] In the CRM, sequential droplet evaporation and Coulombic fission events yield acharged droplet that contains asingle analyte ion, which evaporates to dryness via the loss of neutral solvent molecules.Inthe IEM, the electric field on the surface of ah ighly charged droplet near the moment of ion formation is sufficient to result in the ejection of an analyte ion from the surface of the ionic droplet. Themajority of current evidence indicates that fully desolvated protein ions formed from buffered aqueous solutions are formed by the CRM. Charge carriers such as solvated hydronium ions can be lost via ion evaporation during the ESI process. [6] Recently,the chain-ejection model (CEM), [7] which is related to the IEM, was proposed to explain the formation of protein ions from denaturing solutions based on results from molecular dynamics simulations. [7] In the CEM, ad enatured, disordered protein chain is ejected from ah ighly charged, nanometer-sized ionic droplet. As the protein ion protrudes and is ejected from the droplet, proton transfer to the protein ion can occur. However,t he mechanism by which highly charged protein ions (HCPIs) are formed from denaturing solutions is less well established with evidence supporting both the CRM [8] and CEM/IEM having be...

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Probing Protein Structure and Folding in the Gas Phase by Electron Capture Dissociation

Cited by 24 publications

References 83 publications

Unfolding and Folding of the Three-Helix Bundle Protein KIX in the Absence of Solvent

Unfolding and Folding of the Three-Helix Bundle Protein KIX in the Absence of Solvent

Radical solutions: Principles and application of electron‐based dissociation in mass spectrometry‐based analysis of protein structure

Highly Charged Protein Ions: The Strongest Organic Acids to Date

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